™ Voyager ™ Biospectrometry Workstation with Delayed Extraction™ Technology User’s Guide Version 5 Series Software PerSeptive Biosystems, Inc. 500 Old Connecticut Path Framingham, MA 01701 USA A subsidiary of PE Corporation Part Number V900112-02, Rev.
NOTICE PerSeptive Biosystems, Inc. supplies or recommends certain configurations of computer hardware, software, and peripherals for use with its instrumentation. PerSeptive Biosystems reserves the right to decline support for or impose charges for supporting non-standard computer configurations that have not been supplied or recommended by PerSeptive Biosystems.
Table of Contents Table of Contents Safety and Compliance Information ..................................... How to Use This Guide ........................................................... xiii xxvii Chapter 1 Introducing the Voyager™ Biospectrometry™ Workstations 1.1 1.2 1.3 1.4 1.5 1.6 1.7 ................... 1-2 Voyager-DE STR System Overview ............................................. 1-5 MALDI-TOF MS Technology Overview .........................................
Table of Contents Chapter 2 Installing the Voyager™ Biospectrometry™ Workstations 2.1 2.2 2.3 2.4 2.5 iv ............................................................... 2-2 Selecting the Site .................................................................... 2-2 Installing the System 2.2.1 Voyager-DE and Voyager-DE PRO Workstations ................... 2-2 2.2.2 Voyager-DE STR Workstation ................................................ 2-6 Connecting Voyager-DE and Voyager-DE PRO Workstations .
Table of Contents Chapter 3 Preparing Samples 3.1 Preparing Samples 3.1.1 Selecting a Matrix .................................................................. 3-3 3.1.2 Preparing Matrix .................................................................... 3-4 3.1.3 Matrix Information .................................................................. 3-6 3.1.4 Preparing Sample .................................................................3-17 3.1.5 Sample Cleanup ...............................
Table of Contents Chapter 4 Voyager Instrument Control Panel Basics 4.1 4.2 4.3 Instrument Control Panel .......................................................... 4-2 4.1.1 Parts of the Instrument Control Panel .................................... 4-2 4.1.2 Manual and Automatic Control Modes .................................... 4-6 4.1.3 Accessing Sequence Control Panel and Data Explorer .......... 4-7 Using the Control Pages ...........................................................
Table of Contents 5.2 5.3 5.4 Instrument Settings Parameter Descriptions ................................. 5-14 5.2.1 Instrument Settings Page ......................................................5-15 5.2.2 Mode/Digitizer Settings Dialog Box .......................................5-24 5.2.3 Automatic Control Dialog Box ...............................................5-31 Impact of Changing Instrument Settings Parameters ...................... 5-39 5.3.1 Summary of Parameters ...................
Table of Contents Chapter 6 Acquiring Mass Spectra 6.1 6.2 6.3 6.1.1 Overview of Acquisition Options ............................................. 6-2 6.1.2 Guidelines for Acquiring ......................................................... 6-4 6.1.3 Calibrating the Mass Scale .................................................... 6-7 Acquiring in Manual Mode from the Instrument Control Panel ........... 6-11 6.2.1 Manually Acquiring, Evaluating, and Saving Spectra ............. 6-11 6.2.
Table of Contents 6.6.6 6.7 Process that Occurs when Accumulating Spectra from Multiple Search Pattern Positions .................................6-56 6.6.6.1 Process that Occurs when Accumulating All Spectra .....................................6-57 6.6.6.2 Process that Occurs when Accumulating Passing Spectra .............................6-58 Acquiring Spectra from the Sequence Control Panel ...................... 6-60 6.7.1 Understanding Settings, Macros, and Calibration ..................
Table of Contents Chapter 7 PSD Analysis 7.1 7.2 7.3 7.4 7.5 Overview of PSD Analysis ......................................................... 7-2 7.1.1 Post-Source Decay Analysis .................................................. 7-2 7.1.2 Segments and Composite Spectra ......................................... 7-6 7.1.3 PSD Data Files ...................................................................... 7-7 7.1.4 Mass Calculation for Fragment Ions .......................................
Table of Contents Appendix A Specifications ................................................................. A-1 Appendix B Warranty/Service Information .............................. B-1 Appendix C Matrices .............................................................................. C-1 Appendix D Log Sheets ........................................................................ D-1 Appendix E Grid Voltage% and Delay Time Settings ....... E-1 Appendix F Reference Standard Information .................
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Safety and Compliance Information Safety and Compliance Information In this section This section includes: • Instrument safety • Safety and EMC standards • Laser safety Instrument Safety In this section This section includes: • • • • • Notes, Hints, Cautions, and Warnings Notes, Hints, Cautions, and Warnings Safety symbols Before operating this instrument Material Safety Data Sheets (MSDSs) General Warnings Notes, Hints, Cautions, and Warnings are used in this document as follows.
1 Safety and Compliance Information A Caution provides information to avoid damage to the system or loss of data and appears as: CAUTION Do not touch the lamp. This may damage the lamp. A Warning provides specific information essential to the safety of the operator and appears as: WARNING CHEMICAL HAZARD. Wear appropriate personal protection and always observe safe laboratory practices when operating your system.
Safety and Compliance Information Safety symbols The following symbols may be displayed on the system. These symbols may also appear next to associated warnings in this document. Electrical Symbols The following chart is an illustrated glossary of electrical symbols that may be displayed on your instrument. Whenever such symbols appear on instruments, please observe appropriate safety procedures. This symbol indicates the on position of the main power switch.
1 Safety and Compliance Information WARNING: This symbol indicates the presence of high voltage and warns the user to proceed with caution. WARNING: This symbol alerts you to consult the manual for further information and to proceed with caution. Non-electrical Symbols The following is an illustrated glossary of non-electrical safety alert symbols that may be displayed on your instrument. WARNING: This symbol illustrates a heater hazard.
Safety and Compliance Information Symboles des alertes de sécurité Les symboles suivants peuvent être affichés sur le système. Dans ce document, ces symboles peuvent aussi apparaître à côté des avertissements auxquels ils s’associent. Symboles électriques Le tableau suivant donne la signification de tous les symboles électriques qui figurent sur les appareils. En présence de l’un de ces symboles, il est impératif de se conformer aux consignes de sécurité appropriées.
1 Safety and Compliance Information AVERTISSEMENT: Indique la présence d’une haute tension et avertit l’utilisateur de procéder avec précaution. AVERTISSEMENT: Avertit l’utilisateur de la nécessité de consulter le manuel pour obtenir davantage d’informations et de procéder avec précaution. Symboles non électriques Le tableau suivant donne la signification des symboles d’alertes de sécurité non électriques qui figurent sur les appareils.
Safety and Compliance Information Before operating this instrument Ensure that anyone involved with the operation of the instrument is instructed in both general safety practices for laboratories and specific safety practices for the instrument. Make sure you have read and understood all related Material Safety Data Sheets. Material Safety Data Sheets (MSDSs) Some of the chemicals that may be used with your system are listed as hazardous by their manufacturer.
1 Safety and Compliance Information General Warnings WARNING FIRE HAZARD. Using a fuse of the wrong type or rating can cause a fire. Replace fuses with those of the same type and rating. AVERTISSEMENT DANGER D’INCENDIE. L’usage d’un fusible de type ou de valeur nominale différents risque de provoquer un incendie. Il convient donc de remplacer les fusibles usagés par des fusibles du même type et de la même valeur nominale. WARNING LASER HAZARD. The laser emits ultraviolet radiation.
Safety and Compliance Information 1 WARNING ELECTRICAL SHOCK HAZARD. Severe electrical shock can result by operating the instrument without the front or side panels. Do not remove instrument front or side panels. High voltage contacts are exposed with front or side panels removed. AVERTISSEMENT RISQUE DE DÉCHARGE ÉLECTRIQUE. Des décharges électriques sérieuses peuvent résulter du fonctionnement de l’appareil lorsque le panneau avant et les panneaux latéraux sont retirés.
1 Safety and Compliance Information WARNING PHYSICAL INJURY HAZARD. Use the Voyager Biospectrometry Workstation only as specified in this document. Using this system in a manner not specified may result in injury or damage to the system. AVERTISSEMENT DANGER DE BLESSURES CORPORELLES.Veuillez suivre avec attention les indications figurant dans ce document lorsque vous utilisez la Station de Travail de Biosptectrométrie Voyager.
Safety and Compliance Information 1 Safety and EMC (Electromagnetic Compliance) Standards US Safety and EMC Standards Safety This instrument has been tested to and complies with standard ANSI/UL 3101-1, “Electrical Equipment for Laboratory Use; Part 1: General Requirements”, 1st Edition. It is an ETL Testing Laboratories listed product. EMC This device complies with Part 15 of the FCC Rules.
1 Safety and Compliance Information Canadian Safety and EMC Standards Safety This instrument has been tested to and complies with standard CSA 1010, “Safety Requirements for Electrical Equipment for Measurement, Control, and Laboratory Use; Part 1: General Requirements”. It is an ETL Testing Laboratories listed product.
Safety and Compliance Information 1 Laser Safety Laser classification The Voyager™ Biospectrometry™ Workstation uses a standard nitrogen laser and an optional Nd:YAG laser. Under normal operating conditions, the instrument laser is categorized as a Class I laser.
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How to Use This Guide 1 How to Use This Guide Purpose of this guide Audience Structure of this guide PerSeptive Biosystems’ Voyager Biospectrometry Workstation User’s Guide details the procedures for installing, using, maintaining, and troubleshooting Voyager™ Biospectrometry™ workstations. This guide is intended for novice and experienced Voyager workstation users who are analyzing biomolecules. PerSeptive Biosystems’ Voyager Biospectrometry Workstation User’s Guide is divided into chapters.
1 How to Use This Guide Chapter 6, Acquiring Mass Spectra Describes how to use the Voyager processing software, including labeling peaks, calibrating, calculating resolution, and calculating signal-to-noise ratio. Chapter 7, PSD Analysis Describes using PSD analysis software, and exploring the impact of system settings on the quality of data obtained. Chapter 8, Maintenance and Troubleshooting Lists routine maintenance procedures performed by PerSeptive Biosystems.
How to Use This Guide 1 Appendix I, Using the Oscilloscope and Control Stick Related documentation Reference documentation Describes using the Control stick to move sample position, start and stop the laser, and start and stop acquisition, These related documents are shipped with your system: • Voyager™ Biospectrometry™ Workstation Getting Started Guide —Use this guide to learn the basics of operating the system. It provides step-by-step information for running your first experiment.
1 How to Use This Guide You also receive the appropriate manual for the digitizer included with your system: • • • • Send us your comments Signatec Digitizer Manual Precision Instruments Digitizer Manual LeCroy ™ Embedded Signal Analysis Products Operator’s Manual LSA1000 Series and LeCroy™ Embedded Signal Analysis Products Remote Control Manual LSA1000 Series GPIB Software Reference Manual + Tek Manuals We welcome your comments and suggestions for improving our manuals.
1 Introducing the Voyager™ Biospectrometry ™ Workstations Chapter 1 This chapter contains the following sections: 1.1 Voyager-DE and Voyager-DE PRO System Overview ..................................... 1-2 1.2 Voyager-DE STR System Overview .............. 1-5 1.3 MALDI-TOF MS Technology Overview 1.4 Voyager-DE™ (Delayed Extraction™) Technology ............................................ 1-11 1.5 Parts of the Voyager-DE and Voyager-DE PRO Systems ........................ 1-17 1.
Chapter 1 Introducing the Voyager™ Biospectrometry™ Workstations 1 1.1 Voyager ™-DE and Voyager-DE PRO System Overview The Voyager™-DE and Voyager-DE PRO Biospectrometry™ Workstations are designed for use by mass spectrometrists, biochemists, molecular biologists, and life scientists. Voyager-DE The PerSeptive Biosystems’ Voyager-DE Biospectrometry Workstation (Figure 1-1) is a benchtop MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) mass spectrometer.
Voyager™-DE and Voyager-DE PRO System Overview Voyager-DE PRO The Voyager-DE™ PRO Biospectrometry™ Workstation (Figure 1-2) is a benchtop MALDI-TOF (matrix-assisted laser desorption time-of-flight) mass spectrometer that includes a reflector analyzer. Voyager Delayed Extraction™ technology provides improved resolution and mass accuracy.
Chapter 1 Introducing the Voyager™ Biospectrometry™ Workstations Features 1 Features of the Voyager-DE and Voyager-DE PRO Biospectrometry Workstations include: • Positive or negative ion detection • m/z range in excess of 300 kDa • Sensitivity to less than 5 femtomoles with dried droplet application • Ion path length: • Voyager-DE—1.2 meter • Voyager-DE PRO in linear mode—1.3 meter • Voyager-DE PRO in reflector mode—2.
Voyager-DE STR System Overview 1.2 Voyager-DE STR System Overview Voyager-DE STR 1 The Voyager™-DE STR Biospectrometry™ Workstation (Figure 1-3) is a floor-standing MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) mass spectrometer that includes a reflector analyzer.
Chapter 1 1 Introducing the Voyager™ Biospectrometry™ Workstations Biospectrometry Biospectrometry is the application of mass spectrometry in the field of the life sciences. This field uses fast chromatographic techniques, enzymatic chemistries, and surface chemistries and combines them with mass spectrometry and advanced software to better enable biomolecular research and facilitate data interpretation.
MALDI-TOF MS Technology Overview 1.3 MALDI-TOF MS Technology Overview Mass spectrometry 1 Mass spectrometry measures the mass of molecules by measuring the mass-to-charge ratio (m/z). Mass is a molecular attribute that can help identify or confirm the identity of a molecule. Molecular weight measurements by mass spectrometry are based upon the production, separation, and detection of molecular ions.
Chapter 1 1 Introducing the Voyager™ Biospectrometry™ Workstations Matrix-Assisted Laser Desorption Ionization (MALDI) In Matrix-Assisted Laser Desorption Ionization (MALDI), sample is embedded in a low molecular weight, UV-absorbing matrix that enhances sample ionization. The role of the matrix is to facilitate intact desorption and ionization of the sample. The matrix is present in vast excess of sample, and therefore isolates individual sample molecules.
MALDI-TOF MS Technology Overview For acquiring TOF spectra, time measurement starts: • Delayed Extraction mode—Measurement of the ion signal starts when the extraction pulse is applied. The time at which the extraction pulse is applied is user-settable. See Section 1.4, Voyager-DE™ (Delayed Extraction™) Technology for more information. • Continuous Extraction mode—The extraction field is continuously applied. Measurement of the ion signal starts when the laser pulses.
Chapter 1 Introducing the Voyager™ Biospectrometry™ Workstations Drift time is proportional to the square root of the mass as defined by the following equation: 1 ½ ( ) m t=s (2KE)z where: t s m KE z = = = = = drift time drift distance mass kinetic energy number of charges on ion Approximate ion mass is determined using the equation above.
Voyager-DE™ (Delayed Extraction™) Technology 1.4 Voyager-DE™ (Delayed Extraction ™) Technology In this section This section describes: • • • • • Limitations of MALDI technology Limitations of MALDI technology Delayed Extraction Delayed Extraction versus Continuous Extraction Benefits of Delayed Extraction Velocity focusing In traditional MALDI, ions exhibit a broad kinetic energy distribution which is largely due to the initial velocity imparted to ions during the desorption/ionization process.
Chapter 1 Introducing the Voyager™ Biospectrometry™ Workstations Delayed Extraction 1 With Voyager-DE™ (Delayed Extraction™) technology, ions form in a field-free region, and then are extracted by applying a high voltage pulse to the accelerating voltage after a predetermined time delay. Figure 1-6 compares Delayed and Continuous Extraction.
Voyager-DE™ (Delayed Extraction™) Technology In Continuous Extraction mode: 1 • Accelerating voltage is continuously applied, and the potential gradient exists when sample is ionized • Ions are immediately accelerated Figure 1-7 and Figure 1-8 show the improved resolution obtained in Delayed Extraction mode.
Chapter 1 Introducing the Voyager™ Biospectrometry™ Workstations Benefits of Delayed Extraction 1 Delayed Extraction of ions overcomes many of the adverse effects of Continuous Extraction: Benefits of Delayed Extraction Velocity focusing of ions is controlled by variable-voltage grid in the ion source and the delay time applied to acceleration. See “Velocity focusing” on page 1-15. Adverse Effects of Continuous Extraction Initial velocity distribution of ions.
Voyager-DE™ (Delayed Extraction™) Technology Velocity focusing Delayed Extraction technology facilitates tuning modes, when the time-of-flight of an ion is independent of the initial velocity. After ions are released from the sample surface, their position in the ion source is correlated with their initial velocity. When the extraction voltage pulse is applied, initially slower ions acquire slightly higher energy from the accelerating field than initially faster ions.
Chapter 1 Introducing the Voyager™ Biospectrometry™ Workstations 1 Detection Voltage (U) Extraction delay and the magnitude of the extraction pulse can set that ions of a given massto-charge ratio with different initial velocity reach the detector exactly at the same time. + + t slow = t Detector fast PB100773 Figure 1-10 Velocity Focusing of Ions in Linear Mode —Detection Reflector mode 1-16 In Reflector mode, ions are velocity-focused at the exit of the ion source instead of at the detector.
Parts of the Voyager-DE and Voyager-DE PRO Systems 1.5 Parts of the Voyager-DE and Voyager-DE PRO Systems 1 This section describes: • • • • • • System components Mass spectrometer Vacuum system Vacuum gauge panel Front panel controls and indicators Computer components 1.5.1 System Components Voyager-DE The Voyager-DE Biospectrometry Workstation is shown in Figure 1-11.
Chapter 1 1 Introducing the Voyager™ Biospectrometry™ Workstations Voyager-DE PRO The Voyager-DE PRO Biospectrometry Workstation is shown in Figure 1-12.
Parts of the Voyager-DE and Voyager-DE PRO Systems Parts of the Voyager-DE and Voyager-DE PRO systems include: 1 • Mass spectrometer—A time-of-flight mass spectrometer, described in Section 1.5.2, Mass Spectrometer. The high-vacuum system of the mass spectrometer is described in Section 1.5.3, Vacuum System. • Computer/Data System—A computer that operates the Voyager control software and the Voyager processing software. You control the mass spectrometer using the computer.
Chapter 1 1 Introducing the Voyager™ Biospectrometry™ Workstations 1.5.2 Mass Spectrometer Voyager-DE The parts of the Voyager-DE Biospectrometry Workstation mass spectrometer are shown in Figure 1-13.
Parts of the Voyager-DE and Voyager-DE PRO Systems Voyager-DE PRO The parts of the Voyager-DE PRO Biospectrometry Workstation mass spectrometer are shown in Figure 1-14.
Chapter 1 1 Introducing the Voyager™ Biospectrometry™ Workstations Parts of the mass spectrometer The Voyager-DE and Voyager-DE PRO mass spectrometers include: • Laser, attenuator, and prism—A nitrogen laser that operates at 337 nm and ionizes sample. It produces 3-nanosecond-wide pulses (factory set to 3 Hz). Laser rate is not user-settable. The laser attenuator varies the intensity of the laser beam reaching the sample. The prism deflects the laser beam into the ion source.
Parts of the Voyager-DE and Voyager-DE PRO Systems • Linear detector—A device that detects ions that travel down the flight tube. The linear detector measures ion abundance over time and sends a signal to the digitizer for conversion. On the Voyager-DE PRO system, the linear detector is used in Linear mode only. It is not used in Reflector or PSD mode. Linear detectors are hybrid high-current detectors consisting of a single microchannel plate, a fast scintillator, and a photomultiplier.
Chapter 1 Introducing the Voyager™ Biospectrometry™ Workstations The single-stage design provides high mass resolution across a wide mass range and highly accurate mass measurements. Accurate calibration formulas for the single-stage reflector allow you to vary instrument conditions without degrading mass accuracy. Also, easy calibration of PSD data is facilitated by single-stage reflectors. For more information, see Chapter 7, PSD Analysis.
Parts of the Voyager-DE and Voyager-DE PRO Systems 1.5.3.
Chapter 1 1 Introducing the Voyager™ Biospectrometry™ Workstations Vacuum pumps Two vacuum pumps create the vacuum environment: • Fore pump—Creates a vacuum in the sample loading chamber, creates a lower-than-atmospheric-pressure condition before the turbo pump starts, and provides backing pressure to the turbo pump. • Turbo pump—Creates a high vacuum condition in the main source chamber. Vacuum is maintained in the chambers by valves that isolate the chambers.
Parts of the Voyager-DE and Voyager-DE PRO Systems 1.5.3.
Chapter 1 1 Introducing the Voyager™ Biospectrometry™ Workstations Vacuum pumps Three vacuum pumps create the vacuum environment: • Fore pump—Creates a vacuum in the sample loading chamber, creates a lower-than-atmospheric-pressure condition before the turbo pumps start, and provides backing pressure to the turbo pumps. • Turbo pump 1—Creates a high vacuum condition in the main source chamber. • Turbo pump 2—Creates a high vacuum condition in the mirror chamber.
Parts of the Voyager-DE and Voyager-DE PRO Systems 1.5.4 Vacuum Gauge Panel 1 The Vacuum Gauge Panel (Figure 1-17) is located on the right front of the Voyager-DE and Voyager-DE PRO mass spectrometer cabinet. ATM 1.0 TC .1 1 .001 EMIS 1.2 -7 TORR Chan EMS BA 1 PB100270 Figure 1-17 Vacuum Gauge Panel CAUTION Do not press any other buttons on the panel. Pressing buttons other than the Chan and EMIS buttons can recalibrate the pressure scale of the system.
Chapter 1 1 Introducing the Voyager™ Biospectrometry™ Workstations Gauge Measures Expected Pressure BA2 Pressure in mirror chamber (Voyager-DE PRO only) Less than 2x10-7 TC2 Pressure in sample loading chamber Less than 10-2 during operation. Higher when loading or ejecting sample plate. TC1, TC3, TC4 Not used, displays E03 (indicates gauge not connected) ______ • EMIS—Turns BA1 and BA2 on or off. Used during troubleshooting only.
Parts of the Voyager-DE and Voyager-DE PRO Systems 1.5.5 Computer Components Hardware 1 The Voyager-DE and Voyager-DE PRO Biospectrometry Workstations include the following IBM®-compatible computer hardware components: • Minimum computer configuration of Pentium® II 350 MHz, with 4.3 GB hard disk and 128 MB RAM (random access memory) • 3.5-inch HD (high density) 1.
Chapter 1 Introducing the Voyager™ Biospectrometry™ Workstations 1 1.6 Parts of the Voyager-DE STR System This section describes: • • • • • • System components Mass spectrometer Vacuum system Vacuum gauge panel Front panel indicators Computer components 1.6.1 System Components The Voyager-DE STR Biospectrometry Workstation is shown in Figure 1-18.
Parts of the Voyager-DE STR System Parts of the Voyager-DE STR system include: 1 • Mass spectrometer—A time-of-flight mass spectrometer, described in Section 1.6.2, Mass Spectrometer. The high-vacuum system of the mass spectrometer is described in Section 1.6.3, Vacuum System. • Computer/Data System—A computer that operates the Voyager control software and the Voyager processing software. You control the mass spectrometer using the computer.
Chapter 1 1 Introducing the Voyager™ Biospectrometry™ Workstations 1.6.2 Mass Spectrometer The parts of the Voyager-DE STR Biospectrometry mass spectrometer are shown in Figure 1-19.
Parts of the Voyager-DE STR System Parts of the mass spectrometer The Voyager-DE STR mass spectrometer includes: • Laser, attenuator, and prism—A nitrogen laser that operates at 337 nm and ionizes sample. It produces 3-nanosecond-wide pulses (factory set to 3 Hz). Laser rate is not user-settable. The laser attenuator varies the intensity of the laser beam reaching the sample. The prism deflects the laser beam into the ion source. • Ion Source—A high voltage region used to accelerate ions.
Chapter 1 Introducing the Voyager™ Biospectrometry™ Workstations • Vacuum system—A pumping system and a sealed enclosure that creates and maintains a high-vacuum environment for unobstructed ion drift. For more information, see Section 1.6.3, Vacuum System. 1 • Flight tube and beam guide wire—A field-free region (no additional accelerating forces are present) in which ions drift at a velocity inversely proportional to the square root of their masses.
Parts of the Voyager-DE STR System The single-stage design provides high mass resolution across a wide range and highly accurate mass measurements. Accurate calibration formulas for the single-stage reflector allow the user to vary instrument conditions without degrading mass accuracy. Also, easy calibration of PSD data is facilitated by single-stage reflectors. For more information, see Chapter 7, PSD Analysis. • Reflector detector—The reflector detector measures ions reflected by the mirror.
Chapter 1 1 Introducing the Voyager™ Biospectrometry™ Workstations 1.6.3 Vacuum System Overview The Voyager-DE STR Biospectrometry Workstation provides a high-vacuum environment for time-of-flight analysis.
Parts of the Voyager-DE STR System 1 BA2 Mirror chamber (high-vacuum) Turbo pump 2 Foreline valve 2 TC2 Fore pump Foreline valve 1 BA1 Turbo pump 1 Main source chamber (high-vacuum) Sample loading chamber (low-vacuum) Figure 1-20 Voyager-DE STR Biospectrometry Workstation Vacuum Chambers (Top View) Voyager™ Biospectrometry™ Workstation User’s Guide 1-39
Chapter 1 1 Introducing the Voyager™ Biospectrometry™ Workstations Vacuum gauges The Voyager-DE STR Biospectrometry Workstation include three vacuum gauges: • BA1 (Bayard-Alpert Gauge)—Monitors pressure in the main source chamber. Called BA1 on Vacuum Gauge Panel. • BA2 (Bayard-Alpert Gauge)—Monitors pressure in the mirror chamber. Called BA2 on Vacuum Gauge Panel. • TC2—Monitors pressure in the sample loading chamber. Readings from the vacuum gauges are displayed: • On the Pressure Gauge Panel.
Parts of the Voyager-DE STR System 1 CAUTION Do not press any other buttons on the panel. Pressing buttons other than the Chan and EMIS buttons can recalibrate the pressure scale of the system. You use two buttons on the pressure gauge panel: • Chan—Toggles through readings for: Gauge Measures Expected Pressure BA1 Pressure in main source chamber Less than 5 x 10 -7 BA2 Pressure in mirror chamber Less than 5 x10-8 TC2 Pressure in sample loading chamber Less than 10-2 during operation.
Chapter 1 1 Introducing the Voyager™ Biospectrometry™ Workstations 1.6.5 Front Panel Indicators The front panel of the Voyager-DE STR system is shown in Figure 1-22. PerSeptive Biosystems Vestec Mass Spectrometery Products LASER Nd YAG N2 TURBO PUMPS SOURCE START UP ENABLED NORMAL SYSTEM HIGH VOLTAGE LOGIC INTERLOCK ENABLE FAULT REFLECTOR PB100267 Figure 1-22 Front Panel Indicators Indicator When Lit Laser Laser N2 (Red) Indicates that the laser power is on.
Parts of the Voyager-DE STR System Indicator When Lit 1 System High Voltage Indicates high voltage is on. Interlock Indicates an interlock error (door open or panel off). Automatically disables laser and high voltage. Logic Internal control board in mass spectrometer has been powered up. Enabled Computer is controlling mass spectrometer. 1.6.
Chapter 1 Introducing the Voyager™ Biospectrometry™ Workstations Software 1 The Voyager-DE STR Biospectrometry Workstation includes the following software components: • Microsoft Windows NT version 4.
Software Overview 1.7 Software Overview 1 The Voyager Biospectrometry Workstation software includes control software (Voyager Instrument Control Panel, Voyager Sequence Control Panel) and post-processing software (Data Explorer software): • Voyager Instrument Control Panel—Controls the mass spectrometer for calibration and acquisition of single samples.
Chapter 1 Introducing the Voyager™ Biospectrometry™ Workstations • Ability to zoom in on up to four different areas of a trace. 1 • Ability to acquire single samples in Manual or Automatic Control mode. • Manual accumulation of mass spectra from multiple acquisitions into a single data file. The Instrument Control Panel (Figure 1-23) is displayed when you start the Voyager Control Panel software. The Instrument Control software is described in Chapter 4, Voyager Instrument Control Panel Basics.
Software Overview Sequence Control Panel The Sequence Control Panel works in conjunction with the Instrument Control Panel to allow you to acquire multiple samples using different instrument settings (.BIC) files. The Sequence Control Panel (Figure 1-24) is displayed when you start the Voyager Sequence Control software, or click a toolbar button in the Instrument Control Panel.
Chapter 1 1 Introducing the Voyager™ Biospectrometry™ Workstations 1.7.2 Post-Processing Software (Data Explorer) Data Explorer software is a powerful software module that allows you to graphically and interactively manipulate spectral data. Using the Data Explorer software, you can: • Automatically and manually calibrate spectrum data. • Set peak detection parameters and custom labels for regions of the trace.
2 Installing the Voyager™ Biospectrometry™ Workstations Chapter 2 This chapter contains the following sections: 2.1 Installing the System........................................ 2-2 2.2 Selecting the Site............................................. 2-2 2.3 Connecting Voyager-DE and Voyager-DE PRO Workstations ....................... 2-7 2.4 Connecting the Voyager-DE STR Workstation........................ 2-19 2.5 Installing Software ......................................... 2-24 2.
Chapter 2 Installing the Voyager™ Biospectrometry™ Workstations 2.1 Installing the System Your Voyager Biospectrometry Workstation is initially installed by a PerSeptive Biosystems Technical Representative. Do not use the Voyager system before it is properly installed. Use the information in this chapter if you move the Voyager system. 2 2.2 Selecting the Site This section includes: • Voyager-DE and Voyager-DE PRO Workstations • Voyager-DE STR Workstation 2.2.
