LTQ FT Ultra - Thermo Fisher Scientific · Welcome to the Thermo Electron, LTQ FT Ultra™ system!...

Analyze • Detect • Measure • Control™

LTQ FT Ultra™Getting Started 2.2

119 4571 Revision A - September 2006

For Research Use OnlyNot for use in Diagnostic Procedures

For Research Use OnlyNot for use in Diagnostic Procedures

LTQ FT Ultra™

Getting Started 2.2

119 4571 Revision A - September 2006

© 2006 Thermo Electron Corporation. All rights reserved.

“Swagelok” is a registered trademark of the Crawford Fitting Company. “Teflon” is a registered trademark of E. I. du Pont de Nemours & Co.

All other trademarks are the property of Thermo Electron Corporation and its subsidiaries.

Technical information contained in this publication is for reference purposes only and is subject to change without notice. Every effort has been made to supply complete and accurate information; however, Thermo Electron Corporation assumes no responsibility and will not be liable for any errors, omissions, damage, or loss that might result from any use of this manual or the information contained therein (even if this information is properly followed and problems still arise).This publication is not part of the Agreement of Sale between Thermo Electron Corporation and the purchaser of an Thermo Electron system. In the event of any conflict between the provisions of this document and those contained in Thermo Electron Corporation’s Terms and Conditions, the provisions of the Terms and Conditions shall govern.System Configurations and Specifications supersede all previous information and are subject to change without notice.

Printing History: Revision A printed in September 2006.

WEEE KonformitätDieses Produkt muss die EU Waste Electrical & Electronic Equipment (WEEE) Richtlinie 2002/96/EC erfüllen. Das Produkt ist durch folgendes Symbol gekennzeichnet:

Thermo Electron hat Vereinbarungen getroffen mit Verwertungs-/Entsorgungsanlagen in allen EU-Mitgliederstaaten und dieses Produkt muss durch diese Firmen wiederverwertet oder entsorgt werden. Mehr Informationen über die Einhaltung dieser Anweisungen durch Thermo Electron, die Verwerter und Hinweise die Ihnen nützlich sein können, die Thermo Electron Produkte zu identifizieren, die unter diese RoHS Anweisung fallen, finden Sie unter www.thermo.com/WEEERoHS.

WEEE ComplianceThis product is required to comply with the European Union’s Waste Electrical & Electronic Equipment (WEEE) Directive 2002/96/EC. It is marked with the following symbol:

Thermo Electron has contracted with one or more recycling/disposal companies in each EU Member State, and this product should be disposed of or recycled through them. Further information on Thermo Electron’s compliance with these Directives, the recyclers in your country, and information on Thermo Electron products which may assist the detection of substances subject to the RoHS Directive are available at www.thermo.com/WEEERoHS.

Conformité DEEECe produit doit être conforme à la directive européenne (2002/96/EC) des Déchets d'Equipements Electriques et Electroniques (DEEE). Il est marqué par le symbole suivant:

Thermo Electron s'est associé avec une ou plusieurs compagnies de recyclage dans chaque état membre de l’union européenne et ce produit devrait être collecté ou recyclé par celles-ci. Davantage d'informations sur la conformité de Thermo Electron à ces directives, les recycleurs dans votre pays et les informations sur les produits Thermo Electron qui peuvent aider la détection des substances sujettes à la directive RoHS sont disponibles sur www.thermo.com/WEEERoHS.

Read This First

Welcome to the Thermo Electron, LTQ FT Ultra™ system! The LTQ FT Ultra is a member of the LTQ™ family of mass spectrometer (MS) detectors.

This LTQ FT Ultra Getting Started manual provides you with information on how to set up, calibrate, and tune the LTQ FT Ultra. All of these procedures can be performed from the Xcalibur® Tune Plus window. LTQ FT Ultra Getting Started includes the following chapters:

• Chapter 1: “Introduction” provides general information about this manual.

• Chapter 2: “Tune Plus Window” provides information on the Tune Plus window.

• Chapter 3: “Calibrating the LTQ FT Ultra for FT Measurements” provides procedures to calibrate your LTQ FT Ultra for FTmeasurements.

• Chapter 4: “Performing Diagnostics/Checks” describes several diagnostic procedures.

• Chapter 5: “Data Dependent Settings” describes the FT relevant topics of the data dependent settings in the Instrument Setup.

• Chapter 6: “Instrument Configuration” gives instructions about configuring your instrument.

• Chapter 7: “ECD” describes the changes in the software when the ECD option is installed on your instrument.

• Chapter 8: “IRMPD” describes the changes in the software when the IRMPD option is installed on your instrument.

• Appendix A: “Miscellaneous Information” gives additional information about various topics.

• Appendix B: “Safety Information for Optional Hardware”gives safety information for the optional hardware that is available for the LTQ FT Ultra MS detector.