Selecting the Site Allow 4 inches (10 cm) on the right side of the mass spectrometer for cables. Allow an additional 40 inches (102 cm) to the right side of the mass spectrometer for: • • • • Weight Video monitor Computer, monitor, control stick, and keyboard Printer Optional oscilloscope or external digitizer The Voyager-DE system weighs approximately 250 pounds (113 kg). The Voyager-DE PRO system weighs approximately 350 pounds (159 kg).
Chapter 2 Installing the Voyager™ Biospectrometry™ Workstations To select input voltage: 1. Remove the power cord from the mass spectrometer. 2. Carefully remove the voltage selector/fuse holder from the system (Figure 2-1) using a small flat-blade screw driver. 3. Carefully remove the voltage selector from the holder and insert the selector with the proper voltage displayed in the window of the holder. 2 CAUTION The plastic tabs that hold the voltage selector in place are fragile.
Selecting the Site WARNING FIRE HAZARD. Using a fuse of the wrong type or rating can cause a fire. Replace fuses with those of the same type and rating. 4. Insert two fuses of the proper rating for the selected voltage. 2 Electrical Rating Volts/Amps Fuse (5 x 20 mm) 100 V~10A T10A 250V 120 V~10A T10A 250V 220 V~6.3A T6.3A 250V 240 V~5A T5A 250V 5. Insert the voltage selector/fuse holder into the receptacle. 6. Plug in the mass spectrometer.
Chapter 2 Installing the Voyager™ Biospectrometry™ Workstations 2.2.2 Voyager-DE STR Workstation In this section This section includes: • Required space • Weight • Power/voltage requirements Required space 2 The Voyager-DE STR system is a floor-standing unit that measures: • 34 inches (87 cm) deep • 94 inches (239 cm) wide • 46 inches (117 cm) high The Voyager-DE STR Workstation is constructed on a rolling base.
Connecting Voyager-DE and Voyager-DE PRO Workstations CAUTION Before operation, internal jumpers must be set to accommodate your power source. Do not plug in or power up the Voyager-DE STR Biospectrometry Workstation unless it has been configured correctly by a PerSeptive Biosystems Technical Representative. In addition, you need grounded outlets for: • • • • 2 Computer CPU Computer monitor External digitizer (if your system includes one) Printer (optional) 2.
Chapter 2 Installing the Voyager™ Biospectrometry™ Workstations 2.3.1 Side Panel Diagrams for Mass Spectrometer and Computer This section includes diagrams for: • Mass spectrometer • Computer Mass spectrometer 2 Figure 2-2 shows the connections on the right side panel of the Voyager-DE and Voyager-DE PRO mass spectrometer cabinet.
Connecting Voyager-DE and Voyager-DE PRO Workstations Computer Depending on your digitizer option, the computer will have one of the following installed when you receive it: • Signatec 500 MHz digitizer board • Dedicated ethernet board for LeCroy® LSA 1000 digitizer • GPIB board for oscilloscope option Figure 2-3 shows the rear panel of the computer. Figure 2-4 shows the boards that may be installed in your computer, depending on the digitizer option on your system.
Chapter 2 Installing the Voyager™ Biospectrometry™ Workstations PB100784 CH1 CH2 TRIG Signatec Board Not used Tektronix Oscilloscope IEEE-488 (GPIB) 2 LSA 1000 LeCroy Dedicated ethernet network connection Figure 2-4 Digitizer Options 2-10 PerSeptive Biosystems
Connecting Voyager-DE and Voyager-DE PRO Workstations 2.3.2 Connecting the Mass Spectrometer to the Computer Refer to the following table when connecting the mass spectrometer to the computer.
Chapter 2 Installing the Voyager™ Biospectrometry™ Workstations 2.3.3 Connecting the Signatec 500 MHz Digitizer Board If you have an oscilloscope or an external digitizer on your system, disregard this section. CAUTION Do not use the Signatec 500 MHz digitizer board without digitizer signal protection circuits (blue boxes on cables). Operation without digitizer signal protection circuits will result in damage to the internal digitizer.
Connecting Voyager-DE and Voyager-DE PRO Workstations 2.3.4 Connecting the LSA1000 LeCroy Digitizer If you have an oscilloscope or an internal digitizer on your system, disregard this section. This section describes the features of the LSA1000 LeCroy digitizer that has been previously installed by a PerSeptive Biosystems Technical Representative.
Chapter 2 Installing the Voyager™ Biospectrometry™ Workstations Refer to the following table when connecting the LSA1000 LeCroy digitizer: Connection on digitizer (see Figure 2-5) 2 10/100 Base-T Connection on right side panel of Mass Spectrometer (see Figure 2-2) None Connection on rear panel of computer (see Figure 2-4) Dedicated digitizer network card NOTE: Connect the digitizer to the dedicated network card installed in the slot, not the network connector located below the slots.
Connecting Voyager-DE and Voyager-DE PRO Workstations 2.3.5 Connecting the Tektronix Oscilloscope If you have an internal digitizer board or external digitizer in your computer, disregard this section. Figure 2-6 shows the front panel of the oscilloscope.
Chapter 2 Installing the Voyager™ Biospectrometry™ Workstations Refer to the following table when connecting the oscilloscope.
Connecting Voyager-DE and Voyager-DE PRO Workstations 2.3.6 Connecting the Video Monitor Figure 2-7 and Figure 2-8 show the rear panels of available models of video monitors.
Chapter 2 Installing the Voyager™ Biospectrometry™ Workstations Refer to the following table when connecting the video monitor to the Voyager-DE and Voyager-DE PRO workstations. Connection on rear panel of video monitor (see Figure 2-7 or Figure 2-8) 2 Connection on side panel of Mass Spectrometer (see Figure 2-2) Cable Video INPUT VIDEO BNC with video adapter Power receptacle Video monitor power receptacle (or wall power receptacle) Power 2.3.
Connecting the Voyager-DE STR Workstation 2.4 Connecting the Voyager-DE STR Workstation Figure 2-9 shows the connections and the on/off switch (main power circuit breaker) on the rear panel of the mass spectrometer cabinet.
Chapter 2 Installing the Voyager™ Biospectrometry™ Workstations I/O CTRL Printer (LPT1 or parallel) Serial or COM 1 2 I/O POWER VAC GAUGE CTRL (Serial 2 or COM 2) Control stick Mouse STP MTR CTRL Audio in/out (not used) Keyboard PB100776 VGA Braided Ground Cable (to rear of mass spec) Network Digitizer options (see Figure 2-4) Figure 2-10 Computer Connections for Voyager-DE STR Mass Spectrometer Device 2-20 Connection Keyboard 5-pin round connector VGA monitor 12-pin connector (3 row
Connecting the Voyager-DE STR Workstation NOTE: The computer layout may change without notice. Boards may be located in different slots than those shown in Figure 2-10. The braided ground cable connection may be located in a different position. Oscilloscope Figure 2-11 shows the front panel of the oscilloscope. The CH1, CH2 and Ch3 (or Aux 1) input cables are all brought out through the center hole in the front panel of the Voyager-STR workstation.
Chapter 2 Installing the Voyager™ Biospectrometry™ Workstations Video monitors Figure 2-12 and Figure 2-13 show the rear panels of available models of video monitors.
Connecting the Voyager-DE STR Workstation NOTE: In the US only, you can plug the video monitor into a grounded wall outlet or into the receptacle on the mass spectrometer.
Chapter 2 Installing the Voyager™ Biospectrometry™ Workstations 2.5 Installing Software All necessary software is installed on your Voyager workstation when it is shipped to you. Use these instructions to reinstall software or install a new version of software. The Voyager software requires a total of 100 MB of free disk space plus additional space for data files.
Installing Software 2.5.1 Installing the Voyager Software Installing To install the Voyager software: 1. Insert the Voyager CD into the CD drive in the computer. The installation routine automatically starts and the Welcome dialog box appears. NOTE: If the installation routine does not automatically start, click Start on the Windows NT desktop, click Run, type D:\VOYAGER\SETUP (or the drive designation for your CD drive), and click OK. 2. Click Next.
Chapter 2 Installing the Voyager™ Biospectrometry™ Workstations • User Guides—Includes PDF (portable document format) versions of the Voyager documentation that you can view online using Adobe® Acrobat® Reader. 3. Click Next. 4. A series of dialog boxes in which you specify the hardware options and serial number for your system are displayed. Leave settings at the defaults, or change the settings if needed. Click Next. 2 The Select Program Folder dialog box appears. 5.
Installing Software 2.5.2 Starting the Software Starting Instrument Control Panel To start the Voyager Instrument Control Panel from the Windows NT desktop, double-click the Voyager Control Panel icon on the desktop. The Instrument Control Panel is displayed (Figure 2-14). NOTE: If the Instrument Control Panel is not displayed as shown in Figure 2-14, select Instrument Page Control from the View menu, then select Default Layout for control mode.
Chapter 2 Installing the Voyager™ Biospectrometry™ Workstations Starting Sequence Control Panel To start the Sequence Control Panel from the Windows NT desktop: 1. Make sure the Instrument Control Panel is running. NOTE: If you start the Instrument Control Panel using the Sequence Control Panel icon, it functions the same way as if you started it using the Instrument Control Panel icon, with two exceptions.
Installing Software 2.5.3 Exiting the Software CAUTION If you are using the Voyager Workstation and you exit the Voyager Instrument Control software, you can no longer control the workstation. Do not exit the Voyager software until you have finished using the workstation. Sequence Control Panel To exit the Sequence Control software: 1. In the Sequence Control window, select Exit from the File menu. A message is displayed. Click Yes. The Sequence Control software closes.
Chapter 2 Installing the Voyager™ Biospectrometry™ Workstations 2.6 Hardware Configuration CAUTION Do not alter the Hardware Configuration unless instructed to do so by a PerSeptive Biosystems Technical Representative. Altering these settings may cause your Voyager Biospectrometry Workstation to function improperly.
Hardware Configuration . 2 Figure 2-16 Vacuum Configuration 3. Check the following values as needed: • Source Chamber (BA1) Max Operating Pressure—Pressure (Torr) above which the high voltage power supplies are automatically turned off to prevent damage to the instrument. Valid range is 10-5 to 10-9. Default is 9x10-6. If the Source Chamber pressure is above the Max Operating Pressure, an error message is displayed and the high voltage cannot be turned on.
Chapter 2 Installing the Voyager™ Biospectrometry™ Workstations • Source Chamber (BA1) Wait Time—Time (seconds) that the software waits for the instrument to reach the Source Chamber Maximum Operating Pressure, after the sample plate is loaded. If the wait time is exceeded, an error message is displayed which gives you the option of an additional wait time or ejecting the plate. Valid range is 0 to 300 seconds. Default is 120 seconds.
Hardware Configuration High voltage configuration To check high voltage configuration: 1. In the Instrument Control Panel, select Hardware Configuration from the Instrument menu. 2. Click the High Voltage tab to display the High Voltage page (Figure 2-17). 2 Figure 2-17 High Voltage Configuration 3. Check the following values as needed: • Maximum Accelerating Voltage—Maximum value in volts that the Accelerating Voltage is configured (25,000 V).
Chapter 2 Installing the Voyager™ Biospectrometry™ Workstations • Idle Power Off/Idle Time—When enabled, the number of minutes after which the high voltage power supplies automatically turn off, if the instrument is not used. Default is 60 minutes. If this value is zero, the high voltage remains on until any of the following occur: you select Instrument/Turn off High Voltage, Source Pressure exceeds Maximum Operating Pressure, you click Load or Eject, you align a sample plate, or you exit the software.
Hardware Configuration 3. Check the following values as needed: • Flight Length to Deflector (Read-only)— Distance in millimeters from the grid to the deflector. • Deflector Gate Width— Distance in millimeters that the Timed Ion Selector is on. 4. Instrument configuration Click OK to exit. 2 To check the instrument configuration: 1. In the Instrument Control Panel, select Hardware Configuration from the Instrument menu. 2. Click the Instrument tab to display the Instrument page (Figure 2-19).
Chapter 2 Installing the Voyager™ Biospectrometry™ Workstations 3. Check the following fields as needed: • Instrument type (Read-only)—Displays your instrument type. • Delayed Extraction (Read-only)—Reflects whether your system has delayed extraction hardware installed. • Laboratory Name—You can edit this field to display your laboratory name. Names listed in this field are included in .DAT files and on printouts. 2 • Instrument Name—You can edit this field to display your instrument name.
Hardware Configuration 2 Figure 2-20 Laser Configuration 3. Check the following fields as needed: • External laser (Read-only)—Checked if an external laser is installed. • Manual Intensity Adjustment—Determines the increments in which the laser attenuator moves when using the Fine and Coarse laser controls on the Manual Laser/Sample Positioning control page: Small—Determines laser adjustment increments when you click the Fine laser controls or press Ctrl+PgUp/Ctrl+PgDn.
Chapter 2 Installing the Voyager™ Biospectrometry™ Workstations Digitizer configuration To check the configuration of the digitizer: 1. In the Instrument Control Panel, select Hardware Configuration from the Instrument menu. 2. Click the Digitizer tab to display the Digitizer page (Figure 2-21). . 2 Figure 2-21 Digitizer Configuration Check the Digitizer type field. This is a read-only value that displays the digitizer type installed. 3. 2-38 PerSeptive Biosystems Click OK to exit.
Aligning the Sample Plate 2.7 Aligning the Sample Plate In this section This section describes: • • • • • • • • • • • Overview When to align Overview When to align .PLT files and multiple alignments How the system aligns a plate Overview of video monitor display What you need Corner positions in .
Chapter 2 Installing the Voyager™ Biospectrometry™ Workstations .PLT files and multiple alignments A .PLT file defines the sample positions on a sample plate. For example, 100.PLT may define a 100-well plate. If you have more than one 100-well plate, you may need to align each plate. The Voyager software allows you to assign a unique Plate ID to each plate that allows customized alignment of more than one plate that uses the sample .PLT file (Figure 2-22). Plate A Plate ID 1A 100well.
Aligning the Sample Plate Overview of video monitor display The following examples show how the sample positions and laser spot may be displayed on the video monitor during sample plate alignment. Example Perimeter of sample position Laser spot Figure 2-23 Ideal Sample Position Alignment Description Ideal sample position alignment—The center of the sample position is aligned with respect to the laser spot.
Chapter 2 Installing the Voyager™ Biospectrometry™ Workstations What you need To align the sample plate you need the following materials: • Sample plate with α-cyano-4-hydroxycinnamic acid (CHCA) matrix spotted in four corner sample positions, as described in Table 2-1. For more information, see Section 3.5.4, Adjusting the Laser Position for a Custom .PLT File.
Aligning the Sample Plate Using the control stick Before aligning To align a sample plate, you must use the control stick. For details on using the control stick, see Appendix I, Using the Oscilloscope and Control Stick. Before aligning the sample plate: 1. Spot the sample plate with matrix as described in “What you need” on page 2-42. 2.
Chapter 2 Installing the Voyager™ Biospectrometry™ Workstations 2 Figure 2-26 Sample Plate Alignment Wizard 3. Click Next. The sample plate moves to the first alignment position on the sample plate. See Table 2-1, “Four-Corner Positions on Sample Plates,” on page 2-42, to determine your plate positions. 2-44 4. Start the laser using the control stick. 5. Mark the laser position on the transparency. Do not mark the sample position.
Aligning the Sample Plate The software calculates the alignment and uses the settings to ensure all sample positions are centered under the laser. If the alignment is successful, a message is displayed. 10. Do one of the following: Click Finish To Save the alignment for the specified Plate ID Cancel End the Sample Plate procedure without saving the alignment for the plate Back Repeat the alignment procedure NOTE: A message is displayed if the alignment is outside the preferred tolerance.
Chapter 2 Installing the Voyager™ Biospectrometry™ Workstations 2.8 Startup and Shutdown In this section This section describes: • • • • • 2 Powering up Powering up Initializing Reinitializing Powering down system components Powering down the mass spectrometer To power up the Voyager Workstation: 1. Turn on the main power switch. The power switch is located: • On the right side panel of the mass spectrometer cabinet on Voyager-DE and Voyager-DE PRO systems.
Startup and Shutdown If your system includes a LeCroy digitizer, wait approximately one minute until the digitizer completes its internal calibration before starting the Instrument Control Panel. 4. Press the EMIS button on the Vacuum Gauge Panel to turn on the gauges.
Chapter 2 Installing the Voyager™ Biospectrometry™ Workstations Initializing The hardware is automatically initialized when you start the software. During initialization, the video image on the sample stage is displayed. The sample stage moves to the home position, and then to the load position. If problems occur, an error message is displayed when you log onto the workstation. Further details on any problem can be obtained by viewing the Windows NT Event Log.
Startup and Shutdown Powering down the mass spectrometer If you need to perform maintenance on internal parts or move the system, power down the spectrometer: 1. Power down the system components. 2. Turn off the main power switch. The power switch is located: • On the right side panel of the mass spectrometer cabinet on Voyager-DE and Voyager-DE PRO systems. • On the back panel of the mass spectrometer cabinet on the Voyager-DE STR system.
Chapter 2 Installing the Voyager™ Biospectrometry™ Workstations 2.9 Checking System Status Display the System Status page for complete status information. In the Instrument Control Panel, select System Status from the View menu to display the System Status page (Figure 2-27). 2 Figure 2-27 System Status Control Page System and acquisition status are represented by colored indicators and read-only text fields described in Table 2-2.
Checking System Status Table 2-2 System Status Parameters Parameter Description System Status Instrument State High Voltage Possible states are: • ON—Instrument is initialized and high voltage is on. • OFF—Instrument is not initialized and high voltage is off. • FAULT—Indicates a fault condition. Follow the instructions displayed to correct the fault. 2 Possible states are: • • • • RAMPING—Voltage is turning on. ON—High voltage is on. OFF—High voltage is off. FAULT—Indicates a fault condition.
Chapter 2 Installing the Voyager™ Biospectrometry™ Workstations 2 2-52 PerSeptive Biosystems
Chapter 3 Preparing Samples 3 This chapter contains the following sections: 3.1 Preparing Samples ......................................... 3-2 3.2 Loading Samples on Sample Plates ............................ 3-28 3.3 Cleaning Sample Plates................................. 3-36 3.4 Loading Sample Plates in the Mass Spectrometer........................................ 3-39 3.5 Sample Plate Types Supported ......................
Chapter 3 Preparing Samples 3.1 Preparing Samples NOTE: Sample preparation is critical to obtain good results in MALDI-TOF applications. Sample preparation technique has a direct impact on the quality of the data you obtain.
Preparing Samples 3.1.1 Selecting a Matrix Purpose of matrix In MALDI-TOF, the matrix plays a key role in the ionization process. The well-developed crystals of matrix material assist in ionizing the molecules you are analyzing.
Chapter 3 Preparing Samples Mixtures Specialized applications If you are examining a sample mixture, it may be necessary to prepare the mixture with several different matrices.
Preparing Samples NOTE: HPLC-grade water may vary in salt concentration and may produce adduct ions in mass spectra. A high salt concentration may interfere with some applications, particularly oligonucleotide analysis. Use double-deionized water, such as Milli-Q™ grade 18 mΩ, which is appropriate for most applications. WARNING CHEMICAL HAZARD. Refer to the Material Safety Data Sheet (MSDS) provided by the chemical manufacturer before handling solvents or matrices. Preparing matrix To prepare matrix: 1.
Chapter 3 Preparing Samples 5. Cap the tube and vortex thoroughly for approximately 1 minute or until dissolved. You can shake the tube by hand if you do not have a vortex mixer. 6. Microcentrifuge the tube for 30 seconds at 2,000 to 5,000 rpm. Alternatively, allow the solution to settle for about 10 minutes. You may see a precipitate at the bottom of the tube. When applying matrix, use the supernatant, not the precipitate. 3.1.
Preparing Samples Sinapinic acid Use sinapinic acid for peptides and proteins >10,000 Da. Matrix concentration 10 mg/ml Final sample concentration 0.1–5 pmol/µl Solvents Acetonitrile, 0.1% TFA in deionized water Preparation Follow the procedure in “Preparing matrix” on page 3-5 and combine 700 µl of 0.1% TFA solution in water, and 300 µl acetonitrile to 10 mg of solid matrix. If sample is contaminated with buffer, salt, or detergent, instead of the proportions listed above, combine 500 µl 0.
Chapter 3 Preparing Samples CHCA Useα-cyano-4-hydroxycinnamic acid (CHCA) for: • Dried drop application—Peptides/proteins <10,000 Da • Thin layer application—Peptides <~3,000 Da1 CHCA for dried droplet application Use for peptides/proteins <10,000 Da. Matrix concentration 10 mg/ml Final sample concentration 0.1–5 pmol/µl Solvents Acetonitrile, 0.1% TFA in deionized water Preparation Follow the procedure in “Preparing matrix” on page 3-5 and combine one part 0.
Preparing Samples CHCA for thin layer application Use for peptides <3,000 Da. Matrix concentration 20 mg/ml Final sample concentration Up to 0.1 pmol/µl Materials and solvents • • • Preparation1 Pure nitrocellulose (for example, Bio-Rad Laboratories Trans-Blot® 162-0146) Acetone Isopropanol 1. Dissolve nitrocellulose to a concentration of 20 mg/ml in acetone by vortexing for 15 minutes. 2. Add isopropanol at a ratio of 1:1. 3.
Chapter 3 Preparing Samples THAP Use THAP for small oligonucleotides <3,500 Da, acidic carbohydrates, acidic glycopeptides, acid sensitive compounds. THAP provides a more even response than 3-HPA. Matrix concentration • • Oligonucleotides—10 mg/ml Acidic carbohydrates—2 mg/ml Additive concentration 50 mg/ml diammonium citrate in deionized water Final sample concentration 1–10 pmol/µl Solvents 50 percent acetonitrile and deionized water NOTE: HPLC-grade water may vary in salt concentration.
Preparing Samples 3-HPA Use 3-HPA for large oligonucleotides >3,500 Da. Matrix concentration 50 mg/ml Additive concentration 50 mg/ml diammonium citrate in deionized water Final sample concentration 1–10 pmol/µl Solvents 50 percent acetonitrile and deionized water NOTE: HPLC-grade water may vary in salt concentration. Do not use for oligonucleotide analysis. Preparation Follow the procedure in “Preparing matrix” on page 3-5 and combine 8:1 3-HPA:diammonium citrate.
Chapter 3 Preparing Samples DHB Use DHB for: • Neutral carbohydrates • Small molecules DHB for neutral carbohydrates 3 Matrix concentration 10 mg/ml Final sample concentration 10 pmol/µl Solvents Deionized water Preparation Follow the procedure in “Preparing matrix” on page 3-5. Dry down quickly under vacuum for even response. If you allow to air dry, you will see uneven response during analysis. Crystals Milky amorphous appearance for promoting cationization (see Figure 3-3 on page 3-35).
Preparing Samples DHB for small molecules Matrix concentration 10 mg/ml Final sample concentration Highly sample-dependent. Ideally a minimum of 10–200 pmol/µl (10–20 ng/µl). With lower concentrations, it may be difficult to distinguish sample signal from matrix signal. Solvents Any solvent in which molecules are soluble (deionized water to 100% methanol or acetone). Preparation Follow the procedure in “Preparing matrix” on page 3-5. Dry down quickly under vacuum for even response.
Chapter 3 Preparing Samples DHBs Use DHBs for peptides and proteins >10,000 Da, and glycosylated proteins. Matrix concentration 10 mg/ml Additive concentration 10 mg/ml 5-methoxysalicylic acid Final sample concentration 10 pmol/µl to 100 fmol/µl Solvents 80% 0.1% TFA in deionized water:20% acetonitrile (DHB) + 50: acetonitrile:50% deionized water (5-methoxysalicylic acid) Preparation Follow the procedure in “Preparing matrix” on page 3-5 and combine 9:1 DHB:5-methoxysalicylic acid solutions.
Preparing Samples Synthetic polymer matrices Choice of matrix depends on the sample you are analyzing: • Aromatic (for example, polystyrene)—Dithranol (25 mg/ml) and 1 mg/ml silver trifluoroacetate (Ag TFA) dissolved in tetrahydrofuran (THF) • Polar—DHB (10 mg/ml) in deionized water • Non-polar—Indole acetic acid (10 mg/ml) or DHB in THF, dimethylformamide (DMF), or acetone Matrix concentration ~0.1 M (10-1 M) Final sample concentration ~0.1 mM (10-4 M) Solvents Sample and matrix dependent.
Chapter 3 Preparing Samples You can load polymer samples on sample plates in two ways: • Thin layer polymer method (yields even response, but provides adequate sample response for only 10 laser shots)—Load 0.3 µl sample/matrix solution in one sample position.
Preparing Samples 3.1.4 Preparing Sample In this section This section includes: • • • • Sample concentration Sample concentration Preparing samples for dried droplet application Preparing samples for thin layer application Internal standards Prepare sample just before loading the sample plate. Refer to Section 3.1.5, Sample Cleanup, to determine if your samples should be cleaned up before preparing.
Chapter 3 Preparing Samples Note the following: • When diluting sample, keep in mind that you will be further diluting when you mix sample with matrix. • If you are unsure of the starting concentration of sample, make a serial dilution to prepare various dilutions of the same sample. • Different samples are soluble in different liquids. Try water first, then add acetonitrile, and then add 0.1% TFA to increase solubility if required.
Preparing Samples 3.1.5 Sample Cleanup Cleaning techniques When to clean samples Use the following techniques to clean samples: • • • • Washing Drop dialysis (floating membrane) Cation exchange ZipTips® Sample cleanup is needed if samples: • Are prepared in phosphate buffers. Ammonium salts or derivatives of organic amines (ammonium bicarbonate, TRIS HCl) do not usually cause a problem in low concentrations (less than 50mM). • Contain salt, for example, from cation or anion exchange purification.
Chapter 3 Preparing Samples 3.1.5.1 Washing When to use What you need Use this technique when you know the solubility of the contaminant. You can wash a dried sample directly on the sample plate. Select a solvent in which the contaminant is more soluble than the matrix and the sample. For example, use: • Cold water (to prevent sample dissolving) with 0.
Preparing Samples 3.1.5.2 Drop Dialysis (Floating Membrane) When to use What you need Use this technique on polar compounds when you know contaminants are of low molecular weight. This technique works well for DNA and polar proteins such as glycoproteins. For drop dialysis, you need a membrane: • With a pore size of 0.025 µm or smaller • That does not adhere to your sample Procedure To perform drop dialysis: 1.
Chapter 3 Preparing Samples NOTE: Sample also passes through the membrane during dialysis, particularly low mass samples. Dialyze small molecules for a shorter time than larger molecules. In general, do not dialyze for more than two hours. 9. When the size of the sample spot stabilizes, remove the sample and place it in a microcentrifuge tube. NOTE: The size of the sample drop can increase by a factor of 10 when salt concentration is high.
Preparing Samples 3.1.5.3 Cation Exchange When to use What you need Preparation of beads in ammonium form Procedure Use this technique for non-polar proteins or DNA when you know the sample contains only a salt contaminant. This technique is faster than dialysis but does not remove other contaminants. Use 200-mesh cation exchange beads. Cation exchange beads in the ammonium form work best for MALDI applications. To prepare cation exchange beads in the ammonium form: 1.
Chapter 3 Preparing Samples 3.1.5.4 ZipTips When to use ® Use this technique for peptides, proteins, and oligonucleotides when you know the sample contains salt, buffer, or glycerol contaminants. This technique is faster, easier, and more effective than dialysis for removing contaminants. NOTE: This technique introduces organic solvent into the sample, which is not compatible with thin layer sample application.
Preparing Samples Procedure To clean samples with ZipTips: 1. Wash a C18 ZipTip in the following order with: • • • • 10 µl of ACN 10 µl of 50:50 ACN:0.1%TFA 10 µl of 0.1% TFA Repeat wash of 10 µl of 0.1% TFA To wash, draw a few microliters of a wash solution up into the ZipTip and expel to waste. 2. Draw a few microliters of the sample up and down in the ZipTip several times. 3. Discard the liquid. The sample is bound to the C18 surface in the ZipTip. 4. Wash the ZipTip again with 10 µl of 0.
Chapter 3 Preparing Samples 3.1.6 Mixing Sample and Matrix (Dried Droplet Application) When to use Use dried droplet application if you are analyzing samples with a concentration >0.01 pmol/µl. If you are analyzing samples with a concentration <0.01 pmol/µl, use the thin layer application technique described in Section 3.2.3, Loading Samples (Thin Layer Application).
Preparing Samples Mixing sample and matrix on the sample plate Mix sample and matrix directly on the sample plate when you are: • Working with dilute samples and can make a 1:1 preparation of sample:matrix • Analyzing only a few samples • Concerned about sample adhering to plastic tubes When mixing directly on the plate, you may need to use a higher concentration of organic and matrix for matrix solutions.
Chapter 3 Preparing Samples 3.2 Loading Samples on Sample Plates In this section This section describes: • • • • Overview Loading samples (dried droplet application) Loading samples (thin layer application) Examining crystals on sample plates 3.2.
Loading Samples on Sample Plates Types of sample plates Three types of 100-position reusable sample plates are available: • Polished blank sample plates (with or without sample numbers)—Liquid is held in place by surface tension of sample/matrix mixture. Advantage of this type of plate is that you can observe crystallization pattern, and the actual sample spot is visible. • Laser-etched sample plates—Liquid is held in place by laser-etched indentation in plate.
Chapter 3 Preparing Samples Handling sample plates To prevent contamination of your analysis: • Start with a clean sample plate. See Section 3.3, Cleaning Sample Plates, for more information. • Handle the sample plate by the edges. • Use powder-free gloves if you wear gloves. Guidelines for good crystallization To ensure good crystallization: • Mix sample and matrix before applying.
Loading Samples on Sample Plates 3.2.2 Loading Samples (Dried Droplet Application) NOTE: If you are loading a plate for acquisition or automated sample positioning in Automatic Control mode, use a laser-etched or welled sample plate to provide a reference point for sample application. When to use Location of standards Loading samples Use this technique for most applications, when sample concentration is >0.1 pmol/ µl.
Chapter 3 Preparing Samples Do not touch the tip of the pipette to the sample spot. Make sure the sample is evenly applied to the spot. NOTE: Some organic solvents such as methanol, acetone, and THF spread very easily on metal surfaces. If the sample contains these solvents, try to load smaller volumes (0.5 µl or less). NOTE: Try to place the sample in the center of the sample position.
Loading Samples on Sample Plates 3.2.3 Loading Samples (Thin Layer Application) When to use Loading matrix and samples Use the thin layer application technique1 for increased sensitivity when analyzing peptides with a concentration <0.1 pmol/ µl. To load matrix and samples: 1. Dispense 0.5 µl of matrix prepared for thin layer application on the sample plate to form a thin layer. It will dry in seconds. For information, see “CHCA for thin layer application” on page 3-9. 2. Load 0.
Chapter 3 Preparing Samples 3.2.4 Examining Crystals on Sample Plates Overview If you are using polished blank or laser etched sample plates (see “Types of sample plates” on page 3-29), you can look at the crystallization of sample and matrix under a microscope. A magnification of 30X is sufficient to see crystals. You can also view crystallization on the video monitor on the instrument. The morphology of crystals is a critical element for successful analysis.
Loading Samples on Sample Plates You can acquire data from a well that does not have an ideal crystallization pattern. However, when sample contains unevenly distributed crystalllization, it may be difficult to analyze. This can cause a problem in Automatic mode. Other matrices Typical appearance of other matrices under magnification are shown in Figure 3-2 and Figure 3-3. When analyzing 3-HPA crystals, aim the laser at the base of the fan-like crystals for best response.
Chapter 3 Preparing Samples 3.3 Cleaning Sample Plates In this section This section describes procedures for cleaning: • Teflon® plates • Gold and stainless steel plates WARNING CHEMICAL HAZARD. Before handling any chemicals, refer to the Material Safety Data Sheet provided by the manufacturer, and observe all relevant precautions. Cleaning Teflon plates NOTE: Avoid using strong organic solvents such as acetone. Use acetonitrile if a solvent is necessary. Avoid ultrasonic cleaning devices.
Cleaning Sample Plates 6. Examine the plate. If you see any sample or matrix residue, oil, or fingerprints on the plate, soak the sample plate in a working solution of laboratory detergent in water (for example, RBS 35 detergent from Pierce or LIQUI-NOX™ from VWR) for the minimum time required to remove samples. CAUTION Do not leave the sample plate in detergent for longer than 10 minutes. Longer exposure can cause the bottom holders on the sample plate to corrode.
Chapter 3 Preparing Samples CAUTION Do not leave the sample plate in detergent for longer than 10 minutes. Longer exposure can cause the bottom holders on the sample plate to corrode. Do not sonicate sample plates or use acid to clean sample plates. Both can alter the surface of the sample plate, and reduce the quality of the data obtained. 5. If residue remains, wipe the plate with a lint-free tissue or cotton swab. A soft toothbrush works well. 6. Rinse the plate thoroughly in deionized water. 7.
Loading Sample Plates in the Mass Spectrometer 3.4 Loading Sample Plates in the Mass Spectrometer This section describes: • Ejecting the sample holder • Loading sample plates NOTE: If you load the sample plate into the Voyager Biospectrometry Workstation before the plate is dry, the pressure in the sample chamber rises, and a “TC2 pressure too high” error code may be displayed in the Instrument Control Panel. Wait a few minutes for the chamber to reach pressure.
Chapter 3 Preparing Samples Table 3-1 Sample Plate Information Parameter Description Plate ID Unique identifier (up to 32 alphanumeric characters) that you can assign to a plate, to allow customized alignment of more than one physical plate using the same .PLT file. For more information, see “.PLT files and multiple alignments” on page 2-40. Plate Type .PLT file which contains plate configuration. .PLT files are located in the C:\VOYAGER directory. For more information on .PLT files see, “.
Loading Sample Plates in the Mass Spectrometer 3.4.1 Ejecting the Sample Holder In the Voyager Instrument Control Panel, select Eject from the Sample Plate menu. The Load/Eject dialog box is displayed. . Figure 3-4 Load/Eject Sample Plate Dialog Box The following also occurs: • A Load Status dialog box is displayed during the ejection sequence which displays hardware status. • High voltage is turned off. • The video monitor displays the sample plate moving.
Chapter 3 Preparing Samples 3.4.2 Loading Sample Plates This section describes loading sample plates in: • Voyager-DE and Voyager-DE PRO • Voyager-DE STR Voyager-DE and Voyager-DE PRO To load sample plates: 1. Eject the sample holder as described in Section 3.4.1, Ejecting the Sample Holder. 2. Hold the sample plate with the bottom of the numbers facing toward the analyzer (for standard 100-well plate) and with the slanted underside of the plate facing to the left. 3.
Loading Sample Plates in the Mass Spectrometer 84 74 85 75 65 86 76 66 PB100278 Figure 3-5 Loading the Sample Plate in a Voyager-DE or Voyager-DE PRO WARNING PHYSICAL INJURY HAZARD. Fingers can get caught in the sample holder. To avoid injury, do not click Load to retract the sample holder when your fingers are near the sample holder. 4. From the Sample Plate menu select Load to retract the sample plate and insert it into the main source chamber.
Chapter 3 Preparing Samples 5. Select a Plate ID. The .PLT file and alignment information associated with the Plate ID are automatically loaded. Alternatively you can specify a new Plate ID and select a .PLT file. For more information, see “Assigning Plate IDs” on page 3-39. 6. Click OK. The sample plate is loaded and aligned as needed. For more information, see “How the system aligns a plate” on page 2-40. It takes a minute or two for the sample plate to reach the correct position.
Loading Sample Plates in the Mass Spectrometer 64 65 66 74 75 76 84 85 86 PB100277 Figure 3-7 Loading the Sample Plate in a Voyager-DE STR 3 WARNING PHYSICAL INJURY HAZARD. Fingers can get caught in the sample holder. To avoid injury, do not click Load to retract the sample holder when your fingers are near the sample holder. 4. From the Sample Plate menu, select Load to retract the sample plate and insert it into the main source chamber.
Chapter 3 Preparing Samples 5. Select a Plate ID. The .PLT file and alignment information associated with the Plate ID are automatically loaded. Alternatively you can specify a new Plate ID and select a .PLT file. For more information, see “Assigning Plate IDs” on page 3-39. 6. Click OK. The sample plate is loaded and aligned as needed. For more information, see “How the system aligns a plate” on page 2-40. It takes a minute or two for the sample plate to reach the correct position.
Sample Plate Types Supported 3.5 Sample Plate Types Supported In addition to 100-position reusable sample plates, the Voyager Instrument Control software supports other reusable and disposable plates. You now can: • Customize other available plate types by copying and editing their .PLT files. See Section 3.5.3, Guidelines for Defining Custom Plate Types. NOTE: These additional plate types are custom options. Contact PerSeptive Biosystems for more information.
Chapter 3 Preparing Samples 3.5.1 Sample Plates Types and Applications The Voyager Instrument Control software supports several types of reusable and disposable plates. See Table 3-2 for the applications of different sample plates. Table 3-2 Sample Plate Types, Applications, and Benefits Sample Plate Type Applications/Benefits Welled Sample Plates Gold 3 • GPC-MALDI or HPLC fractions when high concentration of organic solvent provides no surface tension.
Sample Plate Types Supported Table 3-2 Sample Plate Types, Applications, and Benefits (Continued) Sample Plate Type Applications/Benefits Special Sample Plates Stainless steel, polished blank • High throughput. • Allows customized sample positioning and preparation using an automated sample preparation device. Disposable (gold-coated) • Derivatizing the surface of the sample plate for protein or enzyme immobilization1, 2. • Long term storage of samples. • Eliminating cross-contamination.
Chapter 3 Preparing Samples 3.5.2 Editable-Configuration Plate (.PLT) Types Provided with the System Selecting plate types (.PLT files) You can select plate types (.PLT files) in two ways: • When loading the plates in the mass spectrometer • From the Sample Plate menu in the Instrument Control Panel For an example of how to select plate types, see Section 3.4.2, Loading Sample Plates. Customizing .PLT files 3 You can select and use the plate types described below.
Sample Plate Types Supported .PLT files provided The editable-configuration plate types provided with the system are described in Table 3-3. Table 3-3 Editable-configuration Plate Types (Dimensions in Microns, µm) Plate Type Position Positions Plate Position Positions Diameter Center-to-Center Description Number Arrangement (µm) Distance (µm) 64 well disposable plate.PLT Disposable with wells 64 8 x 8 (subset of 10 x 10) 2,540 5,080 x 5,080 100 well plate.
Chapter 3 Preparing Samples 3.5.3 Guidelines for Defining Custom Plate Types You can define custom plate types of unlimited positions by creating your own .PLT files. This section describes: • • • • .PLT file format 3 .PLT file format Guidelines for creating .PLT files Guidelines for creating search pattern (.SP) files How to create a .PLT file A .PLT file is an ASCII text file in which each line represents an individual position on the plate. As an example, Figure 3-10 shows how the 96 well plate.
Sample Plate Types Supported A .PLT file describes each position on the plate as discussed in Table 3-4. Table 3-4 .PLT File Parameters Parameter WellUnits (optional) Description Defines the units of the sample positions that are displayed in the Manual Laser/Sample Positioning control page (see Figure 4-9 on page 4-31). If you do not include the WellUnits parameter, the software uses the default.
Chapter 3 Preparing Samples Table 3-4 .PLT File Parameters (Continued) Parameter X and Y Description Coordinates of the position center in reference to the lower left corner of the sample plate. To determine the coordinates to enter in the .PLT file, display the Sample View (see Figure 4-9 on page 4-31) and record the Absolute X, Absolute Y (logical coordinates) pairs for each position specified. See “Logical Coordinates on Plate View” on page 3-61 for information on displaying logical coordinates.
Sample Plate Types Supported Table 3-4 .PLT File Parameters (Continued) Parameter Description Width Use if you are customizing a .PLT file. Defines the width of the sample positions that are displayed in the Manual Laser/Sample Positioning control page. This value is optional. If you enter a Width value for a customized .PLT file, the WellWidth value is overridden. If you do not specify a value, the software defaults to WellWidth. Height Use if you are customizing a .PLT file.
Chapter 3 Preparing Samples Figure 3-11 illustrates the coordinates for the four corner positions that define the edges of the area of the sample plate that provides optimum mass accuracy. Position 1 For optimum mass accuracy, do not specify coordinates or spot sample on the outer edges of a plate (shown in gray) Home position (Absolute X=0, Absolute Y=0) X 6667.5 X 42227.5 Y 42227.5 Y 42227.5 Load position (Absolute X= 50,800, Absolute Y=25,400) y X 6667.5 Y 6667.5 X 42227.5 Y 6667.
Sample Plate Types Supported Guidelines for creating .PLT files Note the following: • The diameter of a position on a plate is determined by the plate used: • If you are using welled or laser-etched plates, the position diameter is determined by the well or laser-etched position size (2,540 µm on a 100-well plate).
Chapter 3 Preparing Samples NOTE: You can correct for systematic errors introduced by a sample preparation device by aligning the sample plate in the Voyager Workstation. See Section 2.7, Aligning the Sample Plate. If you are using different sample preparation devices, you can compensate for different systematic errors by creating different .PLT files and sample plate alignments for each system.
Sample Plate Types Supported Tolerances and non-systematic errors When creating search pattern files, make sure to compensate for: • Positional tolerance (related to variability in the position of the plate) Positional Tolerance Plate type µm Reusable plates 76.2 Disposable plates 381 NOTE: Due to limited surface area and variability in the position of the disposable inserts, do not specify more than 384 positions for a disposable plate.
Chapter 3 Preparing Samples Center-to-center distance Total allowable tolerance X,Y coordinate Radius available for analysis (half the center-to-center distance) Search pattern radius Figure 3-12 Area Available for Analysis 3 How to create a .PLT file NOTE: Display the Sample View while creating a .PLT file. Move the sample plate to the positions you want analyzed and note the Absolute X, Absolute Y coordinates to enter in the .PLT file. To create a .PLT file: 3-60 1.
Sample Plate Types Supported Figure 3-13 shows the Plate View displaying the logical coordinates for the BLANK.PLT file. Logical Coordinates 3 Figure 3-13 Logical Coordinates on Plate View 4. Use the Manual Laser/Sample Positioning control page or the Control Stick to move to the first position and note the absolute coordinates for that position. 5. Open the Microsoft Windows NT® Notepad text editor. See the Microsoft Windows NT User’s Guide if you need help using Notepad. 6.
Chapter 3 Preparing Samples 8. On the second line, type in absolute coordinates for the first position. Separate absolute x and y coordinates with a comma, and include one X,Y pair per line (see Figure 3-10 on page 3-52, for X,Y pair example). Type in the position name, and the comment if needed; start the comment with a semicolon. Blank lines are allowed. 9.
Sample Plate Types Supported 3.5.4 Adjusting the Laser Position for a Custom .PLT File After you have defined your own sample plate format: 1. Spot sample on the four corner positions defined in the .PLT file. For optimum mass accuracy, do not spot sample on the outer edges of the plate. See Figure 3-11 on page 3-56. 2. Load the sample plate and .PLT file as described in Section 3.4.2, Loading Sample Plates. 3.
Chapter 3 Preparing Samples 3 3-64 PerSeptive Biosystems
4 Voyager Instrument Control Panel Basics Chapter 4 This chapter contains the following sections: 4.1 Instrument Control Panel ................................. 4-2 4.2 Using the Control Pages .................................. 4-8 4.3 Using the Spectrum Window .......................... 4-10 4.4 Customizing the Instrument Control Panel ..... 4-21 4.5 Controlling the Workstation ............................ 4-24 4.6 Sequence Control Panel ................................ 4-32 4.
Chapter 4 Voyager Instrument Control Panel Basics 4.1 Instrument Control Panel The Voyager Instrument Control Panel allows you to directly control the Voyager mass spectrometer to acquire and inspect mass spectra in Manual or Automatic Control mode. NOTE: The Voyager Sequence Control Panel allows you to collect data for multiple samples using different conditions. For more information, see Section 4.6, Sequence Control Panel, and Section 4.7, How the Instrument and Sequence Control Panels Interact. 4.1.
Instrument Control Panel Instrument settings file name Toolbar Data Storage Control page Instrument Settings Control page Manual Laser/ Sample Position Control page Output window Status bar Spectrum window Figure 4-1 Instrument Control Panel Toolbar The toolbar contains buttons that control the software and the instrument. For a description of a toolbar button, place the cursor on it. A brief description of the button (Tooltip) is displayed below the button. For more information, see Section 4.5.
Chapter 4 Voyager Instrument Control Panel Basics Control pages The Instrument Control Panel contains five control pages: • Instrument Settings—Controls settings for instrument mode, voltages, spectrum acquisition, and calibration. For more information, see Chapter 5, Optimizing Instrument Settings. • Data Storage—Controls data storage information such as file location and file name. For more information, see “Setting Data Storage parameters” on page 6-14.
Instrument Control Panel Spectrum window The Spectrum window provides a display of data. The data displayed depends on your digitizer option: • Signatec or LeCroy digitizers—Displays a live real-time spectrum trace as data is acquired. Trace changes from Live to Current when acquisition is complete. • Tektronix oscilloscope—No trace displayed during acquisition. Displays a Current spectrum trace when acquisition is complete. When acquisition is complete, peaks can be detected and labeled.
Chapter 4 Voyager Instrument Control Panel Basics Displaying The Output window is automatically displayed when you: • Acquire data • Store data To display the Output window manually, select Output Window from the View menu. To close the Output window, deselect Output Window from the View menu, or right-click in the Output window and select Hide. 4.1.
Instrument Control Panel Automatic control mode To select Automatic Control mode: 1. Open the Instrument Control Panel. 2. Select Instrument Settings from the View menu. 3. Click the Automatic button on the Instrument Settings control page. 4. Click the Automatic Control button. 5. Set laser controls, sample positioning, and data storage for automatic adjustment and control as described in Section 6.6.2, Setting Instrument Settings for Automatic Control Mode. 4.1.
Chapter 4 Voyager Instrument Control Panel Basics 4.2 Using the Control Pages The Instrument Control Panel allows you to display, organize, and rearrange one or more control pages. This section describes: • Displaying control pages • Types of page control Displaying control pages You can display control pages in several ways: • Select individual control pages from the View menu.
Using the Control Pages Types of page control You can select between two types of page control for the control pages: • Docked—Pages are attached, or “anchored” to other pages or the edge of the Instrument Control Panel. You can do the following with a docked page: • Deselect it from the View menu to close it. • Double-click it to change it to a floating page (described below). This automatically maximizes the page. Double-click it again to dock the page.
Chapter 4 Voyager Instrument Control Panel Basics 4.3 Using the Spectrum Window This section includes: • • • • • Adjusting the display range Zooming on traces Adding traces to a window Annotating traces Previewing and printing traces 4.3.1 Adjusting the Display Range Select commands from the Display menu to set the display range in Spectrum window: 1. Click on the Spectrum window to activate it. 2. From the Display menu, select Range. 3. Select X Range to set the x-axis range.
Using the Spectrum Window 4. Set From and To values for the display range (m/z units) and click OK. 5. From the Display menu, select Range. 6. Select Y Range to scale the y-axis. The Display Y Axis Setup dialog box (Figure 4-4) is displayed.
Chapter 4 Voyager Instrument Control Panel Basics 7. Set as needed: Parameter Description Scaling Mode Display Relative Autoscales the trace to the largest peak in the selected range. Base Peak Relative Autoscales the trace to the base peak in the entire range, not the selected range. Displays a right axis label with the base peak intensity. NOTE: To turn off the right axis, select Graphic Options from the Display menu, click the Graph #1 Setup tab, and deselect the Show Right Axis check box.
Using the Spectrum Window 4.3.2 Zooming on Traces Zooming and unzooming You can expand (zoom) an area of a trace by left-click-dragging a box around the area of interest. You can also click buttons in the toolbar to: • Zoom in • Zoom out to the previous zoom • Full Unzoom Expanding and linking traces When you have more than one trace displayed in the same data file in a window, you can: • Select (click on) a trace, then click in the toolbar to expand the selected trace for closer examination.
Chapter 4 Voyager Instrument Control Panel Basics Types of traces The Spectrum window can contain two types of traces (see Figure 4-5 on page 4-15): • Live/Current—A live, real-time trace of data. The display updates as you view or acquire data. When acquisition is complete, the trace name changes from Live to Current. NOTE: On systems with LeCroy or Signatec digitizers, the Live Trace dynamically updates as data is acquired.
Using the Spectrum Window Hint: Resize the window to view all added traces. Figure 4-5 Adding Traces When you add a specific type of trace, the label of the trace changes from Not Used to the label for the type of trace created. Removing traces To remove a trace from the Spectrum window: 1. Select (click on) the trace. 2. Click in the toolbar. The trace is removed.
Chapter 4 Voyager Instrument Control Panel Basics 4.3.4 Annotating Traces This section describes: • Two ways to annotate • Copying text from ASCII source • Annotating the trace Two ways to annotate You can add text annotation to traces by: • Copying ASCII text from any source, then pasting on the trace • Typing text on the trace Copying text from ASCII source To copy ASCII text: 1. Open ASCII text file. 2. Select the line of text to copy, then right-click and select Copy from the menu displayed.
Using the Spectrum Window Hint: To move the text, left-click and hold on the text, then drag to the desired position. 3. To customize the appearance of the annotated text, see Section 4.4, Customizing the Instrument Control Panel. NOTE: Text annotations are associated with the Spectrum window, not the trace. Text annotations remain in the window after the trace is overwritten by a new trace. Text annotations are not saved in the data file. 4.
Chapter 4 Voyager Instrument Control Panel Basics 4.3.5 Previewing and Printing Traces This section includes: • Previewing and printing traces • Dedicating a printer to landscape orientation • Print Setup Previewing and printing Setting trace colors manually To preview and print traces: 1. Display the traces as desired. For a clearer printout, you can set the Trace Color and other attributes to black before printing traces: • Select Graphic Options from the Display menu.
Using the Spectrum Window NOTE: If you set Landscape printing orientation within Instrument Control Panel, this setting is lost when you close Instrument Control Panel. To permanently set the printer, see “Dedicating a printer to landscape orientation” on page 4-20. 4. From the File menu, select Print Preview to view the traces before printing. NOTE: To print without previewing, select Print Spectrum from the File menu. 5. Click Print.
Chapter 4 Voyager Instrument Control Panel Basics Dedicating a printer to landscape orientation To dedicate the printer to landscape orientation, set the orientation from the Windows desktop: 1. Click Start, then select Settings. 2. Click Printers. 3. Select the printer name in the list displayed. 4. Click on File and select Document Defaults. 5. In the Page Setup Tab, select Landscape orientation.
Customizing the Instrument Control Panel 4.4 Customizing the Instrument Control Panel Undocking toolbars The toolbar at the top of the Instrument Control Panel is divided into sections. A section is preceded by a double vertical bar. You can “undock” each section of the toolbar and move it anywhere within the Instrument Control Panel by click-dragging the double bar at the left of the toolbar section.
Chapter 4 Voyager Instrument Control Panel Basics Accessing graphic options To access the graphic options: 1. Display the trace of interest. 2. From the Display menu, select Graphic Options, then click a Graph Setup tab in the Graph and Plot Options dialog box (see Figure 4-6 on page 4-23). 3. Set Setup parameters. 4. Set colors, line widths, data cursors, and graphic compression. See the Data Explorer Software User’s Guide , Section 1.5, Setting Graphic Options, for more information.
Customizing the Instrument Control Panel Turn grid on and off Turn right axis on and off Change line type of trace Change color of peak labels Turn cursor on and off 4 Figure 4-6 Graph and Plot Options Dialog Box For additional graphic and plot option descriptions, see the Data Explorer Software User’s Guide, Section 1.5, Setting Graphic Options.
Chapter 4 Voyager Instrument Control Panel Basics 4.5 Controlling the Workstation This section includes: • Using toolbar buttons and instrument menu commands • Adjusting laser intensity and selecting sample position 4.5.1 Using Toolbar Buttons and Instrument Menu Commands Instrument buttons in the toolbar (Figure 4-7) and Instrument menu commands allow you to control the software and the Voyager mass spectrometer.
Controlling the Workstation Turning high voltage on and off Click in the toolbar to turn the high voltage on and off. You can also control high voltage by selecting Turn On/Off High Voltage from the Instrument menu. NOTE: High voltage is automatically turned on when an acquisition is started. High voltage is automatically turned off when exiting the Instrument Control Panel or ejecting a sample plate. Loading and ejecting the sample plate Click in the toolbar to load or eject the sample plate.
Chapter 4 Voyager Instrument Control Panel Basics Accumulating spectra Click in the toolbar to manually accumulate spectra. You can also accumulate spectra by selecting Accumulate Spectrum from the Acquisition menu. For more information, see Section 6.2.2, Manually Accumulating Spectra from Multiple Acquisitions. Clearing an accumulated spectrum Click in the toolbar to manually clear an accumulated spectrum.
Controlling the Workstation 4.5.2 Adjusting Laser Intensity and Selecting Sample Position This section describes: • • • • • • Displaying the Manual Laser/Sample Position page Manually adjusting laser intensity Selecting the active sample position in Plate view Displaying coordinates of active position Switching between Plate view and Sample view Adjusting sample position in Sample view For information on automatically controlling the laser and sample position see Section 5.2.
Chapter 4 Voyager Instrument Control Panel Basics The Manual Laser/Sample Position control page allows you to: • • • • • Manually adjusting laser intensity Manually adjust laser intensity Select the active sample position Display coordinates of active position Switching between Plate view and Sample view Adjusting sample position in Sample view You can adjust the laser intensity using any of the following: • Slider control—Use to set laser intensity to an exact setting.
Controlling the Workstation Selecting the active sample position in Plate view The active sample position is the sample position from which data is acquired. Select the active sample position (from the Plate view) by doing any of the following: • Type a position name or number in the Active Position field. • Select a number from the drop-down list in the Active Position field. • Click on a sample position (ToolTip displays the position number). • Use the control stick to move to a sample position.
Chapter 4 Voyager Instrument Control Panel Basics You can use these coordinates when you create a search pattern file. For information on creating an .SP file, see “Creating a search pattern file” on page 6-47. Switching between Plate and Sample View You can change the view of the sample plate between the whole sample plate and a single sample position. Refer to the following table for plate view choices: In this view... Plate (Figure 4-8 on page 4-27) If you... The view...
Controlling the Workstation Hint: If you right-click on a position, you can change between Sample View and Plate View. Laser position Scroll bars Coordinates of Active Position Figure 4-9 Manual Laser/Sample Position Control Page—Sample View Adjusting sample position in the Sample view Adjust the sample position (in the Sample view display) by doing any of the following: • Click the up/down and left/right scroll bars.
Chapter 4 Voyager Instrument Control Panel Basics 4.6 Sequence Control Panel Sequence Control Panel The Voyager Sequence Control Panel (Figure 4-10) allows you to collect data for multiple samples using different conditions. Toolbar Run list Sequence Status Figure 4-10 Sequence Control Panel The Sequence Control Panel includes: • Toolbar—Contains buttons that control the software and the instrument. For a description of a toolbar button, place the cursor on it.
How the Instrument and Sequence Control Panels Interact When you start the Sequence Control Panel, the Instrument Control Panel is automatically started and tiled horizontally at the bottom of the screen. You can hide the Instrument Control Panel by deselecting Instrument Control Panel from the View menu. For more information, see Section 4.7, How the Instrument and Sequence Control Panels Interact, and Section 6.7, Acquiring Spectra from the Sequence Control Panel. 4.
Chapter 4 Voyager Instrument Control Panel Basics If you start the Instrument Control Panel using the Sequence Control Panel icon, it functions the same way as if you started it using the Instrument Control Panel icon, with the following exceptions: • Warning and error messages are not displayed during operation. • The Instrument Control Panel will close when you close the Sequence Control Panel.
How the Instrument and Sequence Control Panels Interact Keeping both control panels open You can keep the Sequence Control Panel and the Instrument Control Panel open at the same time. However, if you do not need Sequence Control Panel functions, close the Sequence Control Panel to improve system performance. CAUTION If you started the Instrument Control Panel by double-clicking the Sequence Control Panel icon, the Instrument Control Panel closes when you close the Sequence Control Panel.
Chapter 4 Voyager Instrument Control Panel Basics 4 4-36 PerSeptive Biosystems
] 7 5 Optimizing Instrument Settings Chapter 5 This chapter contains the following sections: 5.1 Loading, Modifying, and Saving Instrument Settings .............................. 5-2 5.2 Instrument Settings Parameter Descriptions ................................................... 5-14 5.3 Impact of Changing Instrument Settings Parameters ..................... 5-39 5.4 Optimizing Instrument Settings Parameters .....................
Chapter 5 Optimizing Instrument Settings 5.1 Loading, Modifying, and Saving Instrument Settings This section includes: • • • • • • Using instrument settings (.BIC) files Standard instrument settings (.BIC) files provided Opening and viewing instrument settings Modifying an instrument settings (.BIC) file Saving and printing instrument settings Setting instrument settings files to “read-only” status 5.1.1 Using Instrument Settings (.BIC) Files NOTE: Instrument settings and .
Loading, Modifying, and Saving Instrument Settings NOTE: Data storage parameters are not stored in .BIC files. See “Setting Data Storage parameters” on page 6-14, for more information. 5.1.2 Standard Instrument Settings (.BIC) Files Provided This section includes: • • • • Standard instrument settings files Standard instrument settings files Location of .BIC files .BIC files and control mode List of .BIC files Standard read-only instrument settings files are provided on your system.
Chapter 5 Optimizing Instrument Settings List of .BIC files Table 5-1 through Table 5-3 list the standard .BIC files provided on your system for the following modes: • Linear mode • Reflector mode • PSD mode Table 5-1 Linear Mode .BIC Files .BIC File 5 Mass Range in .BIC Optimized at (Da)* Sample Test Angiotensin_Linear.BIC Low mass peptide mix1 Calibration and Resolution (angiotensin I) 500–2,000 ACTH_Linear.
Loading, Modifying, and Saving Instrument Settings Table 5-2 Reflector Mode .BIC Files .BIC File Mass Range in .BIC Optimized at (Da)* Sample Test Angiotensin_Reflector.BIC Low mass peptide mix1 Calibration and Resolution (angiotensin I) 500–2,000 ACTH_Reflector.BIC Peptide mix2 Resolution across mass range 1,000 to 4,000 (optimized at 2,500) Insulin_Reflector.BIC Peptide mix2 Resolution (insulin) 5,000–7,000 Peptide_Sensitivity_Reflector.
Chapter 5 Optimizing Instrument Settings Table 5-3 PSD Mode .BIC Files .BIC File Sample Test Mass Range in .BIC Optimized at (Da)* PSD_Precursor.BIC Angiotensin Mirror ratio 1 for Precursor ion in Reflector mode 1,000–1,400 Angiotensin_PSD.BIC Angiotensin Mirror ratio varies for PSD analysis PSD ions for precursor mass 1296.69 Substance_P_PSD.BIC Substance P Mirror ratio varies for PSD analysis PSD ions for precursor mass 1347.74 * Mass Range specified for acquisition may be wider.