Thermo Electron Corporation LTQ FT Ultra Getting Started i

PrefaceChanges to the Manual

Changes to theManual

To suggest changes to this manual, please send your comments to:

Editors, Technical DocumentationThermo Electron (Bremen) GmbHAdvanced Mass SpectrometryHanna-Kunath-Str. 11

28199 Bremen

Germany

[email protected]

You are encouraged to report errors or omissions in the text or index.Thank you.

ii LTQ FT Ultra Getting Started Thermo Electron Corporation

PrefaceTypographical Conventions

TypographicalConventions

Typographical conventions have been established for Thermo Electron manuals for the following:

• Data input

• Admonitions

• Topic headings

Data Input Throughout this manual, the following conventions indicate data input and output via the computer:

• Messages displayed on the screen are represented by capitalizing the initial letter of each word and by italicizing each word.

• Input that you enter by keyboard is identified by quotation marks: single quotes for single characters, double quotes for strings.

• For brevity, expressions such as “choose File > Directories” are used rather than “pull down the File menu and choose Directories.”

• Any command enclosed in angle brackets < > represents a single keystroke. For example, “press <F1>” means press the key labeled F1.

• Any command that requires pressing two or more keys simultaneously is shown with a plus sign connecting the keys. For example, “press <Shift> + <F1>” means press and hold the <Shift> key and then press the <F1> key.

• Any button that you click on the screen is represented in bold face letters. For example, “click on Close”.

Thermo Electron Corporation LTQ FT Ultra Getting Started iii

PrefaceTypographical Conventions

Admonitions Admonitions contain information that is important, but not part of the main flow of text.

Admonitions can be of the following types:

• Note – information that can affect the quality of your data. In addition, notes often contain information that you might need if you are having trouble.

• Caution – information necessary to protect your instrument from damage.

• Warning – hazards to human beings. Each Warning is accompanied by a Warning symbol.

Topic Headings The following headings are used to show the organization of topics within a chapter:

Chapter Name

The following headings appear in the left column of each page:

Second Level Topics

Third Level Topics

Fourth Level Topics

iv LTQ FT Ultra Getting Started Thermo Electron Corporation

PrefaceSafety and EMC Information

Safety and EMCInformation

In accordance with our commitment to customer service and safety, these instruments have satisfied the requirements for the European CE Mark including the Low Voltage Directive.

Designed, manufactured and tested in an ISO9001 registered facility, this instrument has been shipped to you from our manufacturing facility in a safe condition.

Caution This instrument must be used as described in this manual. Any use of this instrument in a manner other than described here may result in instrument damage and/or operator injury.

Identifying SafetyInformation

The LTQ FT Ultra Getting Started contains precautionary statements that can prevent personal injury, instrument damage, and loss of data if properly followed. Warning symbols which alert the user to check for hazardous conditions. These appear throughout the manual, where applicable, and are defined in Table i.

Table i. Warning Symbols

Symbol Description

General This general symbol indicates that a hazard is present, which if not avoided, could result in injuries.

The source of danger is described in the accompanying text.

Cold Burns Hazard Wear protective clothing.

Electric Shock High voltages capable of causing personal injury are used in the instrument. The instrument must be shut down and disconnected from line power before service or repair work is performed.

Hot Surface / Heat Allow heated components to cool down before servicing them!

Laser Radiation Avoid eye or skin exposure to direct or scattered radiation!

Thermo Electron Corporation LTQ FT Ultra Getting Started v


Instrument-SpecificHazards

Every instrument has specific hazards, so be sure to read and comply with the following precautions. They will help ensure the safe, long-term use of your system.

1. Before plugging in any of the instrument modules or turning on the power, always make sure that the voltage and fuses are set appropriately for your local line voltage.

2. Only use fuses of the type and current rating specified. Do not use repaired fuses and do not short-circuit the fuse holder.

3. The supplied power cord must be inserted into a power outlet with a protective earth contact (ground). When using an extension cord, make sure that the cord also has an earth contact.

4. Do not change the external or internal grounding connections.Tampering with or disconnecting these connections could endanger you and/or damage the system.

Caution The instrument is properly grounded in accordance with regulations when shipped. You do not need to make any changes to the electrical connections or to the instrument’s chassis to ensure safe operation.

5. Never run the system without the housing on. Permanent damage can occur.

6. Do not turn the instrument on if you suspect that it has incurred any kind of electrical damage. Instead, disconnect the power cord and contact a Service Representative for a product evaluation. Do not attempt to use the instrument until it has been evaluated. (Electrical damage may have occurred if the system shows visible signs of damage, or has been transported under severe stress.)

7. Damage can also result if the instrument is stored for prolonged periods under unfavorable conditions (e.g., subjected to heat, water, etc.).

vi LTQ FT Ultra Getting Started Thermo Electron Corporation


8. Always disconnect the power cord before attempting any type of maintenance.

9. Capacitors inside the instrument may still be charged even if the instrument is turned off.

10. The superconducting magnet is still charged even if the instrument is turned off.

11. Never try to repair or replace any component of the system that is not described in this manual without the assistance of your service representative.