Loading, Modifying, and Saving Instrument Settings 5.1.3 Opening and Viewing Instrument Settings Overview There are two ways to open an instrument settings file: • Directly open a .BIC file • Select a .DAT file that contains the instrument settings of interest, and the software loads the .BIC Opening From the Instrument Control Panel: 1. Select Open Instrument Settings from the File menu. The Open dialog box is displayed (Figure 5-1).
Chapter 5 Optimizing Instrument Settings Opening from .DAT To open an instrument settings file from a .DAT file, select .DAT from the Files of Type drop-down list, select the .DAT that contains the .BIC of interest, and click OK. The instrument settings file is loaded. The currently loaded instrument settings file name is displayed in the title bar of the Instrument Control Panel. Viewing To view all of the instrument settings in a .
Loading, Modifying, and Saving Instrument Settings Modifying for Manual Control mode To modify the instrument settings file for Manual Control mode: 1. If the Instrument Settings control page (Figure 5-2) is not displayed, select Instrument Settings from the View menu. Figure 5-2 Instrument Settings Control Page 2. Click Mode/Digitizer to select settings. For parameter descriptions, see “Linear/Reflector Digitizer parameters” on page 5-26.
Chapter 5 Optimizing Instrument Settings Optimizing 3. Select Manual Control mode. For parameter descriptions, see Section 5.2.1, Instrument Settings Page. 4. Adjust the mass range if needed. 5. To include matrix peaks in the spectrum for calibration, deselect Low Mass Gate and set the mass to a mass below the matrix peak mass. For matrix masses, see, Appendix C, Matrices. 6. Select a calibration file (.CAL), or if you are screening samples, use the default calibration.
Loading, Modifying, and Saving Instrument Settings 5.1.5 Saving and Printing Instrument Settings Saving To save instrument settings: 1. Set all parameters as needed. 2. To save the changes under the current instrument settings file name, select Save Instrument Settings from the File menu. The name of the current instrument settings (.BIC) file is displayed in the title bar of the Instrument Control Panel.
Chapter 5 Optimizing Instrument Settings Saving .BIC files for different modes When saving .BIC files for use in different operating modes, make sure to create an identifier so that you will know which instrument settings (.BIC) files are optimized for which experiments. For example: • Linear mode—Use _LIN.BIC • Reflector mode—Use _REF.BIC • PSD mode—Use _PSD.BIC Printing To print instrument settings: 1. Open the instrument settings file in the Instrument Control Panel. 2.
Loading, Modifying, and Saving Instrument Settings 5.1.6 Setting Instrument Settings Files to “Read-Only” Status Standard instrument settings files are “read-only” files. Read-only files cannot be changed and saved. However, they can be changed temporarily and used without saving them, or saved with a new name. You can set any instrument settings file to “read-only” status. To set an instrument settings file to read-only status: 1. Display the Windows NT Explorer. 2. Select the instrument settings (.
Chapter 5 Optimizing Instrument Settings 5.2 Instrument Settings Parameter Descriptions This section describes the parameters on the Instrument Settings control page and associated dialog boxes that are stored in a .BIC file.
Instrument Settings Parameter Descriptions 5.2.1 Instrument Settings Page Select Instrument Settings from the View menu to display the Instrument Settings page (Figure 5-3).
Chapter 5 Optimizing Instrument Settings Instrument settings parameters are described in Table 5-4. Table 5-4 Instrument Settings Parameters Parameter Instrument Mode Description Displays: • • Reflector, Linear, or PSD operating mode Positive or Negative polarity For more information, see Section 1.4, Voyager-DE™ (Delayed Extraction™) Technology. Mode/Digitizer settings Click to display Instrument Mode/Digitizer dialog box. See Section 5.2.2, Mode/Digitizer Settings Dialog Box.
Instrument Settings Parameter Descriptions Table 5-4 Instrument Settings Parameters (Continued) Parameter Description Voltages Accelerating Voltage Voltage applied to the first stage ion source. Valid range is 0 to 25,000 V. For information on settings for different mass ranges, see Section 5.4.4.2, Setting Accelerating Voltage. NOTE: The calibration of the mass scale changes significantly when you change the Accelerating Voltage. Default calibration adjusts for these changes.
Chapter 5 Optimizing Instrument Settings Table 5-4 Instrument Settings Parameters (Continued) Parameter Guide Wire Voltage% Description Voltage applied to the beam guide wire. Overcomes the dispersion effect from the source and refocuses ions on the detector. The valid range for Guide Wire Voltage% is 0.000 to 0.300% of the Accelerating Voltage: • Linear mode—Use 0.05 to 0.3% as suggested by the standard instrument settings, and increase the Grid Voltage% with increasing mass. • Reflector mode—Use 0.
Instrument Settings Parameter Descriptions Table 5-4 Instrument Settings Parameters (Continued) Parameter Shots/Spectrum Description Determines the number of laser shots that each spectrum will contain. For more information, see Section 5.4.4.4, Setting Shots/Spectrum.
Chapter 5 Optimizing Instrument Settings Table 5-4 Instrument Settings Parameters (Continued) Parameter Description Spectrum Acquisition (continued) Low Mass Gate (Da) Turns on the detector voltage after the ions of the Mass specified have passed the detector. Ion masses below this entry are not considered during analysis. Suppresses matrix peaks that can interfere with ion detection, and saturate the detector as laser intensity increases.
Instrument Settings Parameter Descriptions Table 5-4 Instrument Settings Parameters (Continued) Parameter Description Calibration (continued) Default Enables default calibration. For more information, see “Default calibration” on page 6-9. External file Specifies calibration using a specified external (.CAL) file. Click to select a .CAL file previously generated in the Data Explorer software. For more information, see the Data Explorer Software User’s Guide , Section 5.3.2, Manually Calibrating. 1.
Chapter 5 Optimizing Instrument Settings Matrix influence The initial velocity is the average speed at which matrix ions desorb. The initial velocity of matrix contributes to the higher order terms in the calibration equation (see Figure 6-1 on page 6-9). The software allows you to correct the calibration equation for matrix initial velocity by selecting a matrix in instrument settings (see page 5-20).
Instrument Settings Parameter Descriptions Modifying the matrix reference file Matrix options are located in the Matrix field in the Instrument Settings control page. You can add or delete information in the matrix reference file using Microsoft Notepad Editor. You can add information to the matrix reference file by doing the following: 1. Open the Microsoft Windows NT ® Notepad text editor. See the Microsoft Windows NT User’s Guide if you need help using Notepad. 2.
Chapter 5 Optimizing Instrument Settings 5.2.2 Mode/Digitizer Settings Dialog Box Click Mode/Digitizer Settings in the Instrument Settings control page (see Figure 5-3 on page 5-15) to display the Mode/Digitizer dialog box (Figure 5-4).
Instrument Settings Parameter Descriptions Instrument Mode parameters Click the Instrument Mode tab to display the Instrument Mode page (Figure 5-4). Instrument Mode parameters are described in Table 5-6. Table 5-6 Instrument Mode Parameters Parameter Operation Mode Description Specifies Operation Mode: • • • Linear Reflector PSD NOTE: Reflector and PSD modes are not available on the Voyager-DE system. If you have a Voyager-DE system, buttons for these two modes are not displayed.
Chapter 5 Optimizing Instrument Settings Linear/Reflector Digitizer parameters Click the Linear or Reflector Digitizer tab to display the Linear Digitizer or Reflector Digitizer page (Figure 5-5). NOTE: The Reflector Digitizer tab is not displayed on the Voyager-DE system. . Figure 5-5 Mode/Digitizer Settings Dialog Box with Linear Digitizer Tab Displayed Linear and Reflector Digitizer parameters are described in Table 5-7.
Instrument Settings Parameter Descriptions Table 5-7 Linear and Reflector Digitizer Parameters Parameter Description Horizontal Settings Optimization Bin Size (nsec) Determines the time (nanoseconds) interval between subsequent data points (see Figure 5-11 on page 5-49). Use this setting to optimize resolution. Bin size and Number of Data Points are dependent values. The Bin sizes available on your system depend on the frequency (500 MHz to 4 GHz) of your digitizer.
Chapter 5 Optimizing Instrument Settings Table 5-7 Linear and Reflector Digitizer Parameters (Continued) Parameter Description Vertical Settings Vertical Scale Specifies the input range of the digitizer in millivolts. To take full advantage of the dynamic range of the digitizer, set the Vertical Scale slightly higher than the signal intensity. Choices depend on the digitizer model. For more information, see Section 5.3.5, Understanding Digitizer Settings.
Instrument Settings Parameter Descriptions Advanced parameters Click the Advanced tab to display the Advanced page (Figure 5-6). Figure 5-6 Mode/Digitizer Settings Dialog Box with Advanced Tab Displayed Advanced parameters are described in Table 5-8.
Chapter 5 Optimizing Instrument Settings Table 5-8 Advanced Parameters Parameter Mirror to Accelerating Voltage Ratio (not available in Linear mode) Description Specifies the ratio between the Mirror Voltage and the Accelerating Voltage in Reflector mode, to adjust the voltage of the mirror so that it is slightly higher than the Accelerating Voltage. A higher voltage is needed at the mirror to reflect ions. If the voltage at the mirror is equal to the Accelerating Voltage, ions will pass the mirror.
Instrument Settings Parameter Descriptions 5.2.3 Automatic Control Dialog Box On the Instrument Settings control page (see Figure 5-3 on page 5-15), select Automatic Control mode and click the Automatic Control button to display the Automatic Control Settings dialog box (Figure 5-7). NOTE: The Automatic Control button is dimmed if Automatic Control is not selected.
Chapter 5 Optimizing Instrument Settings For information on setting Instrument Settings for Automatic Control mode, see Section 6.6.2, Setting Instrument Settings for Automatic Control Mode. Table 5-9 Automatic Control Parameters—Laser Parameter Description Automatic Laser Intensity Adjustment Use Automated Laser Intensity Adjustment Enables or disables automated laser intensity adjustment.
Instrument Settings Parameter Descriptions Table 5-10 Automatic Control Parameters—Spectrum Accumulation Parameter Description Spectrum Accumulation Number to Acquire Number of spectra to save or accumulate. This field is dependent on the condition selected in the conditions field. For example, if you select Save First or Save Best, the number to acquire is restricted to one.
Chapter 5 Optimizing Instrument Settings Table 5-10 Automatic Control Parameters—Spectrum Accumulation (Continued) Parameter Save Conditions Description • (continued) Save all spectra that pass acceptance criteria— Each spectrum that meets the specified acceptance criteria is saved in a separate data file. Acquisition is performed on the same search pattern position* until Acceptance Criteria fail. The number of .DAT files created is equal to the number of spectra that pass acceptance criteria.
Instrument Settings Parameter Descriptions Table 5-10 Automatic Control Parameters—Spectrum Accumulation (Continued) Parameter Save Conditions Description • (continued) Save first spectrum to pass acceptance criteria— The first spectrum that meets the selected acceptance criteria is saved. Acquisition is performed on each search pattern position* until a spectrum passes or until the end of the search pattern is reached. If no spectra pass acceptance criteria, no data file is saved.
Chapter 5 Optimizing Instrument Settings Table 5-10 Automatic Control Parameters—Spectrum Accumulation (Continued) Parameter Accumulation Conditions Description The following Accumulation conditions create one data file that contains one spectrum: • Accumulate all—All spectra acquired are accumulated into one .DAT file. One spectrum is acquired from a search pattern position*.
Instrument Settings Parameter Descriptions Table 5-10 Automatic Control Parameters—Spectrum Accumulation (Continued) Parameter Description Accumulation Conditions Accumulate all passing (continued)—Example: If Number to Acquire=5, Number of positions in .SP=7, the number of positions analyzed is determined by whether acceptance criteria fail: (continued) • If all fail, 7 positions analyzed (total number of positions in .SP), no .
Chapter 5 Optimizing Instrument Settings Table 5-11 Automatic Control Parameters—Sample Positioning Parameter Description Automated Sample Positioning Use Automated Sample Positioning Enables or disables automated sample positioning. Search Pattern File Determines the search pattern used when Use Automated Sample Positioning is enabled. See Section 6.6.4, Search Pattern Files, for more information. Number of Positions Displays the number of positions in the currently selected search pattern file.
Impact of Changing Instrument Settings Parameters 5.3 Impact of Changing Instrument Settings Parameters This section includes: • • • • • Summary of parameters Understanding Grid Voltage% Understanding Delay Time Understanding Guide Wire Voltage% Understanding Digitizer settings 5.3.1 Summary of Parameters Optimizing parameters in a specific order Changing instrument settings parameters can impact the sensitivity, resolution, or signal-to noise ratio in different ways.
Chapter 5 Optimizing Instrument Settings Parameter Mode Impact Grid Voltage% Linear/ Reflector Critical parameter with optimum value for maximum resolution. Digitizer Bin size (nanoseconds) Linear/ Reflector Decreasing improves resolution. Digitizer Input Bandwidth (not available with Signatec digitizers) Linear/ Reflector Accelerating Voltage Linear Increasing improves sensitivity and resolution, but is limited by other factors such as the digitization rate.
Impact of Changing Instrument Settings Parameters 5.3.2 Understanding Grid Voltage% NOTE: You must calibrate the mass scale for each Grid Voltage% you use. See Data Explorer Software User’s Guide, Section 5.3.2, Manually Calibrating, for more information. Grid Voltage% works in conjunction with Accelerating Voltage (described in Section 5.4.4.2, Setting Accelerating Voltage) to define an adjustable potential gradient or electric field in the ionization region of the ion source.
Chapter 5 Optimizing Instrument Settings Potential gradient The potential gradient in the ionization region (Figure 5-8) is determined by the voltages applied to the sample plate (Accelerating Voltage) and the variable-voltage grid (Grid Voltage%). Variable-voltage grid at % of Accelerating Voltage Sample plate Accelerating Voltage at 25,000 V Ground grid Potential gradient 2.
Impact of Changing Instrument Settings Parameters For example (Figure 5-8), with a 25,000 V Accelerating Voltage and a Grid Voltage of 56%, the potential gradient is: Potential gradient = 25,000 - 14,000 V 2.8 mm = 11,000 V /2.8 mm = 3,928 V /mm Vary the potential gradient by varying the Grid Voltage% and use the recommended Accelerating Voltage for the mass range. For more information, see Section 5.4.4.2, Setting Accelerating Voltage.
Chapter 5 Optimizing Instrument Settings 5.3.3 Understanding Delay Time Delay Time is the time in nanoseconds after the laser ionizes the sample at which full Accelerating Voltage is applied. This creates the potential gradient that accelerates ions. Delay Time corrects the dependence of ion flight time on initial velocity. Observed mass resolution increases in proportion to the effective length of the ion flight path.
Impact of Changing Instrument Settings Parameters Relationship to Grid Voltage% Delay Time and Grid Voltage% are interactive parameters. For each Grid Voltage% there is an optimum Delay Time, and for each Delay Time there is an optimum Grid Voltage%. The best approach for optimizing Delay Time is to leave the Grid Voltage% at a fixed value, and optimize Delay Time until you obtain optimum resolution. For more information, see Section 5.4.3.4, Optimizing Delay Time.
Chapter 5 Optimizing Instrument Settings 5.3.4 Understanding Guide Wire Voltage% By applying voltage to the beam guide wire (Figure 5-10), you can overcome the dispersion effect from the source and refocus ions on the detector. Detector Guide Wire Voltage applied, ions focused on detector Figure 5-10 Beam Guide Wire All models In general: • Increase Guide Wire Voltage to increase sensitivity. • Decrease Guide Wire Voltage to increase resolution.
Impact of Changing Instrument Settings Parameters Reflector mode • To obtain maximum resolution in Reflector mode for isotopically resolved species, set the Guide Wire% to 0. • To increase sensitivity in Reflector mode, increase the Guide Wire Voltage% to: • Up to 0.02% for <5,000 Da • Up to 0.05% for >10,000 Da PSD mode In PSD mode, use settings between 0.005 and 0.02 percent. For more information, see Chapter 7, PSD Analysis. 5.3.
Chapter 5 Optimizing Instrument Settings Default Number of Data Points/Bin Size settings The software uses the following default digitizer settings: • Number of Data Points—100,000 • Bin size—Smallest possible Bin size to accommodate the mass range selected. Number of Data Points and Bin size are dependent values, if you change one value the other changes. For more information, see “Mass Range (Da)” on page 5-19.
Impact of Changing Instrument Settings Parameters Effects of adjusting Bin size A smaller Bin size (lower number of nanoseconds): • Increases resolution between peaks when peaks are narrow in time width (see Figure 5-11). • Increases the size of the data file because the mass range is recorded with finer sampling and therefore increases the number of data points collected.
Chapter 5 Optimizing Instrument Settings 5.3.5.2 Vertical Digitizer Settings This section describes: • • • • Vertical settings Vertical settings Vertical Scale Vertical Offset Input Bandwidth The digitizer settings include three vertical parameters that affect the signal acquired: • Vertical Scale—Adjusts the dynamic range of the digitizer scale to accommodate the signal you are acquiring.
Impact of Changing Instrument Settings Parameters Decrease if signal is weak Increase if signal exceeds scale Vertical Scale Figure 5-12 Effect of Vertical Scale in Digitizer Settings Suggested settings Use the settings listed below as a starting point for Vertical Scale. Adjust as needed to bring the signal into the proper range. Mass Range (Da) Vertical Scale 0–10,000 1,000 mV 10,000–50,000 200 mV >50,000 50 mV* * Not available on the Signatec digitizer.
Chapter 5 Optimizing Instrument Settings When to increase Increase the Vertical Scale if signal goes offscale (Figure 5-12).The Vertical Scale setting is typically between 50 mV and 1,000 mV. If you set it at 1,000 mV (the maximum Vertical Scale setting) and signal is offscale, decrease the laser intensity to decrease the signal intensity. The offscale signal exceeds 64,000 counts (see the right hand axis).
Impact of Changing Instrument Settings Parameters Input Bandwidth (not available with Signatec digitizers) Decreasing the Input Bandwidth decreases the response time of the detector, and can reduce higher frequency noise. A lower setting can improve resolution and smooth out a baseline, but may also decrease signal-to-noise ratio. Because the Input Bandwidth is an electronic analog filter, it may slightly shift peak centroids toward higher masses relative to peaks recorded with Full Bandwidth.
Chapter 5 Optimizing Instrument Settings 5.4 Optimizing Instrument Settings Parameters In this section This section describes: • • • • Before you begin Optimization strategy Determining the laser setting Optimizing resolution Optimizing signal-to-noise ratio Before optimizing instrument settings parameters, be familiar with the information in: • Data Explorer Software User’s Guide, Appendix B, Overview of Isotopes • Section 5.1.2, Standard Instrument Settings (.BIC) Files Provided • Section 5.1.
Optimizing Instrument Settings Parameters 5.4.1 Optimization Strategy This section gives a suggested approach for optimizing instrument settings. For details on changing instrument settings, see Section 5.1.4, Modifying an Instrument Settings File (.BIC). Standard .
Chapter 5 Optimizing Instrument Settings Strategy To optimize instrument settings, do the following: 1. Open a .BIC file for the mass range you are analyzing. For information on mass ranges in .BIC files, see Section 5.1.2, Standard Instrument Settings (.BIC) Files Provided. If a .BIC file for the mass range you are analyzing does not exist, open a standard .BIC file with the closest higher mass. Hint: To optimize a wide mass range, select a .
Optimizing Instrument Settings Parameters 5.4.2 Determining the Laser Setting This section includes: • • • • Overview Overview Laser intensity and matrix Adjusting laser intensity Signal saturation Adjust laser intensity as described below to obtain a setting that allows you to optimize resolution and signal-to-noise ratio, as described in the following sections. See Section 6.3.2, Laser Intensity, for more information.
Chapter 5 Optimizing Instrument Settings Adjusting laser intensity To efficiently optimize the laser setting, increase or decrease the laser settings to the mid-setting of a continuously decreasing laser window. To adjust the laser settings, click-drag the slider bar on the Manual Laser Intensity/Sample Positioning control page. For more information, see “Manually adjusting laser intensity” on page 4-28. Adjusting laser intensity depends on the following: If... Then... You are using a .
Optimizing Instrument Settings Parameters • If the spectrum you obtain is not acceptable, increase or decrease the laser again in 50 to 100-step increments or decrements and reacquire. If you pass the optimum laser setting, increase or decrease using a setting that is midway between the previous two step increments or decrements. Signal saturation If the laser intensity is too high, the signal may be saturated (Figure 5-16).
Chapter 5 Optimizing Instrument Settings NOTE: Saturated signal in any region of the spectrum may suppress peaks in the Mass Range of interest. Decreasing the laser should optimize the signal (Figure 5-17).
Optimizing Instrument Settings Parameters 5.4.3 Optimizing Resolution This section includes: • • • • • Overview Acceptable resolution in Delayed Extraction Mode Optimizing Guide Wire Voltage% Optimizing Delay Time Optimizing Grid Voltage% For information on measuring resolution, see Section 6.5.2, Calculating Mass Resolution, and Data Explorer Software User’s Guide , Section 6.4, Using the Mass Resolution Calculator.
Chapter 5 Optimizing Instrument Settings 5.4.3.1 Overview This section includes: • • • • Parameters to adjust Parameters to adjust DE parameters Relationship between DE parameters Readjusting the laser after optimization You can set the following parameters to optimize resolution: • Guide Wire Voltage% • Delay Time • Grid Voltage% The following sections give guidelines for setting these parameters.
Optimizing Instrument Settings Parameters DE parameters Improved resolution in Delayed Extraction technology is achieved by velocity-focusing ions. See “Velocity focusing” on page 1-15, for more information. Two instrument settings parameters affect the velocityfocusing of ions in Delayed Extraction: • Delay Time—Time in nanoseconds (after the laser ionizes the sample) at which full Accelerating Voltage is applied, creating the potential gradient that accelerates ions.
Chapter 5 Optimizing Instrument Settings Effects of mass and matrix Note the following: • At a fixed Delay Time, higher masses require a lower Grid Voltage%. See Appendix E, Grid Voltage% and Delay Time Settings, for a graphic representation of the relationship between mass, Grid Voltage%, and Delay Time. • At a fixed Grid Voltage%, higher masses require a higher Delay Time.
Optimizing Instrument Settings Parameters 5.4.3.2 Acceptable Resolution in Delayed Extraction Mode Isotope resolution You should be able to partially resolve isotopes up to the following masses: • Linear mode— ~2,000 Da • Reflector mode— ~6,000 Da Guidelines for acceptable resolution Mass Range Acceptable resolution is determined by the mass range you are analyzing.
Chapter 5 Optimizing Instrument Settings If you cannot obtain the resolution listed for the mass range of interest, consider the following possible causes: • • • • • • Laser intensity is too high Sample oxidized, fresh sample needed Sample does not ionize well in the matrix Sample is too dilute or too concentrated Impurities are suppressing ionization of sample There are heterogeneous components in the peak 5.4.3.3 Optimizing Guide Wire Voltage% Start with the .
Optimizing Instrument Settings Parameters 5.4.3.4 Optimizing Delay Time This section includes: • • • • Overview Overview Inherent Delay Time offset Optimizing Delay Time Setting Delay Time to resolve isotopes across a broad mass range Use this procedure when operating in Linear mode or Reflector mode. Start with a standard .BIC file, optimize the Delay Time, and leave the Grid Voltage% unchanged. Before optimizing, read “Setting Delay Time to resolve isotopes across a broad mass range” on page 5-71.
Chapter 5 Optimizing Instrument Settings Optimizing Delay Time NOTE: If you are analyzing a broad mass range and need optimum resolution in all mass regions, you may need to acquire smaller portions of the mass range and set the Delay Time to optimize resolution for each mass range. To optimize Delay Time: 1. Open a standard .BIC file for the mass range you are acquiring. For more information, see “Selecting a .BIC file” on page 5-8. 2. Acquire a spectrum and observe the resolution.
Optimizing Instrument Settings Parameters 3. If the resolution improves by at least 20 percent (±10 percent), continue increasing the Delay Time in 100 nsec for Linear mode, or 50 nsec for Reflector mode increments. Table 5-12 through Table 5-14 list valid Delay Time settings for different systems and mass ranges. If the resolution does not improve, decrease the Delay Time (by 100 nsec for Linear mode, or 50 nsec for Reflector mode), acquire a new spectrum and observe.
Chapter 5 Optimizing Instrument Settings Table 5-13 Voyager-DE PRO Reflector Mode—Delay Time and Grid Voltage% Values (Continued) Reflector Mode Mass Range (Da) Delay Time (nsec) Grid Voltage% 2,000–10,000 100–500 72–78 10,000–100,000 300–600 72–78 >100,000 No data available No data available Table 5-14 Voyager-DE STR Reflector Mode—Delay Time and Grid Voltage% Values Reflector Mode Mass Range (Da) Delay Time (nsec) Grid Voltage% 500–2,000 50–100 70–80 2,000–10,000 50–500 70–80 10,000
Optimizing Instrument Settings Parameters 4. Continue increasing (or decreasing) the Delay Time in 100 nsec for Linear mode, or 50 nsec for Reflector mode increments (or decrements) until optimum resolution is obtained. Hint: If you obtain close to acceptable resolution at one setting, but less acceptable resolution at the next setting, you may have adjusted the Delay Time too far. Increase or decrease in smaller increments until you obtain optimum resolution. 5.
Chapter 5 Optimizing Instrument Settings 5.4.3.5 Optimizing Grid Voltage% Overview You can use this procedure as an alternative to the procedure in Section 5.4.3.4, Optimizing Delay Time. However, optimizing Delay Time is the recommended procedure. Optimizing the Delay Time ensures maximum stability of the high voltage power supplies, and therefore results in better mass accuracy. You can use this procedure when operating in Linear mode or Reflector mode. Start with a standard .
Optimizing Instrument Settings Parameters Optimizing Grid Voltage% To optimize Grid Voltage%: 1. Open a standard .BIC file for the mass range you are acquiring. For more information, see “Selecting a .BIC file” on page 5-8. 2. Acquire a spectrum and observe the resolution. For more information, see Section 6.5.2, Calculating Mass Resolution. If the resolution is not acceptable, increase the Grid Voltage% (by 0.5 percent for Linear mode, or 1.
Chapter 5 Optimizing Instrument Settings If the resolution does not improve, decrease the Grid Voltage% (by 0.5 percent for Linear mode, or 1.0 percent for Reflector mode), acquire a new spectrum and observe. 4. Continue increasing (or decreasing) the Grid Voltage% in increments (or decrements) of 0.5 percent (Linear mode) or 1.0 percent (Reflector mode) until optimum resolution is obtained.
Optimizing Instrument Settings Parameters 5.4.4 Optimizing Signal-to-Noise Ratio This section includes: • • • • • Overview Setting Accelerating Voltage Setting Guide Wire Voltage% Setting Shots/Spectrum Setting Low Mass Gate For more information on measuring Signal-to-Noise, see Section 6.5.3, Calculating Signal-to-RMS Noise Ratio, and the Data Explorer Software User’s Guide, Section 6.5, Using the Signal-to-Noise Ratio Calculator.
Chapter 5 Optimizing Instrument Settings 5.4.4.1 Overview You can set the following parameters to optimize signal-to-noise ratio: • • • • Accelerating Voltage Low Mass Gate Shots per Spectrum Guide Wire Voltage% The following sections give guidelines for setting these parameters. .
Optimizing Instrument Settings Parameters 5.4.4.2 Setting Accelerating Voltage Overview Accelerating Voltage defines the energy of ions as they travel in the flight tube and reach the detector. Efficiency of detection, particularly for high mass ions, increases with higher ion energy. Therefore, the maximum Accelerating Voltage typically yields optimum performance when analyzing masses above 10,000 Da. However, a lower Accelerating Voltage can increase flight times, and can improve resolution in spectra.
Chapter 5 Optimizing Instrument Settings A lower Accelerating Voltage setting does the following: • Provides more data points across a peak for better peak definition when analyzing low mass ions (Linear mode only). • Improves resolution when analyzing compounds below 2,000 Da, and the resolution is limited by the digitization rate of the system (Reflector mode). 5.4.4.3 Setting Guide Wire Voltage% To optimize sensitivity, you can adjust the Guide Wire Voltage%: • Linear mode—Increase in increments of 0.
Optimizing Instrument Settings Parameters 5.4.4.4 Setting Shots/Spectrum Overview A higher number of Shots/Spectrum can improve signal-to-noise ratio, and also improve the dynamic range of the acquisition. However, acquiring a higher number of Shots/Spectrum increases acquisition time and file size. When random noise is present in a spectrum, the improvement in signal-to-noise ratio is approximately proportional to the square root of the number of Shots/Spectrum taken.
Chapter 5 Optimizing Instrument Settings When to use Turn on Low Mass Gate when: • Analyzing masses greater than 2,000–3,000 Da • Matrix signal is more intense than the sample signal Optimum setting for starting mass You will need to experiment with the optimum setting for the starting mass (threshold) for Low Mass Gate.
Optimizing Instrument Settings Parameters Low Mass Gate spike Figure 5-20 Low Mass Gate Spike The spike occurs at a time that corresponds to just below the Mass for the Low Mass Gate entered in the .BIC file. For example, if the Mass is set to 400 Da, you would see the spike at approximately the time that corresponds to 370 Da. You can identify the Low Mass Gate spike by its sharp lift-off, its narrow width, and the noise as it returns to baseline.
Chapter 5 Optimizing Instrument Settings 5 5-82 PerSeptive Biosystems
Chapter 6 Acquiring Mass Spectra 6 This chapter contains the following sections: 6.1 Before You Begin ............................................. 6-2 6.2 Acquiring in Manual Mode from the Instrument Control Panel ................. 6-11 6.3 Obtaining Good Spectra in Delayed Extraction Mode ........................... 6-20 6.4 Making Accurate Mass Measurements........... 6-25 6.5 Evaluating Data in the Instrument Control Panel ..................... 6-27 6.
Chapter 6 Acquiring Mass Spectra 6 6.1 Before You Begin This section includes: • Overview of acquisition options • Guidelines for acquiring • Calibrating the mass scale 6.1.1 Overview of Acquisition Options NOTE: You cannot operate the mass spectrometer with the front or side panels off. Safety interlocks prevent operation when panels are not in place.
Before You Begin Table 6-1 Voyager Acquisition Options (Continued) Acquisition Option Automatic Control mode in Instrument Control Panel Batch mode in Sequence Control Panel Description • Single sample • Automatic/manual control of laser intensity or sample position • Automatic control of data accumulation and data storage • Automatic data evaluation based on acceptance criteria you specify • Default or external calibration • Multiple samples • Uses instrument settings previously defined for
Chapter 6 6 Acquiring Mass Spectra 6.1.2 Guidelines for Acquiring This section includes: • • • • • • High voltage warmup for improved mass accuracy High voltage warmup for improved mass accuracy Initial laser setting Using optimized instrument settings When acquiring a spectrum Obtaining acceptable data Obtaining maximum mass accuracy For maximum mass accuracy, allow the high voltage power supplies to warm up for a short period of time before acquisition.
Before You Begin When acquiring a spectrum When acquiring a spectrum, note the following: • For the first spectrum you acquire, the laser setting saved in the .BIC file selected is applied. If the laser intensity in the .BIC is not optimized for the mass range, you may not see a signal. • Make subsequent laser adjustments as described in “Adjusting laser intensity” on page 5-58.
Chapter 6 Acquiring Mass Spectra • Observe the Spectrum window or Oscilloscope screen to determine if data is acceptable. See Section 4.3, Using the Spectrum Window, or Appendix I, Using the Oscilloscope and Control Stick, for more information. 6 Obtaining acceptable data The quality of the data you acquire is directly affected by: • Ionization properties of the sample • Sample preparation, discussed in Section 3.
Before You Begin 6.1.3 Calibrating the Mass Scale 6 This section describes: • • • • • • Types of calibration Types of calibration When to calibrate Calibration equations Default calibration Generated calibration Acquiring calibration standards The Voyager software includes a default calibration routine that provides adequate mass accuracy for many applications.
Chapter 6 Acquiring Mass Spectra • 6 Sequence Control calibration–Provides external, internal, and internal with automatic updating calibration options during acquisition from the Sequence Control Panel. For more information, see Section 6.7.7.1, Calibration Options in a Sequence. For the mass accuracy specifications for your instrument, refer to Appendix A, Specifications.
Before You Begin Calibration equations The general equation that the Voyager software uses for calibration with a .CAL file is described in Figure 6-1. 6 t (nsec) = t 0 + A √m/z + (higher order terms) where: t = t0 = A ~ ~ Flight time of the ion Difference in time between the start time of the analysis and the time of ion extraction in Delayed Extraction, or the time of ionization in Continuous Extraction. Also called Effective Delay.
Chapter 6 Acquiring Mass Spectra Generated calibration 6 If you are performing an internal standard calibration, the software determines the constants as listed below: Internal Standard Calibration One-point Constant Value Used A Calculates from standard mass t0 Uses value from default calibration Two-point t0 and A Calculates from standard mass Three-point t0 and A Calculates by linear least-squares fit of the data points from standard mass Acquiring calibration standards Use standards t
Acquiring in Manual Mode from the Instrument Control Panel 6.2 Acquiring in Manual Mode from the Instrument Control Panel This section includes: • Manually acquiring, evaluating, and saving spectra • Manually accumulating spectra from multiple acquisitions Before acquiring spectra, become familiar with the information in Section 6.1, Before You Begin. 6.2.
Chapter 6 6 Acquiring Mass Spectra Name of .BIC file Instrument Settings Data Storage Laser/ Sample Positioning Calibration info Figure 6-2 Instrument Control Panel Before Acquiring NOTE: If the Instrument Settings control page is not displayed, select Instrument Settings from the View menu. 2. 6-12 PerSeptive Biosystems In the Instrument Settings control page, click Manual.
Acquiring in Manual Mode from the Instrument Control Panel 3. Specify calibration information: • Calibration Matrix—Select the matrix used for your application. For more information, see “Matrix influence” on page 5-22. • Default or External—Select Default, or select an external .CAL file. For more information, see “Types of calibration” on page 6-7.
Chapter 6 Acquiring Mass Spectra 6 Hint: You can double-click on the control page to “undock” it, and click-drag it to any location on the screen. Double-click again to re-dock the page. 3. Set the laser intensity by click-dragging the slider or clicking the arrows. For more information, see Section 4.5.2, Adjusting Laser Intensity and Selecting Sample Position, and Section 5.4.2, Determining the Laser Setting. Hint: You can also set laser intensity by pressing Ctrl+PgUp and Ctrl+PgDn on the keyboard.
Acquiring in Manual Mode from the Instrument Control Panel 3. Type a filename. 4. Select Autosequence Filenames if you want the software to determine the next available sequential filename. 6 NOTE: If you deselect Autosequence Filenames, the software uses the name in the Filename field and will overwrite an existing data file. If Autosequence is enabled, a 4-digit suffix starting at 0001 is automatically appended to the filename specified. For example, SAMPLE0001.
Chapter 6 Acquiring Mass Spectra During acquisition 6 During acquisition: • The Live/Current Spectrum trace in the Spectrum window updates to display the spectrum that results from each laser shot. NOTE: If your system includes an oscilloscope, the Current Spectrum does not display a trace until acquisition is complete. The spectra that result from each laser shot are displayed on the oscilloscope screen. • The system averages all spectra acquired since you started acquisition.
Acquiring in Manual Mode from the Instrument Control Panel 6 Check for signal saturation Figure 6-4 Checking for Signal Saturation 3. At this point, you can: • Save the data, described in “Saving data” on page 6-18. • Acquire additional spectra and create an accumulated spectrum, described in Section 6.2.2, Manually Accumulating Spectra from Multiple Acquisitions. CAUTION If you do not save data before starting a new acquisition, the data in the Current trace is lost.
Chapter 6 Acquiring Mass Spectra Saving data 6 To save the data when you have more than one trace displayed in the Spectrum window: 1. Select (click on) the Current trace. 2. Select Save Spectrum from the Acquisition menu. The data is saved using the file name specified in the Data Storage control page. Information about the data file is displayed in the Data Storage tab in the Output window at the bottom of the Instrument Control Panel.
Acquiring in Manual Mode from the Instrument Control Panel 6.2.2 Manually Accumulating Spectra from Multiple Acquisitions 6 You can manually accumulate spectra from different acquisitions to improve resolution and signal-to-noise ratio of your data. Accumulating To manually accumulate spectra from multiple acquisitions: 1. Acquire a spectrum and evaluate the data as described in Section 6.2.1, Manually Acquiring, Evaluating, and Saving Spectra. 2. When acquisition is complete, do not save the data.
Chapter 6 Acquiring Mass Spectra Obtaining Good Spectra 6 6.3 in Delayed Extraction Mode This section describes: • What is a good spectrum? • Laser intensity • Parameters affecting resolution and signal-to-noise ratio 6.3.1 What is a Good Spectrum? A good spectrum is one that is acceptable for your analysis.
Obtaining Good Spectra in Delayed Extraction Mode 6 High laser power causing the following: Baseline noise Mass is fairly accurate Poor separation between molecular ion and matrix adduct ion Signal near saturation point Dimer Figure 6-6 Example of Poor Mass Spectrum for Myoglobin Using Sinapinic Acid Lower laser power producing: Correct intensity ratio between singly-charged ion [M+H]+ and double-charged ion [M+2H]2+ Good separation between molecular ion and matrix adduct ion Resolved to over 1,00
Chapter 6 6 Acquiring Mass Spectra High laser power causing the following: Mass is accurate Matrix peaks and sample saturated Signal is saturated Broad peak Poor resolution Minor components or fragments of major components ionized Baseline noise Figure 6-8 Example of Poor Mass Spectrum for Angiotensin Lower laser power producing: Peak not saturated Matrix peaks minimized Sharp, narrow peak resolved to isotopes No minor components or fragments Figure 6-9 Example of Good Mass Spectrum for Angiote
Obtaining Good Spectra in Delayed Extraction Mode 6.3.2 Laser Intensity Overview 6 Laser intensity does not have a major impact on resolution or signal-to-noise ratio. You need to find the laser setting that gives you an acceptable signal-to-noise ratio and acceptable resolution (optimum is not necessary), and then fine-tune. If adjusting Grid Voltage% and Delay Time causes the signal to saturate, you may need to make additional laser adjustments.
Chapter 6 6 Acquiring Mass Spectra Prompt fragments PSD fragments Prompt fragments appear in the spectrum at masses that correspond to the theoretical masses of the fragments, because they are formed in the source. PSD fragments appear in the spectrum at masses slightly higher than the theoretical masses of the fragments because they are formed after the source, but travel at the same speed as the precursor until they reach the reflector.
Making Accurate Mass Measurements 6.4 Making Accurate Mass Measurements Overview 6 Accurate mass measurements are typically made by including reference compounds (internal standards) in the same spectrum as the analyte, and then recalibrating the spectrum. In Reflector mode (Voyager-DE PRO and Voyager-DE STR), internal calibration provides mass spectra with a mass accuracy of 10 to 20 ppm routinely. This section describes the factors that influence mass accuracy at this level.
Chapter 6 Acquiring Mass Spectra • Signal intensity of reference and analyte peaks is in the same range—Signal intensity of reference and analyte peaks should be of similar magnitude. If they are not in the same range, you may see weak analyte peaks with poor signal-to-noise ratio while the internal standard is adequate, or saturated internal standard peaks with adequate analyte peaks. • No contaminants present—Unresolved contaminants can affect peak shape.
Evaluating Data in the Instrument Control Panel 6.5 Evaluating Data in the Instrument Control Panel 6 This section describes: • Detecting, integrating, and labeling peaks • Calculating mass resolution • Calculating signal-to-RMS noise ratio 6.5.1 Detecting, Integrating, and Labeling Peaks Detecting peaks Peaks are not detected in the Spectrum window until acquisition is complete. To adjust peak detection when acquisition is complete 1. From the Tools menu, select Peak Detection.
Chapter 6 Acquiring Mass Spectra NOTE: The parameters in this dialog box correspond to the Advanced Settings tab in the Peak Detection dialog box in the Data Explorer software. 6 2. Select a detection range and set parameters as described in the Data Explorer Software User’s Guide , “Advanced Settings” on page 3-24. 3. Click Apply to accept the parameters and leave the dialog box open, or click OK to accept the parameters and close the dialog box.
Evaluating Data in the Instrument Control Panel 6.5.2 Calculating Mass Resolution 6 You can calculate mass resolution for up to four peaks in the spectrum currently being acquired. The resolution values are displayed in the trace next to the mass value for the peak. Calculating resolution for live data To calculate mass resolution: 1. When acquisition is complete, select the Current spectrum. 2. From the Tools menu, select Resolution Calculator. 3.
Chapter 6 6 Acquiring Mass Spectra 5. Type in up to four Mass/Charge values for which to calculate resolution. 6. For each Mass/Charge, enter the window for calculation (± AMU). NOTE: To label isotopes, set the ± AMU value low enough to prevent the calculation windows for each isotope peak from overlapping. If the calculation windows overlap, only the highest peak is labeled. If you set too low, the peak of interest may not be labeled.
Evaluating Data in the Instrument Control Panel 6 Resolution result Figure 6-13 Resolution Calculator Results The table below lists a general rating scale for resolution and molecular weight ranges for compounds acquired in Linear mode.
Chapter 6 6 Acquiring Mass Spectra 6.5.3 Calculating Signal-to-RMS Noise Ratio Overview A signal-to-RMS noise ratio is typically used to describe how well a mass of interest in a spectrum is distinguished from background chemical and electronic noise. The Control Panel software measures the signal-to-noise ratio in a user-defined region of a mass spectrum. Calculating signal-to-RMS noise ratio To calculate a signal-to-RMS noise ratio: 1.
Evaluating Data in the Instrument Control Panel 4. For each Mass/Charge, enter the window for calculation (± AMU). NOTE: To label peaks, set the ± AMU value low enough to prevent the calculation windows for each peak from overlapping. If the calculation windows overlap, only the first peak is labeled. However, if you set ± AMU too low, the peak of interest may not appear in the window, and signal-to-noise will not be calculated. 5. Click OK.
Chapter 6 Acquiring Mass Spectra Acquiring in 6 6.6 Automatic Mode from the Instrument Control Panel This section includes: • Before acquiring in Automatic Control mode • Setting Instrument Settings for Automatic Control mode • Automatically acquiring, evaluating, and saving spectra • Search Pattern files • Process that occurs during acquisition in Automatic Control mode • Process that occurs when accumulating spectra from multiple search pattern positions 6.6.
Acquiring in Automatic Mode from the Instrument Control Panel See Section 2.7, Aligning the Sample Plate, to determine if sample plate alignment is necessary. If you do need to align the sample plate, you must do so for each sample plate you use. 6.6.2 Setting Instrument Settings for Automatic Control Mode This section includes: • Setting Automatic Control settings • Setting spectrum acceptance and laser adjustment criteria • Saving the instrument settings (.
Chapter 6 Acquiring Mass Spectra 6 NOTE: Make sure the instrument settings yield acceptable results in Manual Control mode before setting to Automatic Control mode. 2. In the Instrument Settings control page, select Automatic Control mode. 3. Click Automatic Control to display the Automatic Control dialog box (Figure 6-15). NOTE: The Automatic Control button is dimmed if Automatic Control is not selected.
Acquiring in Automatic Mode from the Instrument Control Panel Laser 4. Select Use Automated Laser Intensity Adjustment. NOTE: To manually control the laser intensity when acquiring in Automatic Control mode, deselect Use Automated Laser Intensity Adjustment. For more information, see Section 4.5.2, Adjusting Laser Intensity and Selecting Sample Position. 5. If you enabled Use Automated Laser Intensity Adjustment, specify the Minimum and Maximum Laser Intensity and the Step Size to use.
Chapter 6 6 Acquiring Mass Spectra Spectrum 8. accumulation and saving Specify Spectrum Accumulation parameters: • Acquire X Spectra—Specifies the number of spectra to acquire. The software compares the value you enter with the possible number of positions in the selected search pattern and displays a message if you enter a value greater than the number of positions. • Under Conditions—Specifies saving or accumulation and data evaluation.
Acquiring in Automatic Mode from the Instrument Control Panel Sample positioning 10. Select Use Automated Sample Positioning. 6 NOTE: To manually adjust sample positioning when acquiring in Automatic Control mode, deselect Use Automated Sample Positioning. For more information, see Section 4.5.2, Adjusting Laser Intensity and Selecting Sample Position. 11. If you enabled Use Automated Sample Positioning, select a search pattern (.SP) file. The number of positions in the selected .SP file is displayed.
Chapter 6 6 Acquiring Mass Spectra Setting spectrum acceptance and laser adjustment criteria If you selected an accumulation condition that uses acceptance criteria (see step 9 on page 6-38): 1. In the Automatic Control dialog box (see Figure 6-15 on page 6-36), click the Spectrum Acceptance Criteria button. The Spectrum Acceptance Criteria dialog box (Figure 6-16) is displayed. Figure 6-16 Spectrum Acceptance Criteria Dialog Box 2.
Acquiring in Automatic Mode from the Instrument Control Panel Table 6-3 Spectrum Acceptance Criteria Parameters Parameter 6 Description Acceptance Criteria Minimum Signal Intensity (spectrum acceptance and laser adjustment) Sets the minimum signal intensity accepted for the most abundant peak (local base peak) within the mass range of interest. Maximum Signal Intensity Sets the maximum signal intensity for the most abundant peak (local base peak) within the mass range of interest.
Chapter 6 Acquiring Mass Spectra Table 6-3 Spectrum Acceptance Criteria Parameters (Continued) 6 Parameter Signal-to-Noise (spectrum acceptance and laser adjustment) (continued) Resolution Description Also used to determine laser adjustment. When the system adjusts the laser, it checks that the signal-to-noise ratio is above the value entered. If it is not, the system increases the laser intensity.
Acquiring in Automatic Mode from the Instrument Control Panel 6.6.3 Automatically Acquiring, Evaluating, and Saving Spectra 6 To automatically acquire a spectrum: 1. In the Instrument Control Panel, open or create an instrument settings file with the appropriate parameters. See Section 6.6.2, Setting Instrument Settings for Automatic Control Mode, for information. 2. Set Data Storage parameters as described in “Setting Data Storage parameters” on page 6-14.
Chapter 6 Acquiring Mass Spectra During acquisition 6 During acquisition: • Laser is adjusted, sample position is adjusted, and Spectrum Acceptance Criteria are applied to each search pattern position in a spectrum. For more information, see “Spectrum Accumulation” on page 5-33, and Section 6.6.5, Process that Occurs During Acquisition in Automatic Mode. • Each spectrum is saved or accumulated as determined by the conditions selected in Spectrum Accumulation in the Automatic Control dialog box.
Acquiring in Automatic Mode from the Instrument Control Panel A search pattern file is an ASCII text file that contains a list of relative X,Y position pairs with respect to the center of the current sample position measured in microns, that represent points in the sample position. On a 100-position plate, a sample position is 2,540 µm in diameter with the origin (0,0) at the center of the position. The centers of the sample positions are 5,080 µm apart.
Chapter 6 Acquiring Mass Spectra Default search pattern file 6 X coordinate (µm) The default search pattern file for a 100-position plate (DEFAULT.SP) causes a serpentine crossing of the sample position determined by the following 20 X,Y coordinates: Y coordinate (µm) X coordinate (µm) Y coordinate (µm) 1. -952.5 158.75 11. -635 -635 2. -635 317.5 12. -238.125 -396.875 3. -317.5 476.25 13. 158.75 -238.125 4. 0 635 14. 476.25 0 5. 317.5 793.75 15. 873.125 238.125 6.
Acquiring in Automatic Mode from the Instrument Control Panel Spiral search pattern file Search pattern for custom plates The SPIRAL.SP file provided is a 20-point search pattern that begins searching at the center of the sample position and spirals outward. This is the best search pattern for uneven matrix crystals. If you create a custom plate type for a plate without laser-etched sample position or wells, with position diameter larger or smaller than 2,540 microns, create a .
Chapter 6 Acquiring Mass Spectra 6 Hint: To determine coordinates, spot a sample plate with standard, load the plate, start the laser, observe where the laser strikes the sample position, move the sample position under the laser, and note the relative coordinates in the Sample view of the Manual Laser/Sample Positioning control page. For more information, see Section 4.5.2, Adjusting Laser Intensity and Selecting Sample Position. 5. Save the file with an SP extension (for example, 4POINT.
Acquiring in Automatic Mode from the Instrument Control Panel 6.6.5 Process that Occurs During Acquisition in Automatic Mode 6 NOTE: This process occurs when you acquire using a .BIC file that has User Automated Laser Intensity Adjustment enabled.
Chapter 6 Acquiring Mass Spectra In Prescan mode, the system does the following: 6 1. The system sets the laser to the maximum setting specified in the Automatic Control dialog box and acquires a spectrum. The system starts acquiring data at the first point specified in the search pattern file (described in “Search pattern files” on page 6-44).
Acquiring in Automatic Mode from the Instrument Control Panel 2. The system sets the laser to the minimum setting specified in the Automatic Control dialog box and acquires a spectrum. 6 If the signal intensity is: • Too low with the laser at minimum—The system continues with step 3. • Too high with the laser at minimum—The system begins acquiring in Acquisition mode. • Within range—The system begins acquiring in Acquisition mode. 3.
Chapter 6 6 Acquiring Mass Spectra Acquisition mode Acquisition mode starts after Prescan mode determines the laser setting, or immediately if Prescan mode is disabled. NOTE: Each time the system begins acquiring in Acquisition mode, it acquires the number of Shots/Spectrum specified in the Instrument Settings control page. In Acquisition mode, the system does the following: 1.
Acquiring in Automatic Mode from the Instrument Control Panel NOTE: If no minimum or maximum signal intensity criteria is specified, the laser is not adjusted. The mid-range laser setting specified in the Automatic Control dialog box is used. • Within range—The system saves the data file if Signal-to-Noise is not enabled, or continues with step 2 if Signal-to-Noise is enabled. 2.
Chapter 6 Acquiring Mass Spectra 4. 6 If Save All or Accumulate All is selected for accumulation, the system moves to the next search pattern position. For all other accumulation conditions, the system repeats step 1 through step 3 until one of the following is true: • If the Acceptance criteria selected are met (the signal is in range, the minimum Signal-to-Noise ratio and resolution are achieved). • The Number of Spectra to Acquire are acquired.
Acquiring in Automatic Mode from the Instrument Control Panel If Acceptance Criteria are not met, if an accumulation condition that does not use Acceptance Criteria is selected, or if the laser can no longer be adjusted, the system begins a new acquisition from the next search pattern position, depending on the Spectrum Accumulation conditions and Spectrum Acceptance Criteria. Acceptance criteria are defined in “Setting spectrum acceptance and laser adjustment criteria” on page 6-40.
Chapter 6 6 Acquiring Mass Spectra 6.6.6 Process that Occurs when Accumulating Spectra from Multiple Search Pattern Positions NOTE: These processes occurs when you acquire using a .BIC file that has User Automated Sample Positioning enabled. You can obtain a single spectrum from multiple positions within a single sample position by specifying a search pattern.
Acquiring in Automatic Mode from the Instrument Control Panel 6.6.6.1 Process that Occurs when Accumulating All Spectra 6 When accumulating all spectra (Accumulation mode is determined by the Spectrum Accumulation conditions described in “Automatic Control Dialog Box” on page 5-31), the system does the following during acquisition: 1. Sets the laser intensity as described in Section 6.6.5, Process that Occurs During Acquisition in Automatic Mode. 2. Turns on the laser. 3.
Chapter 6 6 Acquiring Mass Spectra 6.6.6.2 Process that Occurs when Accumulating Passing Spectra When accumulating only the spectra that meet the Acceptance Criteria (Accumulation mode is determined by the Spectrum Accumulation conditions described in “Automatic Control Dialog Box” on page 5-31), the system does the following during acquisition: 1. Sets the laser intensity as described in Section 6.6.5, Process that Occurs During Acquisition in Automatic Mode. 2. Turns on the laser. 3.
Acquiring in Automatic Mode from the Instrument Control Panel 9. Repeats step 2 through step 8 in subsequent search pattern positions until any of the following is true: • Number of spectra to acquire that you select in the Automatic Control dialog box is reached • All search pattern positions have been scanned • Stop conditions are met 10. Saves the averaged spectrum to disk in the directory designated in the Data Storage control page, described in “Setting Data Storage parameters” on page 6-14.
Chapter 6 Acquiring Mass Spectra Acquiring Spectra from 6 6.7 the Sequence Control Panel Overview The Voyager Sequence Control Panel (Figure 6-19) allows: • Acquisition of multiple samples using different instrument settings (.BIC) files • Selection of macros for advanced processing to apply before or after calibration. You can use the macros supplied or create your own in the Data Explorer software. • External, internal, and internal-update calibration options, described in Section 6.7.
Acquiring Spectra from the Sequence Control Panel 6 Figure 6-19 Sequence Control Panel Voyager™ Biospectrometry™ Workstation User’s Guide 6-61
Chapter 6 6 Acquiring Mass Spectra 6.7.1 Understanding Settings, Macros, and Calibration This section includes: • File types and calibration specified • How file types and calibration specified affect the data File types and calibration specified File Type You specify the following types of files and the Calibration Type in the Sequence Control Panel to determine how data is detected, calibrated, and processed: Description For information see Data Explorer .
Acquiring Spectra from the Sequence Control Panel File Type .CAL Description Contain calibration constants used according to Calibration Type: • External calibration— Constants are applied and saved in .DAT file. • Internal calibration (.CAL optional)—If .CAL specified, constants are applied before the reference masses in the .SET file are matched. Constants are updated within the .DAT file after calibration. • Internal-Update calibration— If .
Chapter 6 No Calibration .DAT file with: . op SE tio T na l • Peak detection settings from .SET or defaults if no .SET • Acquisition calibration specified in .BIC .CAL calibration constants Po s op t m tio ac n a ro l External Calibration Pr e op m a tio cr na o l Data . op SE tio T na l .DAT file with: • Peak detection settings from .SET or defaults if no .SET • Calibration constants settings from .CAL Data Internal Calibration .SET peak detection .
Acquiring Spectra from the Sequence Control Panel 6.7.2 Before Creating a Sequence 6 This section describes: • Optimizing instrument settings (.BIC) files for a sequence run • Creating macros • Creating calibration (.CAL) files • Creating settings (.SET) files Optimizing instrument settings (.BIC) files for a sequence run Select .BIC files based on the compound type and mass range you are analyzing. See Section 5.1.2, Standard Instrument Settings (.BIC) Files Provided. You can use more than one .
Chapter 6 6 Acquiring Mass Spectra Creating macros You can specify macros that execute before and after calibration. You can use the macros supplied or create your own. To create macros for the Sequence Control Panel: 1. Open the Data Explorer software. 2. Create a macro as described in the Data Explorer Software User’s Guide , Section 6.8.2, Recording a Macro. 3. Assign the macro to a button as described in the Data Explorer Software User’s Guide , Section 6.8.3, Assigning Macros to Buttons.
Acquiring Spectra from the Sequence Control Panel Creating settings (.SET) files Create settings (.SET) files in the Data Explorer software if you will be specifying Internal or Internal-Update calibration, or if you want peak detection settings other than default settings stored with the data file. The .SET file can also apply monoisotopic peak filtering.
Chapter 6 6 Acquiring Mass Spectra 6.7.3.1 Setting General Sequence Parameters Set parameters as needed: 1. From the View menu, select General Sequence Parameters. The General Sequence Parameters dialog box is displayed (Figure 6-21). Figure 6-21 General Sequence Parameters Dialog Box 6-68 2. Type or select the Directory name in which to store the data files. 3. Enter text as needed for Author and Comments. This information is stored with the sequence (.SEQ) file. 4.
Acquiring Spectra from the Sequence Control Panel NOTE: Run logs are overwritten each time you run a sequence of the same name. If you want to keep run logs for future reference, rename the run log each time you run the sequence. The run log file contains the list of lines from the run list that were executed during the sequence, and any errors that occurred. If a line in the run list generated more than one data file, log lines are duplicated and file names are incremented accordingly. 5. Click OK. 6.7.
Chapter 6 Acquiring Mass Spectra 6 Run list Figure 6-22 Sequence Run List Hint: You can show and hide columns by selecting a column, then selecting Show/Hide from the View menu. 2. Click the scroll bar at the bottom of the grid to display columns in the grid that are not in view. 3. Click on a cell to activate it. 4. Enter Run List parameters as described in Table 6-4. Table 6-4 Run List Parameters Field Sample Position Description Position from which to acquire data.
Acquiring Spectra from the Sequence Control Panel Table 6-4 Run List Parameters (Continued) Field Instrument Settings File (required entry) Data Explorer .SET File (required entry for Internal, Internal-Update calibration) 6 Description .BIC file to use for the current row. Click the down arrow and select an instrument settings (.BIC) file that you have optimized for Automatic Control mode. For more information see Section 6.6.2, Setting Instrument Settings for Automatic Control Mode. .
Chapter 6 Acquiring Mass Spectra Table 6-4 Run List Parameters (Continued) 6 Field Internal/External Calibration Description Calibration type to use for this row. Click the down arrow and select one of the following: • Blank—No calibration applied during processing. Acquisition calibration (calibration specified in .BIC) is persisted. • External—Applies the constants in the .CAL file specified to the data file acquired in this row. • Internal—Applies the constants in the .
Acquiring Spectra from the Sequence Control Panel Table 6-4 Run List Parameters (Continued) Field Calibration File (required entry for External, Internal-Update calibration, optional entry for Internal calibration) 6 Description .CAL file to use to calibrate the data file acquired in this row. If you specify External or Internal-Update, a .CAL file name is required, even if a corresponding .CAL file with constants does not exist (see below for further explanation). You can specify an existing .
Chapter 6 Acquiring Mass Spectra Table 6-4 Run List Parameters (Continued) 6 Field Post-Macro Description Macro to execute after calibration. Click the down arrow and select a macro number you have defined in the Data Explorer software. For more information, see “Creating macros” on page 6-66. Hint: Display the Data Explorer software and place the cursor over a macro button to determine the macro assigned to the button. Macro buttons are numbered sequentially from left to right.
Acquiring Spectra from the Sequence Control Panel Modifying and customizing the run list You can modify and customize the run list using the following commands on the Edit menu: • Cut, Copy, Paste—Use to cut, copy and paste information. • Insert Row, Insert Multiple Rows, Delete Row—Use to insert and delete rows. • Fill Down—Automatically fills in run list grid entries. Click-drag to select the rows to fill and select Fill Down from the Edit menu.
Chapter 6 Acquiring Mass Spectra Importing 6 To import a .TXT or .XLS file: 1. Select Import from the File menu. 2. Select a file or type in a file name. 3. Click Import. Information is imported into all columns, even if columns are hidden. Exporting To export a .TXT or .XLS file: 1. Select Export from the File menu. 2. Type in a file name. 3. Select .TXT or .XLS from the Save As Type drop-down list. 4. Click Save. NOTE: The Acquisition Status column is not exported.
Acquiring Spectra from the Sequence Control Panel 6.7.4 Preparing to Run a Sequence 6 This section describes: • Aligning the sample plate • High voltage warmup for improved mass accuracy • Before acquiring Aligning the sample plate Sample plate alignment is necessary for a sequence run if the laser is not striking the center of the sample position. Sample plate alignment may not be necessary on your system, particularly if you use 100-well plates and the SPIRAL.
Chapter 6 6 Acquiring Mass Spectra Before acquiring Before acquiring a sequence: 1. Load a sequence by doing one of the following: • Create a new sequence. See Section 6.7.2, Before Creating a Sequence. • Open an existing sequence by clicking toolbar and selecting an .SEQ file. 2. in the Check system status. See Section 2.9, Checking System Status. 6.7.
Acquiring Spectra from the Sequence Control Panel Pausing and resuming a sequence To pause a sequence, click . The sequence pauses after the current entry is acquired and processed, and Sequence Acquisition Status is “Paused”. To resume the sequence, click again. The sequence resumes with the next row selected to run. Modifying the run list during acquisition To modify the run list after you start the sequence, click to pause the sequence.
Chapter 6 6 Acquiring Mass Spectra 6.7.6 Checking Sequence Status You can check sequence status in three places: • Acquisition Status field in the run list • Sequence Status control page (general status of run) • Instrument Control Panel Checking the Acquisition Status field The Acquisition Status field in the run list displays the status of each acquisition. Possible states are: • Acquiring—Acquiring a sample. • Processing—Applying macros or calibrating the data file.
Acquiring Spectra from the Sequence Control Panel Sequence status parameters include: Field 6 Description Sequence File Name Displays the name of the .SEQ file currently running. Overall Run Status Displays overall run status. Possible states are: • • • • • • Off—Not running. Running—Acquiring a sample. Pausing/Paused—Pause button clicked. Stopping/Stopped—Stop button clicked. Finished—Sample acquired and processed. Error—Error occurred during acquisition that terminated the sequence.
Chapter 6 6 Acquiring Mass Spectra 6.7.7 Automatic Calibration During a Sequence Run This section includes: • • • • Calibration options in a sequence Calibration standard requirements Performing close external calibration Internal standard calibration considerations 6.7.7.1 Calibration Options in a Sequence The Sequence Control Panel allows three types of automatic calibration: Type External Internal InternalUpdate Function Applies calibration constants in a specified .CAL file.
Acquiring Spectra from the Sequence Control Panel 6.7.7.2 Calibration Standard Requirements Mass calibration standards 6 The requirements for mass calibration standards are determined by your application. The following are general guidelines: • Mass calibrate on the same sample plate you will use to analyze samples. • For rapid screening in which high mass accuracy is not needed, one calibration standard located in the center of the plate is adequate.
Chapter 6 6 Acquiring Mass Spectra Sample and standard in separate sample positions If you are acquiring samples and standards from different sample positions, enter the standards in the run list preceding the unknowns that use the calibration. For optimum mass accuracy, place samples in sample positions adjacent to standards. See the example in Figure 6-24.
Acquiring Spectra from the Sequence Control Panel The number and placement of standards needed depend on your application. See “Mass calibration standards” on page 6-83. Hint: You can use the same calibration file more than once in a sequence run. Sample and standard in the same sample position You may see improved mass accuracy by spotting sample and standard in as close together as possible within a sample position (Figure 6-25).
Chapter 6 Acquiring Mass Spectra To acquire sample and standard, create two search pattern files to analyze the sample and standard spots. See “Creating search pattern files for close external calibration on separate spots” on page 6-86, for more information. Create two instruments settings (.BIC) files that contain the same settings, but specify the sample search pattern file and the standard search pattern file.
Acquiring Spectra from the Sequence Control Panel NOTE: Use plates without laser-etched sample positions if you spot standard and sample as shown in Figure 6-25. 2. Load the sample plate, select the position containing sample and standard, and display the Sample View. 3. Note the logical x and y coordinates for a minimum of three positions on the standard spot and a minimum of six positions on the sample spot. 4.
Chapter 6 Acquiring Mass Spectra If standard signal suppresses unknown signal, you have two options for analysis: 6 • Perform internal calibration using separate spots— Spot standard and sample as close to each other as possible. Create a search pattern file that analyzes both spots. Create an instrument settings file that generates a single spectrum and specify internal calibration in Sequence Control. See “Creating a search pattern file for internal calibration on separate spots” on page 6-88.
Acquiring Spectra from the Sequence Control Panel 2. Load the sample plate, select the position containing sample and standard, and display the Sample View. 3. Note the relative x and y coordinates for a minimum of three positions on the standard spot and a minimum of six positions on the sample spot. 4. Create the search pattern file using Windows Notepad as described “Creating a search pattern file” on page 6-47. Figure 6-26 shows an example search pattern (.
Chapter 6 6 Acquiring Mass Spectra 6.7.8 Customizing the Sequence Display Using Workbook mode Workbook mode displays the run list (contains sample information and conditions for acquisition and processing) in tabbed, framed format. To use Workbook mode: 1. Open or create a sequence run list. 2. Open or create another sequence run list. 3. Select Workbook mode from the Window menu. Figure 6-27 displays the Sequence Control Panel in Workbook mode (two tabs at bottom of run list).
Acquiring Spectra from the Sequence Control Panel 5. To set preferences, select Preferences from the File menu and specify: • Autosize—Automatically enabled when you are in Workbook mode, and the run list is automatically resized and displayed appropriately in tabbed frames when you resize the window. When Workbook mode is disabled, you can disable Autosize, and manually resize and move the run list window to any dimensions.
Chapter 6 Acquiring Mass Spectra 6 6-92 PerSeptive Biosystems
Chapter 7 7 PSD Analysis This chapter contains the following sections: 7.1 Overview of PSD Analysis ............................... 7-2 7.2 Enhancing Fragmentation with CID ................ 7-11 7.3 Acquiring PSD Data with Standard Instrument Settings (.BIC) Files ..................... 7-17 7.4 Exploring PSD Mode...................................... 7-36 7.5 Viewing PSD Data .........................................
Chapter 7 PSD Analysis 7.1 Overview of PSD Analysis This section includes: • • • • • 7 Post-source decay analysis Segments and composite spectra PSD data files Mass calculation for fragment ions Optimizing the Precursor Ion Selector NOTE: Analysis of post-source decay is available on Voyager-DE PRO and Voyager-DE STR workstations only.
Overview of PSD Analysis PSD fragment ions At higher laser intensities, some molecular ions decompose into PSD fragment ions in the flight tube after they leave the ion source (the post-source decay process).
Chapter 7 PSD Analysis Reflector AH+ from ion source Mirror Ratio = 1.00 BH+ MH+ MH+ (MW 1,000) correctly focused AH+ (MW 700) poorly focused BH+ (MW 300) poorly focused 7 Figure 7-1 Molecular and Fragment Ion Flight in the Reflector The AH+ fragment has 70 percent of the kinetic energy of the MH+ ion and the BH+ ion has 30 percent. With a Mirror Ratio setting of 1.0000, ions with lower kinetic energy are reflected quickly and are not focused by the mirror.
Overview of PSD Analysis Reflector from ion source Mirror Ratio = 0.7 AH+ BH+ MH+ (MW 1,000) not focused AH+ (MW 700) correctly focused BH+ (MW 300) poorly focused MH+ 7 Reflector Mirror Ratio = 0.3 from ion source BH+ AH+ + MH MH+ (MW 1,000) not focused AH+ (MW 700) not focused BH+ (MW 300) correctly focused Figure 7-2 Effect of Changing Mirror Ratio A Mirror Ratio setting of 1.0000 correctly focuses the original ion. Values of 0.7 and 0.3 correctly focus the lower energy fragments.
Chapter 7 PSD Analysis 7.1.2 Segments and Composite Spectra Overview To obtain the most information about an ion, collect fragment ion spectra across a molecular weight range from the mass of the original precursor ion down to 50 Da (determined by the desire to see immonium ions that indicate the presence of individual amino acids). Each fragment ion spectrum is referred to as a segment, and is collected with a discrete focusing region (controlled by the Mirror Ratio setting).
Overview of PSD Analysis 7.1.3 PSD Data Files PSD data (.DAT) files include: • Precursor ion mass • All segments acquired during a PSD experiment • Composite spectrum Segments are stored in the data file in the order in which they are collected. Precursor ion mass PSD Segment 1 PSD Segment 2 Raw data PSD Segment 3 PSD Composite Results PSD .DAT File Figure 7-3 PSD .
Chapter 7 PSD Analysis 7.1.
Overview of PSD Analysis 7.1.5 Optimizing the Precursor Ion Selector The Precursor Ion Selector (called Timed Ion Selector in Reflector mode) allows you to analyze the ion of interest by deflecting ions until the time that corresponds to the mass of the ion of interest. At the time that corresponds to the mass of the ion of interest, the Precursor Ion Selector voltage is turned off, and the ion of interest passes to the reflector.
Chapter 7 PSD Analysis 7 Figure 7-4 Timed Ion Selector Tab in Hardware Configuration Dialog Box Set the Deflector Gate Width as needed. A lower setting increases specificity, but may decrease sensitivity. CAUTION Do not change the Flight Length to Deflector parameter. This parameter is optimized for your system.
Enhancing Fragmentation with CID 7.2 Enhancing Fragmentation with CID Overview Collision-induced dissociation (CID) is a technology that enhances fragmentation in Post-Source Decay (PSD) analysis. CID is available as an option on the Voyager-DE PRO and Voyager-DE STR workstations. 7 The CID option includes: • A 0.
Chapter 7 PSD Analysis Benefits The benefits provided by CID include: • Fragmentation of ions that does not occur under normal PSD conditions • Side chain fragmentation that may allow you to distinguish between Leucine and Isoleucine isomers • Greater number of immonium ions generated for peptide analysis 7 Figure 7-6 and Figure 7-7 are sample spectra from low and mid mass ranges that illustrate the impact of CID gas.
Enhancing Fragmentation with CID CID off CID on, enhanced peaks labeled 7 Figure 7-7 Mid Masses—Impact of CID (Glu-1-Fibrinopeptide) In the top trace (CID off), typical fragments are seen and labeled. In the bottom trace (CID on), w fragments1 not seen without CID are labeled. Gas requirements You can use room air, helium, argon, or xenon as the collision gas. If you are using a compressed gas source, regulate the gas source between 2 and 5 psi. 1. Meth. Enzymol., McCloskey, J.A, ed.
Chapter 7 PSD Analysis Purging collision gas lines Before turning on the collision gas, purge the lines to prevent disruption of the vacuum. CAUTION If you do not purge the lines, the CID gas introduction may increase the pressure in the vacuum and cause an Interlock error. 7 Perform the following procedure if the CID gas has been off for more than 15 minutes: 1.
Enhancing Fragmentation with CID Turning on collision gas To turn on the collision gas after purging: 1. If the metering valve on the side of the CID box is set at zero, turn the metering valve approximately 1/4 turn. If the metering valve is not set at zero, do not turn the valve. 2. 3. Wait 1 to 2 minutes for BA1 on the vacuum gauge panel to stabilize at 3 x 10-6 Torr, or at the optimum pressure for CID operation that you have determined and recorded for your system.
Chapter 7 PSD Analysis Turning off collision gas Adjusting collision gas 7 To turn off the collision gas: 1. Turn the top 3-way valve on the CID box to the Purge Cell position. 2. Wait approximately 20 seconds for the gas to evacuate. 3. Turn the top 3-way valve on the CID box to the Off position. To optimize fragmentation, adjust the flow of the collision gas. Turn the metering valve on the CID box until you observe the desired fragmentation. If gas pressure is too high, signal is suppressed.
Acquiring PSD Data with Standard Instrument Settings (.BIC) Files 7.3 Acquiring PSD Data with Standard Instrument Settings (.
Chapter 7 PSD Analysis Differences from regular analysis When operating in PSD mode, note the following differences from analysis in non-PSD mode: • Higher laser intensity—In non-PSD mode, you use a laser intensity that yields acceptable performance without fragmentation. You need a higher laser intensity to generate PSD fragments.
Acquiring PSD Data with Standard Instrument Settings (.BIC) Files 7.3.1 Determining the Precursor Ion Mass Overview Before beginning an analysis in PSD mode: • Generate a precursor spectrum in Reflector mode to determine the mass of the precursor ion. A Reflector mode analysis provides optimum resolution and mass accuracy. • Generate an external calibration for the precursor ion to use during the PSD acquisition. Generating the precursor spectrum To generate the precursor spectrum: 1.
Chapter 7 PSD Analysis Generating an external calibration for the precursor ion To obtain maximum mass accuracy for the precursor ion, generate an external calibration file that you will use when you perform the PSD acquisition. This external calibration is used to determine the tp value (precursor ion flight time) needed for the PSD calibration equation (described on page 7-8). The tp value is determined using the standard calibration equation (“t” in the standard equation described on page 6-9).
Acquiring PSD Data with Standard Instrument Settings (.BIC) Files 7.3.
Chapter 7 PSD Analysis Hint: You can use the Windows calculator to determine natural log values. If the ln function is not displayed when you open the Windows calculator, select Scientific from the View menu in the calculator to access advanced functions. Hint: The Angiotensin_PSD.BIC file provided includes 10 segments, which is suitable for many applications. If the mass you are analyzing differs by more than 300 Da from the mass in the Angiotensin_PSD.
Acquiring PSD Data with Standard Instrument Settings (.BIC) Files Size of segments You can acquire segments of different sizes by varying the Mirror Ratio setting. For example, you can set the first Mirror Ratio to collect a 20 percent segment, then set the next Mirror Ratio to collect a 10 percent segment. If you change the default Decrement Ratio, fill-down subsequent existing rows, and the Mirror Ratio values are automatically recalculated.
Chapter 7 PSD Analysis 7.3.3 Setting PSD Acquisition Parameters Setting PSD Acquisition parameters includes: • Displaying the PSD Acquisition control page • Setting voltages and external calibration for the precursor spectrum • Setting precursor mass and PSD calibration for fragment spectra 7 Displaying the PSD Acquisition control page To display PSD Acquisition control page: 1. Open the Angiotensin_PSD.BIC file provided. This is a PSD mode .BIC file.
Acquiring PSD Data with Standard Instrument Settings (.BIC) Files 2. If the PSD Acquisition Settings control page (Figure 7-8) is not displayed, select PSD Acquisition from the View menu. 7 Figure 7-8 PSD Acquisition Settings Control Page NOTE: The Precursor Ion Selector in PSD mode is the same parameter as the Timed Ion Selector in Reflector mode.
Chapter 7 PSD Analysis 4. In the Calibration section of the Instrument Settings control page, select the matrix you are using and the .CAL file you created in “Generating an external calibration for the precursor ion” on page 7-20. NOTE: The calibration you specify on the Instrument Settings control page is used to determine the tp value (precursor ion flight time) needed for the PSD calibration equation (described on page 7-8).
Acquiring PSD Data with Standard Instrument Settings (.BIC) Files NOTE: The calibration you specify on the PSD Acquisition Settings control page is used to determine the values for α , β , and γ needed for the PSD calibration equation (described on page 7-8). The calibration you specified on the Instrument Settings control page in step 4 is used to determine the value for tp (precursor ion flight time) needed for the PSD calibration equation.
Chapter 7 PSD Analysis 7.3.4 Filling in the Segment List This section describes: • • • • 7 Filling in the list If you are using Angiotensin_PSD. BIC Filling in the list Typing or selecting new values Using the Fill Down command Adding or deleting rows To fill in the segment list: 1. If you are using the standard instrument settings (.BIC) file provided, Angiotensin_PSD.BIC, the segment list contains 10 segments with the Mirror Ratio settings listed in “Default Mirror Ratio settings” on page 7-22.
Acquiring PSD Data with Standard Instrument Settings (.BIC) Files Rows are added with the following default values for all columns. Parameter Default Value Segment Sequential number starting at 1 Saved check box Blank until segment is saved (the software automatically places a check mark in this field when you save a segment) Mirror Ratio 1.000 Guide Wire% 0.
Chapter 7 PSD Analysis Typing or selecting new values To type or select new values in the segment list: 1. Type in new values for Mirror Ratio or Guide Wire% in any row in the table. You can specify Mirror Ratios in any order in the table. NOTE: When you click on the Mirror Ratio field, the entry is displayed with more than three-digit precision, which is the precision used to calculate the mass range for the segment during the analysis.
Acquiring PSD Data with Standard Instrument Settings (.BIC) Files NOTE: Do not select Guide Wire% Tracks Mirror Ratio option on Voyager-DE PRO or Voyager-DE STR systems. This parameter is for use with older systems only. 2. Type in values in any row that you want to fill down into selected rows. 3. Click on the Segment number to select the row containing the values to fill down. 4. Click 7 . The following occurs: • The Mirror Ratio is calculated and entered in all rows below.
Chapter 7 PSD Analysis 7.3.5 Acquiring and Saving PSD Segments This section includes: • • • • • • • • • 7 Overview Overview Acquiring PSD segments Selecting and acquiring a segment During acquisition Changing settings Accumulating or saving the segment Selecting and acquiring remaining segments Reacquring a segment Stopping the experiment When you start acquiring in PSD mode, the software automatically opens a PSD experiment.
Acquiring PSD Data with Standard Instrument Settings (.BIC) Files NOTE: If you stop an experiment without saving any segments, no .DAT file is created. NOTE: All instrument settings except Shots/Spectrum are disabled as soon as you start acquisition in PSD mode, until you stop the experiment. Make sure instrument settings are correct before starting acquisition. Acquiring PSD segments 7 To acquire PSD segments: 1.
Chapter 7 PSD Analysis During acquisition The following occurs: • Mass range for the segment is set to: Mass Start Equivalent to (Precursor mass/4) which is equal to 7 (Precursor flight time/2) End (Mirror to Accelerating Voltage Ratio 2 x Precursor mass) which is equal to (Mirror to Accelerating Voltage Ratio x Precursor flight time) • Acquisition starts. • All instrument settings except Shots/Spectrum are disabled.
Acquiring PSD Data with Standard Instrument Settings (.BIC) Files Accumulating or saving the segment 6. Evaluate the spectrum, then do one of the following: • Click in the toolbar to accumulate the spectrum. You can accumulate as many spectra as needed. When the accumulated spectrum is acceptable, click on the Accumulated trace, then click . • Click in the toolbar to add the segment to the .DAT file. After you save the segment, the Saved check box in the segment list is checked.
Chapter 7 PSD Analysis 7.4 Exploring PSD Mode To be successful in PSD analysis, you need to understand how ions behave in PSD mode, and how to optimize acquisition conditions. Before running samples, spend some time practicing with standards.
Exploring PSD Mode 7.4.1 Observing the Effects of Laser Intensity Adjusting laser intensity affects fragment ion production and signal intensity. In this section In this section, you will: • Observe the effects of setting the laser intensity too high and too low • Determine the laser intensity for your system that yields the best signal for PSD spectra Observing effects of high and low laser intensity To observe the effects of laser intensity: 1. Open the Angiotensin_PSD.BIC file provided. 2.
Chapter 7 PSD Analysis 5. Note the behavior of the signal intensity for the first few spectra and subsequent continued samplings at a given laser power. NOTE: Sample preparations that contain high salt contamination or other impurities often yield increased signal intensity after an initial period of low intensity as the top layer of sample is consumed. 7 You typically see signal intensity reach maximum and decrease more quickly than in non-PSD mode as sample is consumed.
Exploring PSD Mode Hint: As you initially experiment, adjust the laser in large steps, for example 100 counts. As you begin to fine-tune the laser, use smaller steps. Fragment ion yield initially increases with higher laser intensities, and then decreases at very high settings. To make sure that the signal decrease is not due to sample exhaustion, move around in the sample position. Determining optimum laser intensity for fragments 7. Decrease the laser intensity and observe the signal.
Chapter 7 PSD Analysis 7.4.2 Observing the Effects of Precursor Ion Selector Turning on the Precursor Ion Selector eliminates prompt fragments in a spectrum.
Exploring PSD Mode Figure 7-12 compares the spectrum above with the spectrum acquired in the previous section so you can more easily see the peaks that appear when the Precursor Ion Selector is turned off. Figure 7-12 also identifies the two types of fragments seen when the Precursor Ion Selector is turned off.
Chapter 7 PSD Analysis Observing prompt and PSD fragments When operating under conditions that yield high resolution for angiotensin I, and when the Precursor Ion Selector is turned off, you should observe the following: • Prompt fragments—Well-resolved fragment ions generated in the source before acceleration. The sharp peak at 1,181.7 Da is the y9 ion.
Exploring PSD Mode Path of prompt fragment ion Path of molecular ion Path of PSD fragment ion Prompt fragment travels faster Ion Source Flight Tube Prompt fragment formed 7 Reflector PSD fragment formed Ion Source Flight Tube Reflector Precursor Ion Selector on Prompt fragment deflected Figure 7-13 Flight Path of Prompt and PSD Fragments Voyager™ Biospectrometry™ Workstation User’s Guide 7-43
Chapter 7 PSD Analysis 7.4.3 Observing the Effects of Grid Voltage% Adjusting Grid Voltage% affects resolution. In this section In this section, you will: • Understand the function of Grid Voltage% in focusing ions • Observe the impact of Grid Voltage% on higher and lower mass ions 7 NOTE: For typical applications, use the Grid Voltage% in the standard .BIC file provided. This section is a demonstration of the impact of Grid Voltage%.
Exploring PSD Mode By fine-tuning the Grid Voltage%, you can alter the point of initial time focus of the ions, which decreases the amount of time the ion spends defocusing. This allows you to balance the defocusing time with the refocusing time in the reflector (Figure 7-15). The goal in optimizing the Grid Voltage% is to find a suitable setting that optimizes resolution in the middle of the mass range of interest.
Chapter 7 PSD Analysis Acquiring 7 NOTE: In this exercise, you will observe the impact of the Grid Voltage% on the precursor ion at 1,297 Da and the fragment ion at 1,181 Da. At the laser intensity you optimized for the fragment ion, the precursor ion will be saturated. To allow you to observe precursor and fragment ions in the spectrum, change the Vertical Scale setting in the .BIC file (Digitizer/Mode dialog box) to 1,000 mV full scale. 1. Enable the Precursor Ion Selector if it is disabled. 2.
Exploring PSD Mode 6. Check the Grid Voltage% setting in the standard .BIC file provided on your system for angiotensin I (Angiotensin_Reflector.BIC). Acquire a spectrum using this setting. At this setting, you should observe optimum resolution on the high mass peak. 7.4.4 Summary 7 The table below summarizes the impact of changing PSD acquisition conditions. Condition Laser intensity Impact • • • Precursor Ion Selector • • • Increase higher than normal to induce fragmentation.
Chapter 7 PSD Analysis 7.5 Viewing PSD Data For information on viewing PSD data, see the Data Explorer Software User’s Guide, Chapter 8, Viewing Voyager PSD Data.
3 8 Maintenance and Troubleshooting Chapter 8 This chapter contains the following sections: 8.1 8.2 Maintenance .................................................... 8-2 8.1.1 Maintenance Schedule ....................8-2 8.1.2 Hardware Maintenance ....................8-3 8.1.3 Backing Up and Archiving Data ........8-6 Troubleshooting ............................................... 8-7 8.2.1 Spectrum Troubleshooting ...............8-7 8.2.2 Software Troubleshooting .............. 8-19 8.2.
Chapter 8 Maintenance and Troubleshooting 8.1 Maintenance This section describes: • Maintenance schedule • Hardware maintenance • Backing up and archiving data 8.1.1 Maintenance Schedule Maintenance schedule Regular preventative maintenance will help keep your Voyager system functioning properly.
Maintenance 8.1.2 Hardware Maintenance WARNING ELECTRICAL SHOCK HAZARD. Severe electrical shock can result by operating the instrument without the front or side panels. Do not remove instrument front or side panels. High voltage contacts are exposed with front or side panels removed. Wear proper eye protection if front or side panels are removed for service. WARNING LASER HAZARD. The laser emits ultraviolet radiation.
Chapter 8 Maintenance and Troubleshooting • Inspect flap valve 1, flap valve 2, linear actuator o-rings • Clean optics and air lines • Inspect grids, compressor, air pressure, turbo pump, and laser power • Adjust laser flash rate, load offsets, sample offsets, detector gain and voltage, and instrument covers • Inspect load and eject cycles, high voltage power supplies, and computer • Calibrate thermocouple gauges • Check that the instrument meets specifications Changing fuses This procedure is required
Maintenance O I Fuses Voltage selector 220 100 240 120 Fuse holder PB100507 8 Figure 8-1 Changing Fuses WARNING FIRE HAZARD. Using a fuse of the wrong type or rating can cause a fire. Replace fuses with those of the same type and rating.
Chapter 8 Maintenance and Troubleshooting 6. Insert two fuses of the proper rating. Electrical Rating Volts/Amps Fuse (5 x 20 mm) 8 100 V~10A T10A 250V 120 V~10A T10A 250V 220 V~6.3A T6.3A 250V 240 V~5A T5A 250V 7. Insert the voltage selector/fuse holder into the receptacle. 8. Plug in the mass spectrometer and power up. 8.1.3 Backing Up and Archiving Data Back up data weekly, or as needed. Archive data as needed.
Troubleshooting 8.2 Troubleshooting This section includes: • Spectrum troubleshooting • Software troubleshooting • Hardware troubleshooting Troubleshooting information is organized according to likelihood of possible cause, from most likely to least likely possible cause. If you are unable to solve your problem using the information in the following tables, call PerSeptive Biosystems Technical Support.
Chapter 8 Maintenance and Troubleshooting Table 8-1 Spectrum Troubleshooting (Continued) Symptom Possible Cause Action Flat signal on oscilloscope or in spectrum window for sample region (matrix peaks seen) Accelerating Voltage too low Adjust. See Section 5.4.4.2, Setting Accelerating Voltage. Sample does not ionize Analyze in negative ion mode. (continued) Before mixing with matrix, chemically derivatize sample with amino-containing chemical group. Use different matrix. See Section 3.1.
Troubleshooting Table 8-1 Spectrum Troubleshooting (Continued) Symptom Flat signal on oscilloscope or in spectrum window for sample and matrix region Possible Cause Action Vertical scaling needs adjustment Adjust. See Section 5.3.5, Understanding Digitizer Settings. Spectrum window needs adjustment See Section 4.3, Using the Spectrum Window. Laser set to 0 Adjust laser by using the slider controls on the Manual Laser/Sample Positioning control page.
Chapter 8 Maintenance and Troubleshooting Table 8-1 Spectrum Troubleshooting (Continued) Symptom Possible Cause Poor resolution/ sensitivity in Delayed Extraction mode Delay Time and Grid Voltage% not optimized Optimize. See Section 5.4, Optimizing Instrument Settings Parameters. Guide Wire Voltage% not optimized Optimize. See Section 5.4, Optimizing Instrument Settings Parameters.
Troubleshooting Table 8-1 Spectrum Troubleshooting (Continued) Symptom Round tops on peaks Possible Cause Action Saturated (flat top) and unsaturated (sharp top) scans averaged Decrease laser setting by using the slider controls on the Manual Laser/Sample Positioning control page until peak tops are sharp. While acquiring, laser power changed. Averaged scan includes saturated (flat top) and unsaturated (sharp top) scans. Reacquire using one laser setting that gives sharp peaks.
Chapter 8 Maintenance and Troubleshooting Table 8-1 Spectrum Troubleshooting (Continued) Symptom Possible Cause Action Poor mass accuracy in Delayed Extraction mode High voltage power supplies not warmed up Start high voltages by clicking (external calibration only) on the toolbar before calibration. Standard and sample of interest not in adjacent sample position Calibrate using standard that is in a sample position adjacent to the sample of interest.
Troubleshooting Table 8-1 Spectrum Troubleshooting (Continued) Symptom Poor signal-to-noise ratio or sensitivity Possible Cause Laser intensity too high Decrease laser intensity to threshold by using the slider controls in the Manual Laser/Sample Positioning control page. Laser intensity too low Increase laser intensity by using the slider controls in the Manual Laser/Sample Positioning control page. Sample contaminated To test, mix the sample with a standard of known sensitivity.
Chapter 8 Maintenance and Troubleshooting Table 8-1 Spectrum Troubleshooting (Continued) Symptom Possible Cause Poor signal-to-noise ratio or sensitivity (continued) Guide Wire Voltage% too high (lower masses) or too low (higher masses) Adjust. See Section 5.3.4, Understanding Guide Wire Voltage%. Accelerating Voltage too low Adjust. See Section 5.4.4.2, Setting Accelerating Voltage. Too much salt or buffer in sample Clean up sample. See Section 3.1.5, Sample Cleanup. Decrease salt or buffer.
Troubleshooting Table 8-1 Spectrum Troubleshooting (Continued) Symptom Possible Cause Action Dimer in spectrum Laser intensity too high Adjust laser by using the slider controls on the Manual Laser/Sample Positioning control page. Dimers, trimers, and tetramers in spectrum Sample concentration too high Prepare sample/matrix with a final sample concentration appropriate for sample and matrix. See Section 3.1.3, Matrix Information.
Chapter 8 Maintenance and Troubleshooting Table 8-1 Spectrum Troubleshooting (Continued) Symptom Peaks not symmetrical 8 Possible Cause Laser intensity too high Decrease laser intensity by using the slider controls on the Manual Laser/Sample Positioning control page. Sample contains more than one component Purify sample before analyzing.
Troubleshooting Table 8-1 Spectrum Troubleshooting (Continued) Symptom Possible Cause On Voyager-DE PRO and Voyager-DE STR systems, cannot see high mass ions in Reflector mode Refer to “Flat signal on oscilloscope or in spectrum window for sample region (matrix peaks seen)” symptom on page 8-7 Refer to “Flat signal on oscilloscope or in spectrum window for sample region (matrix peaks seen)” action on page 8-7.
Chapter 8 Maintenance and Troubleshooting Table 8-1 Spectrum Troubleshooting (Continued) Symptom Large tail on the high mass side of peak 8 8-18 PerSeptive Biosystems Possible Cause Unresolved salt or buffer adducts due to sample contamination Action Clean up sample. See Section 3.1.5, Sample Cleanup.
Troubleshooting 8.2.2 Software Troubleshooting This section includes: • Instrument Control Panel troubleshooting • PSD troubleshooting • Checking the Windows NT Event Log Table 8-2 Instrument Control Panel Troubleshooting Symptom Possible Cause Action Slider control does not change laser setting Voyager Instrument Control Panel is not the active window Click on the Instrument Control Panel to activate the window before using slider controls.
Chapter 8 Maintenance and Troubleshooting Table 8-2 Instrument Control Panel Troubleshooting (Continued) Symptom Possible Cause Calibration (mass) shifted up or down by 10 Da Uneven matrix layer causing hot and cold spots Action Acquire a number of spectra and accumulate scans. Prepare new sample spot. Resolution labels or signal-to-noise not displayed Peaks not detected Apply peak detection when acquisition is complete by clicking in the toolbar.
Troubleshooting Checking the Windows NT Event Log The Windows NT Event Log is a running list of events that automatically starts when you run Windows NT. An event is considered any significant occurrence in the system or application that requires the user to be notified. You can use Event Viewer to monitor the events that occur in your system. To display Event Viewer: 1. Select Administrative Tools from the Program folder on the Windows Start taskbar. 2. Click Event Viewer.
Chapter 8 Maintenance and Troubleshooting 8.2.3 Hardware Troubleshooting This section includes: • Mass spectrometer troubleshooting • Vacuum gauge panel troubleshooting Table 8-4 Mass Spectrometer Troubleshooting Symptom 8 Possible Cause Action Internal stepper motor making noise when the sample plate is moving Normal operation of the sample plate stepper motor No action. Normal occurrence.
Troubleshooting Table 8-4 Mass Spectrometer Troubleshooting (Continued) Symptom Cracking sound in mass spectrometer Possible Cause Action Arcing caused by dirty sample plate Use clean, particulate-free sample plate. Arcing caused by negative ion mode Decrease Accelerating Voltage. See Section 5.4.4.2, Setting Accelerating Voltage. Arcing caused by excess matrix in sample preparation (may be required for ionization of certain samples) Decrease amount of matrix in sample preparation.
Chapter 8 Maintenance and Troubleshooting Table 8-4 Mass Spectrometer Troubleshooting (Continued) Symptom Sample holder empty when you click Eject Possible Cause Action Sample plate too far way from grabber in Load position Call PerSeptive Biosystems Technical Support. Sample plate jammed in system Call PerSeptive Biosystems Technical Support.
Troubleshooting Table 8-5 Vacuum Gauge Panel Troubleshooting Symptom E09 error message displayed on gauge controller for BA1 or BA2 E08 error message displayed on gauge controller Possible Cause Action BA1 or BA2 gauges shut down due to high pressure. May be caused by: Press the EMIS button on Pressure Gauge Control Panel to turn off. Press again to turn on.
Chapter 8 Maintenance and Troubleshooting 8 8-26 PerSeptive Biosystems
Appendix A Specifications A This appendix contains the following sections: A.1 Voyager-DE Specifications ...................... A-2 A.2 .......... A-4 Voyager-DE STR Specifications ........... A-7 A.3 Voyager-DE PRO Specifications NOTE: The specifications for this instrument are subject to change without notice.
Appendix A Specifications A A.
Voyager-DE Specifications Table A-2 Voyager-DE Mass Spectrometer Specifications (Continued) Condition Ion source voltages A Specification Tunable: • Accelerating Voltage—Up to 25,000 V • Grid Voltage—Range determined by Accelerating Voltage Laser Nitrogen, 337 nm, 3 ns pulse Vacuum system Automatic, with turbomolecular pumping for high vacuum Ion detection Positive and negative Sample analysis • 100-well sample plates • Manual control using control stick or software • Sequence control sof
Appendix A Specifications A A.
Voyager-DE PRO Specifications A Table A-5 Voyager-DE PRO Mass Spectrometer Specifications Condition Reflector Flight tube (horizontal) Specification Single-stage with optimized optics for PSD Analysis • • Linear mode—1.3 m Reflector mode—2.
Appendix A Specifications Table A-6 Voyager-DE PRO Miscellaneous Specifications A Condition Specification Operating temperature 20–25°C (68–77°F) Relative Hhumidity 30–80%, non-condensing Computer Minimum configuration: • 2 GHz digitization for enhanced resolution • Pentium® II 350 MHz, with 4.
Voyager-DE STR Specifications A.
Appendix A Specifications Table A-8 Voyager-DE STR Mass Spectrometer Specifications A Condition Reflector Flight tube (horizontal) Specification Single-stage with optimized optics for PSD Analysis • • Linear mode—2.0 m Reflector mode—3.
Voyager-DE STR Specifications Table A-9 Voyager-DE STR Miscellaneous Specifications Condition A Specification Operating temperature 20–25°C Relative humidity 30–80%, non-condensing Computer Minimum configuration: • 2 GHz digitization for enhanced resolution • Pentium ® II 350 MHz, with 4.
Appendix A Specifications A A-10 PerSeptive Biosystems
Appendix B Warranty/Service Information B This appendix contains the following sections: B.1 Limited Product Warranty ..................... B-2 B.2 Damages, Claims, Returns ................... B-5 B.3 Spare Parts ....................................... B-6 PerSeptive Biosystems, Inc. supplies or recommends certain configurations of computer hardware, software, and peripherals for use with its instrumentation.
Appendix B Warranty/Service Information B.1 Limited Product Warranty Limited warranty B PerSeptive Biosystems, Inc. (“PerSeptive”) warrants that all standard components of its Voyager™ Biospectrometry™ Workstations (the “Product”) purchased new will be free of defects in materials and workmanship for a period of one (1) year. PerSeptive will repair or replace, at its discretion, all defective components during this warranty period.
PerSeptive warrants that for a period of ninety (90) days the software designated for use with the Product will perform substantially in accordance with the function and features described in its accompanying documentation when properly installed on the Product. PerSeptive does not warrant that the operation of the instrument or software will be uninterrupted or error free.
Appendix B Warranty/Service Information Warranty limitations B THE REMEDIES PROVIDED HEREIN ARE BUYER’S SOLE AND EXCLUSIVE REMEDIES.
B.2 Damages, Claims, Returns Damages Please examine any shipments promptly after receipt to check for damage. Contact PerSeptive’s Service Department if you have questions about checking for damage. If you discover damage, stop unpacking. Contact the shipping carrier and request inspection by a local agent. Secure a written report of the findings to support any claim.
Appendix B Warranty/Service Information B.3 Spare Parts Standards and matrices The following compounds are available from the listed vendors. We are listing part numbers for your convenience. However, part numbers may change without our knowledge.
Sample plates The following sample plates are available from PerSeptive Biosystems: Description Part Number Welled Sample Plates Gold, 100-well V700401 Gold, 100-well (no pin, for Voyager Workstations manufactured in 1995 or earlier V700208 B Flat Sample Plates (Laser Etched) Stainless steel, 100-position (indicated by numbers only) V700664 Stainless steel, 100-position (indicated by numbers only, no pin, for Voyager Workstations manufactured in 1995 or earlier) V700665 Stainless steel, 100-posi
Appendix B Warranty/Service Information Description Part Number Membrane, Gels V700698 Hydrophobic plastic surface, flat, 400-position V700699 B B-8 PerSeptive Biosystems
Appendix C C Matrices This appendix lists commonly used matrices. For each matrix, it also lists: • • • • • Applications Description of physical appearance Chemical structure Suggested solution concentration Characteristic matrix ions It also includes matrix spectra for all matrices listed. Refer to these figures for characteristic peaks patterns and masses. For additional matrix information, refer to the bibliography.
Appendix C Matrices Figure C-1 Sinapinic Acid Matrix Spectrum C Figure C-2 α-cyano-4-hydroxycinnamic acid (CHCA) Matrix Spectrum C-2 PerSeptive Biosystems
Figure C-3 2,5-dihydroxybenzoic acid (2,5-DHB) Matrix Spectrum C Figure C-4 Mixture of 2,5-dihydroxybenzoic acid and 5-methoxysalicylic acid (DHBs) Matrix Spectrum Voyager™ Biospectrometry™ Workstation User’s Guide C-3
Appendix C Matrices Figure C-5 2-(4-hydroxy-phenylazo)-benzoic acid (HABA) Matrix Spectrum C Figure C-6 3-hydroxypicolinic acid (3-HPA) Matrix Spectrum C-4 PerSeptive Biosystems
Figure C-7 Dithranol Matrix Spectrum C Figure C-8 2,4,6 trihydroxyacetophenone (THAP) Spectrum Voyager™ Biospectrometry™ Workstation User’s Guide C-5
Appendix C Matrices Figure C-9 trans-3-indoleacrylic acid (IAA) Matrix Spectrum C Matrix Sinapinic acid Applications: (see mass spectrum on page C-2) CH3O C-6 Peptides • Proteins White CHCOOH OCH3 OH • Color of crystals/ solution: MW 224.07 Da CH Matrix Solution Concentration Applications/Color PB100251 NOTE: Matrix powder may also contain orange crystals. Do not use crystals when preparing solutions. PerSeptive Biosystems • • 10 mg/ml in 70:30 water/acetonitrile (0.
Matrix Applications/Color Alpha-cyano- Applications: 4-hydroxycinnamic acid (αCHCA) (see mass spectrum on page C-2) • Peptides • Proteins Matrix Solution Concentration Characteristic Matrix Ions (monoisotopic) 10 mg/ml in 50:50 water/acetonitrile (0.1% TFA final conc.) • • • • • • 164.047 195.050 172.040 379.093 212.032 294.076 10 mg/ml in water • • • • 155.034 154.027 137.024 273.040 Color of crystals/ solution: Yellow MW 189.
Appendix C Matrices Matrix Applications/Color 2,5dihydroxybenzoic acid (2,5-DHB) (see mass spectrum on page C-3) Applications: Small molecules Color of crystals/ solution: White MW 154.03 Da COOH OH C HO C-8 PB100253 PerSeptive Biosystems Matrix Solution Concentration 10 mg/ml in solvent in which sample and matrix are soluble Characteristic Matrix Ions (monoisotopic) • • • • 155.034 154.027 137.024 273.
Matrix Applications/Color mixture of 2,5dihydroxybenzoic acid and 5-methoxysalicylic acid (DHBs) (see mass spectrum on page C-3) Applications: Large proteins Color of crystals/ solution: White Matrix Solution Concentration 10 mg/ml in solvent in which sample and matrix are soluble Characteristic Matrix Ions (monoisotopic) • • • • • • • 155.034 154.027 137.024 273.040 151.040 168.042 169.050 MW 154.03 Da + MW 168 Da mixture MW 322.
Appendix C Matrices Matrix 2-(4-hydroxyphenylazo)-benzoic acid (HABA) (see mass spectrum on page C-4) Applications: • Proteins • Lipopolysaccharides Polar and nonpolar synthetic polymers Color of crystals/ solution: • ~1.3 mg/ml in 50:50 water/ acetonitrile or in 40:40:20 water/ acetonitrile/ methanol • 10 mg/ml in ethanol or methanol • MW 242.07 Da COOH N Matrix Solution Concentration Applications/Color N Characteristic Matrix Ions (monoisotopic) • • 243.077 265.059 • • • • • • • • 96.
Matrix Applications/Color Dithranol Applications: (see mass spectrum on page C-5) Nonpolar synthetic polymers MW 226.06 Da OH O OH Color of crystals/ solution: Matrix Solution Concentration 10 mg/ml in tetrahydrofuran + silver trifluoroacetate to minimize Na+ and K+ adduct formation Characteristic Matrix Ions (monoisotopic) • • • • 225.055 226.063 227.071 211.076 • 169.
Appendix C Matrices Matrix Applications/Color trans-3indoleacrylic acid (IAA) (see mass spectrum on page C-6) MW 187.2 Non-polar polymers Color of crystals/ solution: White HC N H C Applications: C H CO 2H Matrix Solution Concentration 10-1 M in solvent appropriate for sample Characteristic Matrix Ions (monoisotopic) • • • • • • • • 187.063 188.071 170.061 144.081 130.066 375.134 329.120 284.131 PB100491 Picolinic acid Tang, K., N.I. Taranenko, S.L. Allman, C.H. Chen, L.Y. Chang, and K.B.
Appendix D D Log Sheets This appendix includes log sheets you can copy and use to log samples before loading.
A Voyager™ Biospectrometry™ Sample Log Plate #: Date: 1 1 2 3 4 5 6 7 8 9 10 2 3 4 5 6 7 8 9 10
. A Voyager™ Biospectrometry™ Sample Log Plate: Samp # 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
A Voyager™ Biospectrometry™ Sample Log Plate: Samp # 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50.
A Voyager™ Biospectrometry™ Sample Log Plate: Samp # 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75.
A Voyager™ Biospectrometry™ Sample Log Plate: Samp # 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100.
A Voyager™ Biospectrometry™ Sample Log Plate: Samp # Date: Page Matrix of Sample Path and File Name Linear Reflector
Appendix D Log Sheets D D-8 PerSeptive Biosystems
Appendix E E Grid Voltage% and Delay Time Settings The following figures illustrate the relationship between Grid Voltage% and Delay Time parameters. These values are not absolute values for all systems. Optimum settings may be slightly different for each system.
Appendix E Grid Voltage% and Delay Time Settings Elite, Voyager-DE STR, linear Linear Mode P ulse D e la y (nse c) m/z=15000 m/z=5000 m/z=2000 6 00 4 00 m/z=50000 m/z=25000 2 00 m/z=1000 0 88 90 92 94 G rid Volta ge (% ) E E-2 PerSeptive Biosystems 96
Appendix F Reference Standard Information F This appendix contains the following sections: F.1 Calibration Compounds ...................... F-2 F.2 Conversion of Mass to Time for Typical Standards ............................. F-4 F.3 Theoretical Cleavages for Angiotensin .... F-6 F.4 Observed PSD Fragments in Angiotensin ...............
Appendix F Reference Standard Information F F.1 Calibration Compounds The table below includes masses for common calibration compounds. NOTE: You can display reference mass information in the Data Explorer software by viewing the VOYAGER.REF file in the mass calibration function. Molecular Weight Compound Monoisotopic F-2 Charge State Average Protonated Molecular Ion [M+H]+ Monoisotopic Average Leucine Enkephalin 555.2693 555.63 +1 556.2771 556.64 des-Arg1 Bradykinin 903.4603 904.
Calibration Compounds Molecular Weight Compound Monoisotopic Charge State Average Protonated Molecular Ion [M+H]+ Monoisotopic Average Insulin B chain, oxidized 3493.6435 3495.95 +1 3494.6513 3496.96 Adrenocortico-tropic hormone (ACTH) clip 7–38 3656.9216 3659.18 +1 3657.9294 3660.19 Insulin, bovine ____ 5733.58 +1 ____ 5734.59 +2 ____ 2867.80 Thioredoxin (E. coli, oxidized) ____ 11673.47 +1 ____ 11674.48 Cytochrome C (horse heart) ____ 12360.5 +1 ____ 12361.
Appendix F Reference Standard Information F F.2 Conversion of Mass to Time for Typical Standards The table below includes mass and time values for standards under different acquisition conditions (Accelerating Voltage and flight tube length). You can use this information when observing the signal on the oscilloscope to determine if the peaks you are seeing are at the correct mass. NOTE: These values are not exact. Use them as a guide. Time (seconds) Standards Z Average MW [M+H]+ 25,000 V 1.
Conversion of Mass to Time for Typical Standards F Time (seconds) Standards Bovine Insulin B, Oxidized Z Average MW [M+H]+ 25,000 V 1.3 m 10,000 V 1.3 m 28,000 V 2.0 m 15,000 V 2.0 m 10,000 V 2.0 m 1 3496.9 3.50E-05 5.53E-05 5.09E-05 6.95E-05 8.51E-05 2 1748.95 2.48E-05 3.91E-05 3.60E-05 4.92E-05 6.02E-05 1 5734.5 4.48E-05 7.09E-05 6.52E-05 8.90E-05 1.09E-04 2 2867.75 3.17E-05 5.01E-05 4.61E-05 6.30E-05 7.71E-05 Insulin-like Growth Factor (IgF) 1 7650.76 5.
Appendix F Reference Standard Information F F.3 Theoretical Cleavages for Angiotensin Monoisotopic masses (Da) for the theoretical cleavages of angiotensin I are listed below as calculated for the positive ion mode. Monoisotopic (M + H)+ for the sequence: DRVYIHPFHL is 1296.685. a F-6 b c Fragment 88.040 116.035 133.061 D 244.141 272.136 289.162 DR 343.209 371.204 388.231 DRV 506.273 534.268 551.294 DRVY 619.357 647.352 664.378 DRVYI 756.416 784.411 801.437 DRVYIH 853.
Observed PSD Fragments in Angiotensin F.4 Observed PSD Fragments in Angiotensin The masses listed below are fragment ions of the (M+H)+ percursor ion. Fragment Mass Fragment Mass Designation (monoisotopic) (average) Designation (monoisotopic) (average) 70.066 70.1 P, R ~426.2 426.5 ? 72.081 72.1 V 489.246 489.6 a4-17 86.097 86.1 I, L ~492.2 492.6 ? 110.072 110.1 H 506.273 506.6 a4 136.076 136.2 Y 513.283 513.6 y4 156.101 156.2 R? 517.241 517.6 b4-17 166.062 166.
Appendix F F Reference Standard Information Fragment Mass Fragment Mass Designation (monoisotopic) (average) Designation (monoisotopic) (average) 272.136 272.3 b2 1000.537 1001.2 a8 285.135 285.3 FH 1046.542 1047.2 b8+H 2O 326.183 326.4 a3-17 ~1068.6 1069.2 ? 354.178 354.4 b3-17 1137.596 1138.3 a9 ~364.2 364.4 ? 1165.591 1166.3 b9 371.204 371.4 b3 1181.658 1182.3 y9 382.188 382.4 PFH 1183.601 1184.3 b9+H2O ~400.2 400.5 ? 1296.685 1297.5 MH+ 416.
Appendix G G Maintenance Log The following page includes a log sheet listing maintenance procedures. Copy this page and keep it by your Voyager system. Instructions for performing maintenance procedures are listed in Chapter 8, Maintenance and Troubleshooting.
Maintenance Log for Voyager System Serial Number ____________ Record the date and your initials when you perform maintenance procedures.
Appendix H Continuous Extraction Mode H This appendix contains the following sections: H.1 Optimizing a Continuous Extraction Standard Instrument (.BIC) Setting ...........H-2 H.2 Obtaining Good Spectra in Continuous Extraction Mode ............... H-8 H.3 Troubleshooting in Continuous Extraction Mode ....................................
Appendix H Continuous Extraction Mode H.1 Optimizing a Continuous Extraction Standard Instrument (.BIC) Setting Before you begin NOTE: Due to the superior results obtained during Delayed Extraction (DE) mode, use Continuous Extraction mode for diagnostic purposes only. Before optimizing a Continuous Extraction .
Optimizing a Continuous Extraction Standard Instrument (.BIC) Setting List of standard instrument settings files This section lists standard instrument settings (.BIC) files for Continuous Extraction in the following modes: • • • Linear mode Reflector mode PSD mode Standard instrument settings files are located in the C:\VOYAGER\DATA\FACTORY directory. Table H-1 Continuous Extraction Linear Mode Standard Instrument Settings (.BIC) Files .
Appendix H Continuous Extraction Mode Table H-2 Continuous Extraction Reflector Mode Standard Instrument Settings (.BIC) Files .BIC File H Sample Test Mass Range (Da) R1000 Angiotensin I Resolution at 20,000 V 100–2,000 R1001 Angiotensin I Resolution at 10,000 V 100–2,000 R1002 Insulin Resolution 5,000–7,000 R1003 E.
Optimizing a Continuous Extraction Standard Instrument (.BIC) Setting Optimization strategy Optimizing When optimizing a Continuous Extraction instrument settings file, you: 1. • Start with a standard instrument settings file • Fine-tune laser setting for major improvement in performance • Optionally adjust Grid Voltage% and Guide Wire Voltage% for slight improvement in performance Open a standard instrument settings file for the mass range you are analyzing.
Appendix H Continuous Extraction Mode 6. Set the Grid Voltage% appropriate for the matrix and mass: Table H-4 Grid Voltage% Settings for Continuous Extraction Mode Matrix α-cyano4-hydroxycinnamic acid Sinapinic acid DHB 3-HPA Mass (Da) Grid Voltage%* <5,000 50–70 >5,000 70–90 <5,000 80–85 >5,000 85–90 <5,000 85–90 >5,000 * In Reflector mode, lower Grid Voltage% settings may yield greater resolution, but may compromise sensitivity. H 7. Save the instrument settings (.BIC) file. 8.
Optimizing a Continuous Extraction Standard Instrument (.BIC) Setting Table H-5 Guide Wire Voltage% Settings for Continuous Extraction Mode Mass Range (Da) Guide Wire Voltage <1,500 0.05% 1,500–4,00 0.1% 4,000–15,000 0.2% >15,000 0.
Appendix H Continuous Extraction Mode H.2 Obtaining Good Spectra in Continuous Extraction Mode This section describes: • • • • Spectra, resolution, signal-to-noise ratio, and laser threshold Determining laser threshold Checking Resolution Fine-Tuning the Laser Setting H.2.1 Spectra, Resolution, Signal-to-Noise Ratio, and Laser Threshold What is a good spectrum? H A good spectrum is one that is acceptable for your purposes.
Obtaining Good Spectra in Continuous Extraction Mode High laser power causing the following: Matrix peaks seen with Low Mass Gate On Sample mass slightly higher than expected due to collision of ions with excess neutrals generated by the laser Broad peak Poor resolution (less than 100) No separation between molecular ion and matrix adduct ion Dimer Excess chemical noise Figure H-1 Example of Poor Mass Spectrum for Myoglobin Using Sinapinic Acid Laser power near threshold producing: Sharp, narrow pea
Appendix H Continuous Extraction Mode High laser power causing the following: Matrix and sample peaks saturated (flat-topped) Sample mass slightly higher than expected due to collision of ions with excess neutrals generated by the laser Broad peak Poor resolution Excess chemical noise H Minor components or fragments of major components ionized Figure H-3 Example of Poor Mass Spectrum for Angiotensin Laser power near threshold producing: Well-resolved matrix peaks at 172, 190, and 379 Da Peaks not s
Obtaining Good Spectra in Continuous Extraction Mode Parameters affecting resolution and signal-to-noise ratio These parameters have a primary impact on resolution and signal-to-noise ratio in Continuous Extraction mode: • • Laser position on the sample (hot or cold spot) Laser intensity These parameters have a secondary impact on resolution and signal-to-noise ratio in Continuous Extraction mode: • • • • Digitizer setting Accelerating Voltage Grid Voltage% Guide Wire Voltage% To obtain suitable mass
Appendix H Continuous Extraction Mode H.2.2 Determining Laser Threshold This section describes: • • • Overview Adjusting the laser intensity Verifying threshold setting H.2.2.1 Overview Definition H Factors affecting threshold H-12 Laser threshold is the minimum laser intensity required to produce a steady detectable signal. Laser intensities above threshold generate a dense plume of desorbed neutrals which cause energy loss during acceleration.
Obtaining Good Spectra in Continuous Extraction Mode Determining threshold Determining laser threshold becomes easier with experience. Sample is consumed when exposed to the laser, so minimize the number of spectra you acquire to determine threshold. H.2.2.
Appendix H Continuous Extraction Mode Figure H-5 Saturated Spectrum H If peak of interest is present 3. Check the spectrum for the peak of interest. Zoom in on the spectrum as needed. See Section 4.3, Using the Spectrum Window. 4. Zoom in on the appropriate mass range to check for the peak of interest. 5. If the peak of interest is present, the current laser intensity is the high setting for the sample class/matrix you are analyzing.
Obtaining Good Spectra in Continuous Extraction Mode Fine-tuning threshold When you determine the high and low setting for the sample class/matrix you are analyzing, you can fine-tune the threshold by setting the laser intensity midway between the high setting and low setting determined above. If signal is present when you decrease the laser, assume that this is the new high setting. If signal is not present when you decrease the laser, assume that this is the new low setting.
Appendix H Continuous Extraction Mode H.2.2.3 Verifying Threshold Setting Move to a new area of the sample well that contains the same sample. Acquire a spectrum to verify that the setting is valid for the laser power selected, and not caused by: • Sample surface excitement caused by the previous higher laser power setting • Sample consumption If the setting is valid, you see a spectrum similar to the one just acquired. You may need to adjust the laser slightly (5 to 10 counts).
Obtaining Good Spectra in Continuous Extraction Mode H.2.3 Checking Resolution After you determine laser threshold, calculate the resolution. Determine if the resolution is acceptable for your application. See Section 6.5.2, Calculating Mass Resolution. Table H-6 lists a general rating scale for resolution and molecular weight ranges for compounds acquired in Linear mode.
Appendix H Continuous Extraction Mode H.2.4 Fine-Tuning the Laser Setting When you find the laser threshold, whether you need to fine-tune the setting depends on your needs: H H-18 PerSeptive Biosystems • If you are looking for an estimate of molecular weight, a laser setting slightly higher than the laser threshold setting may be sufficient. • If you need good peak shapes, you may need to increase the laser setting to improve signal-to-noise ratio.
Troubleshooting in Continuous Extraction Mode H.3 Troubleshooting in Continuous Extraction Mode This section includes: • • Laser threshold troubleshooting Spectrum troubleshooting Refer to the table below if you are having trouble determining laser threshold: Table H-7 Laser Threshold Troubleshooting Symptom Action Signal fades very quickly Increase the laser intensity by 1 to 2 percent while acquisition is occurring.
Appendix H Continuous Extraction Mode Table H-7 Laser Threshold Troubleshooting (Continued) Symptom Action Signal is flat Laser setting may be too low, increase. Sample may be consumed, move to a new position in sample well. Sample may not be present, try new position. Sample may not ionize well, use different matrix. Cannot see ions in Reflector mode Check that you can see ions in Linear mode: • If you can see ions in Linear mode, it indicates that voltages or laser power need adjusting.
Troubleshooting in Continuous Extraction Mode Table H-8 Spectrum Troubleshooting (Continued) Symptom Poor resolution in Continuous Extraction mode (continued on next page) Possible Cause Action Laser intensity too high Adjust laser by using the slider controls on the Manual Laser Control page. Accelerating Voltage incorrect Adjust. Guide Wire Voltage% too high Adjust. See Section 5.3.4, Understanding Guide Wire Voltage%.
Appendix H Continuous Extraction Mode Table H-8 Spectrum Troubleshooting (Continued) Symptom Poor resolution in Continuous Extraction mode (continued) H Poor mass accuracy in Continuous Extraction mode (continued on next page) H-22 PerSeptive Biosystems Possible Cause Action Beam guide wire malfunction Call PerSeptive Biosystems Technical Support. Accelerating Voltage malfunction Call PerSeptive Biosystems Technical Support.
Troubleshooting in Continuous Extraction Mode Table H-8 Spectrum Troubleshooting (Continued) Symptom Poor mass accuracy in Continuous Extraction mode Possible Cause Action Incorrect peaks entered in calibration Recalibrate. See the Data Explorer Software User’s Guide , Section 5.3, Manual Calibration.
Appendix H Continuous Extraction Mode Table H-8 Spectrum Troubleshooting (Continued) Symptom Poor mass accuracy in Continuous Extraction mode Possible Cause Crystals did not form homogeneously on sample spot (continued) Action Prepare new sample spot. See “Guidelines for good crystallization” on page 3-30. Use mass closest to the mean (for external calibration only): 1. Acquire six averaged scans (six .DAT files) from one sample well. 2. Check masses in Voyager processing software. H 3.
I Using the Oscilloscope and Control Stick Appendix I This appendix contains the following sections: I.1 Guidelines for Acquiring ........................... I-3 I.2 Scaling ..................................................... I-4 I.3 Using the Control Stick ............................. I-7 NOTE: If your system includes an internal digitizer board or an external digitizer instead of an external oscilloscope, refer to Section 4.3, Using the Spectrum Window.
Appendix I Using the Oscilloscope and Control Stick Oscilloscope overview I An external oscilloscope (instead of the internal digitizer board or the LeCroy digitizer) is available as an option on Voyager systems. The available oscilloscope options include a 500 MHz, 2 GHz, or 4 GHz models. The oscilloscope converts the signal from the mass spectrometer to a signal that the computer can use. The oscilloscope has its own screen to display the averaged ion signal in real-time.
Guidelines for Acquiring I.1 Guidelines for Acquiring I Consider the following as you acquire a spectrum and use the oscilloscope: • When you start acquiring, you should see a signal that contains matrix peaks and sample peaks. • Make sure the full range of the signal is displayed. Brackets must overlap the end range markers (see Figure I-3). If brackets do not overlap, turn the Horizontal Scale and Horizontal Position knobs until the left bracket overlays the left marker.
Appendix I Using the Oscilloscope and Control Stick I I.2 Scaling Initial scaling Initial scaling of the oscilloscope is determined by the settings in the Mode/Digitizer dialog box. See Section 5.2.2, Mode/Digitizer Settings Dialog Box for more information. You can adjust the initial scaling after acquisition starts by using the knobs on the front panel of the oscilloscope.
Scaling Use these knobs to adjust the signal on the oscilloscope: • • • • • • • I Vertical position—Moves the signal up and down Vertical scale—Adjusts the amplitude of the signal Horizontal scale—Adjusts the width of the signal Horizontal position—Moves the signal left and right Select—Activates the right or left cursor Cursor Control—Moves the active cursor Reset button—Resets the oscilloscope screen For information on using other controls on the oscilloscope, refer to the manual shipped with the osci
Appendix I Using the Oscilloscope and Control Stick The Channel indicator (Ch1 in Figure I-3) displays Ch1 in Linear mode. On Voyager-DE PRO and Voyager-DE STR systems, the Channel indicator displays Ch2 in Reflector mode. I The top of the screen also displays: • • Average—During acquisition Stop—When acquisition is complete CAUTION The oscilloscope does not save spectra. If you acquire a new spectrum before downloading to the Voyager processing software, you lose the previous spectrum.
Using the Control Stick I.3 Using the Control Stick Starting acquisition I After you load samples on the sample plate, and load the plate into the system, start acquiring. To start acquiring, press the left button on the base of the control stick (Figure I-4).
Appendix I Using the Oscilloscope and Control Stick I CAUTION Check to see if acquisition has already stopped automatically before pressing the control stick button. If acquisition has stopped, the Instrument Control Panel status bar is blank (it displays an “Acquiring Data” message during acquisition). If acquisition has stopped and you press a control stick button, you will begin a new acquisition and overwrite the current spectrum.
Bibliography General Mass Spectrometry Beavis, R.C. and B.T. Chait, Chem. Phys. Lett., 1991, 181, 479. Cotter, R.J., Time-of-Flight Mass Spectrometry : Instrumentation and Applications in Biological Research (ACS Professional Reference Book), 1997, Amer. Chem. Society. Feigl, P, B. Schueler, and F. Hillenkamp, Int. J. Mass Spectrom. Ion Phys., 1983, 47, 15. Mamyrin, B. A., V. J. Karatajev, D. V. Smikk, and V. A. Zagulin, Soviet Phys. JETP, 1973, 37, 45–48. Middleditch, B.
Bibliography Karas, M., U. Bahr, F. Hillenkamp, Int. J. Mass Spectrom. Ion Proc. , 1989, 92, 231–242. Nordhoff, E. et al., Rapid Commun. Mass Spectrom., 1992, 6, 771–776. Papac, D.I., A. Wong, A.J.S. Jones, Anal. Chem., 1996, 68, 3215–3223. Pieles, U., W. Zurcher, M. Schar, and H. E. Moser, Nucl. Acids Res., 1993, 21, 3191–3196. Russel D., J. Am. Soc. Mass. Spectrom. 1996, 7, 995–1001. Shevchenko, A., M. Wilm, O. Vorm, M. Mann, Anal. Chem., 1996, 68, 850–858. Strupat, K., M. Karas, F. Hillenkamp, Int. J.
Bibliography Fitzgerald, M.C., L. Zhu, and L.M. Smith, “The Analysis of Mock DNA Sequencing Reactions Using Matrix-assisted Laser Desorption/Ionization Mass Spectrometry”, Rapid Commun. Mass Spectrom., 1993, 7, 895–897. Hillenkamp, F., M. Karas, R.C. Beavis, B.T. Chait, “Matrix-Assisted Laser Desorption/ Ionization Mass Spectrometry of Biopolymers”, Anal. Chem. 1991, 63, 1193–1203. Huberty, M.C., J.E. Vath, W. Yu, and S.A.
Bibliography Peptide Applications Bieman, K., “Mass Spectrometry of Peptides and Proteins”, Annu. Rev. Biochem. 1992, 61, 977–1010. Bieman, K., “Sequencing of Peptides by Tandem Mass Spectrometry and High-Energy Collision-Induced Dissociation”, Meth. Enzymol., 1990, 193, 455. Billeci, T.M., J.T. Stults, “Tryptic Mapping of Recombinant Proteins by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry”, Anal. Chem., 1993, 65, 1709–1716. Chait, B.T., R. Wang, R.C. Beavis, S.B.H.
Bibliography Tang, K., N.I. Taranenko, S.L. Allman, C.H. Chen, L.Y. Chang, and K.B. Jacobson, “Picolinic Acid as a Matrix for Laser Mass Spectrometry of Nucleic Acids and Proteins”, Rapid Commun. Mass Spectrom., 1994, 8, 673-677. Wu, K.J., A. Steding, and C.H. Becker, “Matrix-assisted Laser Desorption Time-of-flight Mass Spectrometry of Oligonucleotides Using 3-Hydroxypicolinic Acid as an Ultraviolet-sensitive Matrix”, Rapid Commun. Mass Spectrom., 1993, 7, 142–146.
Bibliography B I B L I O G R A P H Y Bibliography-6 PerSeptive Biosystems
Glossary a, b, and c ions —Generic description of potential ions that are formed by fragmentation of a parent peptide/protein. a, b, and c ions are fragments that retain the charge at the amino end (n-terminus) of the molecule. See also x, y, z ions. an bn cn Pressure gauges that operate below 1 millitorr by measuring a positive ion current produced by electrons from a hot filament. Voyager-DE systems include BA1 only.
Glossary G L O S S A R Y Delay Time —Used in Delayed Extraction mode. Time in nanoseconds (after the laser ionizes the sample) at which full Accelerating Voltage is applied, creating the potential gradient that accelerates ions. GPMAW —General Protein/Mass Analysis for Windows software, a software program used to identify protein sequences. Digitizer —A device that converts Grid voltage —Secondary voltage an analog signal to a digital word and stores the result in memory.
Glossary Ionization —Conversion of sample Low Mass Gate —Mechanism in solid, gaseous, or liquid phase to ions. for suppressing low mass ions. The voltage in the detector is not turned on until ions below the specified starting mass have passed. Ion intensity —See Intensity. Ion source —Device that produces ions. In a TOF instrument, it refers to the surface of the sample plate, the variable-voltage grid above the plate, and the grounded grid and aperture above the variable-voltage grid.
Glossary G L O S S A R Y Matrix —Low-mass, UV-absorbing substance used in MALDI technology to enable sample ionization. Common matrices include sinapinic acid, dihydroxy benzoic acid, and α-cyano 4hydroxy cinnamic acid. See Appendix C, Matrices, for more information. Metastable ion analysis — See MS/MS analysis. Mirror —A single plate at high Post-Source Decay (PSD) —Fragmentation of an ion during flight, after it leaves the ion source region.
Glossary TC2 (Thermocouple vacuum gauge) —Pressure gauge that operates from 1 millitorr to near atmospheric pressure by measuring the temperature of a heated thermocouple junction. As the pressure rises, more heat is removed, lowering the temperature. Timed Ion Selector — Mechanism for suppressing all but the ion of interest, used in PSD analysis. Voltage is applied to ion selector plates in the flight tube before and after the ion of interest passes the selector plates.
Glossary G L O S S A R Y Glossary-6 PerSeptive Biosystems
Index Numerics 384 well plate PLT file 3-51 position/row diagram 3-50 3-HPA chemical structure and molecular weight C-10 concentration 3-11 crystals 3-11, 3-35 initial velocity setting 5-22 laser intensity, relative 5-57 mass spectrum C-4 preparing 3-11 sample concentration 3-11 stability 3-11 when to use 3-3 A a, b, c ions, angiotensin F-6 Absolute counts displaying on right axis 5-60 scaling to 4-12 Accelerating Voltage changes compensated for by system 5-17, 5-77 description 5-77 effect on calibration 5
Index I N D E X Acquiring data, Instrument Control Panel (continued) Automatic Control mode 6-34 Data Storage, setting 6-14 data, saving 6-18 evaluating data automatically 6-40 evaluating data manually 6-16 guidelines 6-4 laser intensity, setting automatically 6-37 laser intensity, setting manually 6-14 Manual Control mode 6-11 moving position on sample spot 6-5 obtaining maximum mass accuracy 6-6 options 6-2 overview 4-6, 6-2 resolution, calculating during 6-29 sample position, selecting automatically 6-
Index Adobe Acrobat Reader installing from Voyager CD 2-26 opening PDF Voyager files 2-26 Advanced parameters, PSD PSD Mirror to Accelerating Voltage Ratio 5-30 Alpha-cyano-4-hydroxycinnamic acid chemical structure and molecular weight C-7 concentration 3-3, 3-8, 3-9 crystals 3-3, 3-8, 3-9, 3-34 initial velocity setting 5-22 laser intensity, relative 5-57 mass spectrum C-2 organic concentration 3-7 organic concentration, dried droplet application 3-8 preparing 3-5 preparing, dried droplet application 3-3,
Index I N D E X Automatic Control mode (continued) optimizing BIC for Sequence run 6-65 optimizing BIC in Manual Control mode before using 6-36 overview 4-6, 6-3 Prescan mode, description 6-49 process that occurs during acquisition 6-49 resolution filtering 6-42 resolution filtering, peak height used 6-42 sample plate required 3-31 sample plate, aligning 2-39, 6-34 sample position, selecting 6-39 saving conditions 6-38 search pattern (.
Index BA2, Voyager-DE STR description 1-40 E09 error 8-25 pressure range 1-41 pressure, displaying 2-51, 4-5 Back panel, see Rear panel Backing up 8-6 Bandwidth, see Input Bandwidth Base peak intensity, scaling to 4-12 Basics Instrument Control Panel 4-2 Sequence Control Panel 4-32 Batch acquisition 6-3, 6-60 Beam guide wire function 1-22, 1-36, 5-18, 5-46 voltage 5-18 Bench space requirements peripherals 2-3 Voyager-DE and Voyager-DE PRO 2-2 Voyager-DE STR 2-6 Biacore Chip sample plate 3-49 BIC files see
Index I N D E X Calibration (continued) impact of changing Grid Voltage% 5-41 impact of Low Mass Gate 5-79 increasing accuracy 3-18, 3-31 internal standard 6-25 internal, see Calibration, internal manual, see Calibration, manual mass accuracy 3-18, 3-31, 6-7, 6-10 matrix reference file, modifying 5-23 matrix, selecting 5-20, 6-13 overview 6-7 PSD, see Calibration, PSD Sequence Control Panel 6-64, 6-72 smoothing spectra 6-10 standards, see Calibration standards types of 6-7 when to calibrate 6-8 Calibratio
Index Calibration, PSD default 7-8 default, selecting 7-26 equation 7-8 in Instrument Settings 7-26 in PSD Acquisition Settings 7-26 Camera, see Video camera Carbohydrates, matrix for 3-3, C-7 Carbonic anhydrase, molecular weight F-3 Cation exchange, sample cleanup 3-23 Cautions Deflector Gate Width in PSD, changing the setting 7-9 exiting Voyager Control software 2-29 Flight Length to Deflector, do not change 7-10 internal jumpers, setting in STR models 2-7 vacuum disruption in CID 7-14 voltage selector,
Index I N D E X Computer (continued) technical support for altered configuration B-1 troubleshooting 8-6 with Signatec 500 MHz digitizer 2-10 with Tektronix oscilloscope 2-10 Concentration, see Sample concentration Conditions, environmental A-3, A-6, A-9 Configuring Deflector Gate Width 7-9 digitizer 2-38 hardware 2-30 high voltage 2-33 instrument 2-35 instrument type 2-36 laser 2-36 Precursor Ion Selector 7-9 sample plate alignment 2-39 Timed Ion Selector 2-34, 7-9 vacuum 2-30 Continuous Extraction see a
Index Crystallization 3-HPA 3-35 alpha-cyano 3-34 desired pattern 3-34 DHB 3-35 DHBs 3-35 examining 3-34 guidelines for 3-30 sinapinic acid 3-34 THAP 3-35 troubleshooting 8-9 uneven, recommended search pattern for 6-47 Current Spectrum trace definition 4-14 during acquisition 6-16 evaluating 6-16 Cursor Instrument Control Panel, enabling 4-21 oscilloscope, moving I-5 Customizing Instrument Control Panel 4-21 toolbars 4-21 Cytochrome C mass to time conversion F-5 molecular weight F-3 D Damage, reporting B-
Index I N D E X Default calibration description 6-7 equation 6-9 PSD 7-8 selecting 5-10 Default layout, Instrument Control Panel 4-8 DEFAULT.
Index DHBs chemical structure and molecular weight C-9 concentration 3-14 crystals 3-14, 3-35 mass spectrum C-3 preparing 3-14 sample concentration 3-14 stability 3-14 when to use 3-3 Dialysis, sample cleanup 3-21 Digitizer see also Oscilloscope configuring 2-38 description 1-19, 1-33 LSA 1000 LeCroy, connecting 2-13 options, connecting to computer 2-10 Signatec, connecting 2-12 Tektronix oscilloscope, connecting 2-15 type, determining 2-38 Digitizer settings adjusting 5-47 Bin size 5-27 default settings 5
Index I N D E X E F Ejecting sample plates, Instrument Control Panel 4-25 Electromagnetic compliance xxiii EMC standards xxiii EMIS button on vacuum gauge panel 1-30, 1-41 Energy kinetic 1-10, 1-23, 1-36, 7-3 minimizing spread of 1-11, 1-23, 1-36, 5-44 spread of ions, reducing 1-11, 5-18, 5-44, 5-46 Enolase, molecular weight F-3 Environmental conditions A-3, A-6, A-9 Equations calibration 6-9 default theoretical calibration 6-9 drift time for multiply charged ions 1-10 drift time for singly-charged ions
Index Fore pump, Voyager-DE STR function 1-38 vacuum gauge 1-40 Foreline valve location 1-25, 1-27, 1-39 Fragment ions see also Prompt fragments see also PSD fragments see also PSD segments and Grid Voltage% 7-44 and laser intensity 7-39 and Precursor Ion Selector 7-40 calibration 7-8 fast, see Prompt fragments kinetic energy 7-4 optimum resolution observed near Max Stitch Mass 7-5, 7-23 poor yield 7-9 prompt 6-23, 7-41, 7-42 PSD 6-23, 7-3, 7-41, 7-42 PSD, kinetic energy 7-3 Fragmentation and Delay Time 5-
Index I N D E X Guide Wire Voltage% adjusting for CID 7-16 description 5-18, 5-46 effect of changing 5-46 effect on resolution 5-39, 5-46, 5-47, 6-24 effect on sensitivity 5-39, 5-46, 5-74, 5-78 effect on signal-to-noise ratio 5-78, 6-24 in Continuous Extraction mode H-6, H-11 in Delayed Extraction mode 5-74 in PSD mode 7-29, 7-47 optimizing resolution 5-66 optimizing signal-to-noise ratio 5-78 range 5-18 Guidelines for acquiring 6-4 H HABA 3-4 chemical structure and molecular weight C-10 mass spectrum C
Index Hot spots in signal intensity 6-5 How to use this guide xxvii HPA, see 3-HPA HPLC-grade water, use of 3-5, 3-10, 3-11 Humidity, operating A-3, A-6, A-9 Hydroxypicolinic acid, see 3-HPA I IAA chemical structure and molecular weight C-12 concentration 3-15 crystals 3-15 mass spectrum C-6 preparing 3-15 sample concentration 3-15 when to use 3-3 Idle Power 2-34 IgG BIC file 5-4 Immonium ions common 7-5 in PSD mode 7-6 Importing into Sequence run list 6-75 Indoleacrylic acid, see IAA Initial Velocity cor
Index I N D E X Instrument Control Panel basics 4-1 BIC loaded at end of Sequence run 6-79 control buttons 4-24 cursor and grid, displaying 4-21 customizing 4-21 Data Explorer, accessing from 4-7 high voltage, turning on/off 4-25 interaction with Sequence Control Panel 1-47, 4-33 layout, changing 4-8 layout, default 4-8 loading sample plates 3-41 output window 4-5 overview 1-46, 4-2 parts of 4-2 peak detection 6-27 pressures, displaying 2-50 Sequence Control Panel, accessing from 4-7 software, exiting 2-4
Index Instrument Settings parameters (continued) Grid Voltage%, Continuous Extraction H-6 Grid Voltage%, Delayed Extraction E-1 Guide Wire Voltage% 5-39, 5-46 Guide Wire Voltage%, Continuous Extraction H-6 Guide Wire% 5-18 impact of changing, Linear and Reflector mode 5-39 impact of changing, PSD mode 7-47 Instrument mode 5-16 Low Mass Gate 5-20 Manual Control mode 5-9, 5-15 mass range 5-19 Matrix 5-20 matrix and Initial Velocity 6-13 optimizing 5-54 optimizing for Continuous Extraction H-2, H-5 optimizing
Index I N D E X Internal standard calibration 6-7, 6-25 concentration 3-18 mass range 3-18 Internal-Update calibration, see Calibration, internal-update, Sequence Control Panel Ion acceleration description 5-41 impact of Accelerating Voltage 5-77 impact of Grid Voltage% 5-41 in Continuous Extraction mode 1-12 in Delayed Extraction mode 1-12 in MALDI-TOF 1-8 Ion polarity, see Polarity Ion source description, Voyager-DE and Voyager-DE PRO 1-22 description, Voyager-DE STR 1-35 second stage, voltage for 5-17
Index Laser (continued) safety information xxv stopping I-7 threshold, see Laser threshold troubleshooting 8-19 turning on and off 4-25 type, setting 5-25 UV radiation warning xx, 8-3 wavelength, pulse width, and frequency 1-22, 1-35 Laser intensity see also Laser intensity, Automatic Control mode parameters adjusting manually 4-28 adjustment criteria, automatic mode 6-40 displayed in Manual Laser/Sample Position control page 4-27 displayed in status bar 4-5 fine/coarse control 4-28 guidelines for adjustin
Index I N D E X Linking traces 4-13 Live Spectrum trace definition 4-14 during acquisition 6-16 not displayed on oscilloscope systems 6-16 Loading sample plate in mass spectrometer 3-39 sample plates, Instrument Control Panel 4-25 samples on plates 3-28 Log file, Sequence Control Panel 6-68 Log sheet maintenance G-1 sample loading D-1 Logging on to Windows NT Username and password 2-47 without initializing hardware 2-48 Low Mass Gate description 5-20, 5-79 function 5-79 impact on calibration 5-79 improvin
Index Manual Laser/Sample Positioning control page displaying 4-27 laser position 4-31 location 4-27 parameters 4-27 shape of positions 3-53 using 4-27 Mass accuracy and location of standard 3-31, H-24 calibration 6-7, 6-10 effect of charges on 8-17 effect of Na and K on 8-17 factors affecting 6-25 improving 1-14, 3-31, 6-4, 6-5, 6-77, H-23, H-24 improving by deisotoping before calibration 6-66 internal calibration 6-25 maximizing 6-25 obtaining maximum 6-6, 6-25 troubleshooting 3-31, 8-12, 8-17, H-23, H-2
Index I N D E X Matrix (continued) selecting 3-3, C-1 selecting type in calibration 5-20 sinapinic acid 3-3, 3-7, C-6 solutions C-6 spectrum, 3-HPA C-4 spectrum, alpha-cyano-4hydroxycinnamic acid C-2 spectrum, DHB C-3 spectrum, DHBs C-3 spectrum, dithranol C-5 spectrum, HABA C-4 spectrum, IAA C-6 spectrum, Sinapinic acid C-2 spectrum, THAP C-5 stability 3-4 storage conditions 3-4 THAP 3-3, 3-10, C-11 thin film 6-5 Max Stitch Mass definition 7-23, 7-29 optimum focus and resolution observed near this mass 7
Index N Na adduct ion effect on masses 8-17 from buffer 3-18 Name instrument, specifying 2-36 laboratory, specifying 2-36 Nd YAG laser, matrices C-12 Negative ion mode BIC file 5-4, 5-5 selection not in BIC file 8-19 setting 5-25 Switch Delay Time 2-33 Neurotensin, molecular weight F-2 Nicotinic acid C-12 Nitrocellulose in matrix 3-9 Noise, reducing higher frequency 5-28 Nonpolar synthetic polymers, matrix for C-11 Not Used traces 4-14 NT Event log checking 8-21 location 8-21 Number of Data Points, digitiz
Index I N D E X P Page control, types of 4-9 Parent ion, see PSD precursor spectrum Password, obtaining from system administrator 2-47 Path length Voyager-DE 1-4 Voyager-DE PRO 1-4 Voyager-DE STR 1-6 PDF files provided 2-26 Peak centroid shift 5-53 Peak detection overview 6-27 setting 6-27 setting, Sequence Control Panel 6-71 Peak filtering, monoisotopic 6-62, 6-67, 6-71 Peak labels color, changing 4-21 enabling and disabling 6-28 overview 6-27 resolution 6-30 Peak shape and accurate mass measurement 6-25
Index PLT files (continued) search pattern file for 3-57 selecting 3-43, 3-45 x,y coordinates, determining 3-60 Plumbing, CID 7-11 Polarity setting displayed in status bar 4-5 setting Positive or Negative 5-25 Switch Delay in configuration 2-33 Polymers matrix for 3-3 methods for sample loading 3-16 nonpolar synthetic, matrix for C-10 polar synthetic, matrix for C-7, C-10 sample plate to use 3-48 Porcine Trypsin, molecular weight F-2 Positional tolerance, of sample plates 3-59 Positive ion mode setting 5-2
Index I N D E X Printer connecting, Voyager-DE and Voyager-DE PRO 2-18 connecting, Voyager-DE STR 2-20 dedicating to landscape orientation 4-20 Printing changing colors to black before 4-18 instrument settings 5-12 landscape orientation 4-20 traces 4-18 traces do not print 4-19, 4-22 Product spectra see PSD mode see PSD segments Prompt fragments acceleration and flight time 7-42 description 6-23, 7-42 example 7-41 mass 7-42 Proteins Input Bandwidth setting 5-28 matrix for 3-3, C-6, C-10 Starting Mass reco
Index PSD Mirror Ratio (continued) function 7-2 precision displayed when you click on entry 7-30 PSD mode Accelerating Voltage, setting 7-25 accumulating spectra 7-35 acquisition, see PSD acquisition autofill list 7-30 BIC files 5-6 CID option 7-11 comparison to Reflector mode 7-18 constants 7-8 data file not available for viewing until experiment closed 7-32 Decrement Ratio and segment size, correlation 7-22 Decrement Ratio, setting 7-30 default values 7-29 definition 7-2 effect of Grid Voltage% 5-41 enab
Index I N D E X PSD segments (continued) mass range 7-34 number of, and composite spectrum resolution 7-6 number to acquire 7-6, 7-21 optimum resolution observed near Max Stitch Mass 7-5, 7-23, 7-30 reacquiring 7-35 saving 7-35 segment list, defaults 7-28 selecting for acquisition 7-33 size and Decrement Ratio, correlation 7-22 size, collecting different 7-23 size, default 7-23 Pulse width, laser 1-22, 1-35 Pump see Fore pump see Turbo pump Pumping down, time required to reach pressure after venting 2-47
Index Resolution, mass (continued) PSD mode 7-44 PSD segment, optimum observed near Max Stitch Mass 7-30 rating scale for MW ranges 6-31, H-17 results 6-31 troubleshooting 5-66, 8-10, H-21 Resolution, optimizing Accelerating Voltage 5-74, 5-78 Delay Time 5-67 for a mixture 5-68, 5-73 Grid Voltage% 5-72, 5-73 Guide Wire Voltage% 5-46, 5-74 Input Bandwidth 5-53 overview 5-61 parameters affecting 5-62 Results resolution, mass 6-31 signal-to-noise ratio 6-33 Return Authorization (RA) number B-5 Returning damag
Index I N D E X Sample concentration (continued) in IAA 3-15 in sinapinic acid 3-7 in THAP 3-10 low concentration application technique 3-17 thin layer application 3-17 Sample holder ejecting 3-41 loading 3-41 Sample ionization, see Ionization, sample Sample list, Sequence Control Panel saving 6-74 Sample loading dried droplet application 3-31 overview 3-28 techniques 3-28 thin layer application 3-33 Sample loading chamber max load pressure 2-32 wait time 2-32 Sample plate see also PLT files see also Samp
Index Sample plate, types of applications for 3-48 disposable, applications 3-49 disposable, maximum number of spots 3-59 disposable, PLT file for 3-51 gels 3-49 gold, applications 3-48 gold, cleaning 3-37 membranes 3-49 overview 3-48 stainless steel, applications 3-48 stainless steel, cleaning 3-37 Teflon, applications 3-49 Teflon, cleaning 3-36 Teflon, PLT file for 3-51 types supported (.
Index I N D E X Sensitivity (continued) range, Voyager-DE STR 1-6 troubleshooting 8-10 Vertical Scale parameter, effect on 5-28 SEQ files 6-74 Sequence see also Sequence Control Panel acquiring 6-78 before creating 6-65 BIC file loaded in Instrument Control Panel 6-79 creating 6-67 general sequence parameters, setting 6-68 loading 6-78 parameters 6-68 parts of 6-67 pausing and resuming 6-79 run list, see Sequence run list saving 6-74 starting 6-78 status 6-80 stopping 6-79 Sequence Control Panel see also
Index SET file, Sequence Control Panel creating 6-67 defaults used if none specified 6-71 description 6-62 selecting 6-71 Shots/Spectrum description 5-19 does not match number of times laser fires 5-19 for maximum mass accuracy 6-6 impact on signal-to-noise 5-40 improving signal-to-noise ratio 5-79 incorrect number 8-19 maximum number 5-19, 5-79 maximum number, overriding with manual accumulation 5-79 troubleshooting 8-19 Shutting down computer 2-48 mass spectrometer 2-49 Side panel, Voyager-DE and Voyager
Index I N D E X Sinapinic acid chemical structure and molecular weight C-6 concentration 3-7 crystals 3-7, 3-34 initial velocity setting 5-22 laser intensity, relative 5-57 mass spectrum C-2 organic concentration 3-7 preparing 3-5, 3-7 sample concentration 3-7 stability 3-7 when to use 3-3 Singly-charged ions, drift time 1-10 Slow fragments, see PSD fragments Small molecules, matrix for C-8 Smoothing before calibration 6-10 Sodium adduct ion effect on masses 8-17 from buffer 3-18 Software Control Panels 4
Index Spectrum window (continued) Grid, displaying 4-21 live data, definition 4-14 Live Spectrum trace, during acquisition 6-16 Low Mass Gate spike 5-80 mass scale not accurate 8-15 peak detection parameters, setting 6-28 peak labels, enabling and disabling 6-28 resolution, calculating 6-29 right axis, displaying Absolute counts 5-60 scaling signal-to-noise ratio, calculating 6-32 trace, displaying as vertical bars 4-22 traces displayed 4-5 traces do not print 4-19, 4-22 traces, previewing and printing 4-1
Index I N D E X T TC2, Voyager-DE description 1-26 max load pressure 2-32 pressure range 1-30 wait time 2-32 TC2, Voyager-DE PRO description 1-28 max load pressure 2-32 pressure range 1-30 wait time 2-32 TC2, Voyager-DE STR description 1-40 max load pressure 2-32 pressure range 1-41 wait time 2-32 Technical support contacting 8-7 for computers with altered configuration B-1 Teflon plates, see Sample plate, Teflon Tektronix oscilloscope see Digitizer see Oscilloscope Temperature, operating A-3, A-6, A-9 Te
Index Traces (continued) types of 4-14 white, does not print 4-18 zooming 4-13 Trihydroxy acetophenone, see THAP Trimers, troubleshooting 8-15 Troubleshooting active position 8-19 CID 7-16 computer 8-6 continuous mode spectrum H-20 dimers/trimers in spectrum 8-15 laser 8-19 laser threshold H-19 mass accuracy 8-12, 8-17, H-23, H-24 mass spectrometer 8-22 no matrix peaks 8-9 no sample peaks 8-7 peak shape 8-11, 8-16, H-20 poor crystallization on sample plate 3-19 PSD mode 8-20 resolution 5-66, 8-10, H-21 sam
Index I N D E X Vacuum system, Voyager-DE PRO see also Vacuum gauge panel chambers 1-25, 1-27 diagram 1-27 function 1-24 gauges 1-28 Vacuum system, Voyager-DE STR see also Vacuum gauge panel chambers 1-38 diagram 1-39 function 1-38 gauges 1-40 Valves, vacuum 1-25, 1-27, 1-39 Velocity focusing description 1-15 in PSD mode 7-45 Velocity, initial, see Initial velocity Vertical bars displaying traces as 4-22 traces do not print 4-19, 4-22 Vertical Offset, digitizer impact of changing 5-52 setting 5-28 suggest
Index Voyager-DE PRO Biospectrometry Workstation (continued) specifications A-4 startup and shutdown 2-46 weight 2-3 Voyager-DE STR Biospectrometry Workstation features 1-6 mass spectrometer, parts of 1-34 overview 1-5 parts of the system 1-32 power requirements 2-6 space required 2-6 specifications A-7 startup and shutdown 2-46 weight 2-6 X W ZipTips, sample cleanup 3-24 Zooming sample position 6-13 spectrum trace 4-13 Warnings, safety fire hazard and fuse ratings 2-5 high voltage xxi, 8-3 removing in
Index I N D E X Index-40 PerSeptive Biosystems