Optional hardware that is installed in the instrument (IRMPD, for example) may pose additional hazards. For precautions against these hazards, refer to Appendix B: “Safety Information for Optional Hardware”.

Thermo Electron Corporation LTQ FT Ultra Getting Started vii

Contents

Chapter 1 Introduction...................................................................................... 1-1

Chapter 2 Tune Plus Window.......................................................................... 2-1Preliminary Remarks ............................................................... 2-2View Menu ............................................................................. 2-3

Spectrum View..................................................................... 2-3Graph View ......................................................................... 2-6Status View .......................................................................... 2-6

Control Menu......................................................................... 2-9Advanced Calibration Features............................................. 2-9

Scan Mode Menu.................................................................. 2-10Define Scan........................................................................ 2-10Scan Time Settings............................................................. 2-15Centroid/Profile ................................................................. 2-16Positive/Negative ............................................................... 2-16

Display Menu ....................................................................... 2-17Spectrum Averaging ........................................................... 2-17

Setup Menu .......................................................................... 2-18FT Ion Optics .................................................................... 2-18FT Injection Control ......................................................... 2-18FT Cryo Monitor............................................................... 2-20FT Vacuum ....................................................................... 2-21FT ECD ............................................................................ 2-22FT IRMPD........................................................................ 2-22

Tune Methods ...................................................................... 2-23Parameters with Differentiation between Ion Trap and FT Scans ............................................................................ 2-23Parameters without Differentiation between Ion Trap and FT Scans ............................................................................ 2-24Parameters not saved in a Tunefile ..................................... 2-24

Chapter 3 Calibrating the LTQ FT Ultra for FT Measurements .................. 3-1Preliminary Remarks ............................................................... 3-2Calibration Files and their Backups......................................... 3-4Calibration Solutions .............................................................. 3-5

LTQ Calibration Solution.................................................... 3-5LTQ FT Calibration Solution.............................................. 3-7Applicable Calibration Solutions for Automatic Calibration 3-8

Thermo Electron Corporation LTQ FT Ultra Getting Started ix

Contents

Applicable Calibration Solutions for Semi-Automatic Calibration........................................................................... 3-8Applicable Calibration Solutions for FT Manual Calibration3-9Calibration Solution for ECD and/or IRMPD..................... 3-9

Calibration and Tuning of the Ion Trap ............................... 3-10Calibration of the Ion Trap................................................ 3-10Tuning the Ion Trap for Positive Ion Mode....................... 3-10Tuning the Ion Trap for Negative Ion Mode ..................... 3-12

Automatic Calibration Page .................................................. 3-14Semi-Automatic Calibration Page ......................................... 3-16Check Calibration Page......................................................... 3-18FT Manual Calibration Page................................................. 3-20

Mass List Group Box ......................................................... 3-21FT Advanced Calibration Page.............................................. 3-22

What to calibrate................................................................ 3-23How to calibrate ................................................................ 3-23Refresh............................................................................... 3-26Apply ................................................................................. 3-26Save.................................................................................... 3-26

Manual Coarse Two-Point Mass Calibration ........................ 3-27

Chapter 4 Performing Diagnostics/Checks................................................... 4-1System Evaluation Procedures ................................................. 4-2

FT Sensitivity Test ............................................................... 4-3FT Noise Test ...................................................................... 4-3FT Isolation Test ................................................................. 4-4FT Dynamic Range Test ...................................................... 4-4FT Stability Test .................................................................. 4-4FT Transfer Optics Test....................................................... 4-5FT High Mass Range Target Compensation ........................ 4-6

Toggles ................................................................................... 4-7FT Apodization.................................................................... 4-8FT Detect during Excite ...................................................... 4-8FT Excite Amplifier ............................................................. 4-9FT Include Transients.......................................................... 4-9FT Manual Calibration in Diagnostic Mode........................ 4-9FT Multipole 1 and 2 RF..................................................... 4-9FT Multipole 3 RF .............................................................. 4-9FT Profile Mode .................................................................. 4-9FT SIM and MSn Injection Waveforms............................. 4-10FT View Frequency............................................................ 4-10FT Zero Offset................................................................... 4-10

Set Device ............................................................................. 4-11Setting new FT Transfer Ion Optics Parameters................. 4-12Setting a new FT Transfer Time ........................................ 4-13

x LTQ FT Ultra Getting Started Thermo Electron Corporation

Contents

Display Settings .................................................................... 4-14Display FT Calibration Settings ......................................... 4-15Display FT Diagnostics...................................................... 4-15Display FT Instrument Settings ......................................... 4-15

Chapter 5 Data Dependent Settings............................................................... 5-1Data Dependent FT Narrow SIM Scans ................................. 5-2Preview Mode ......................................................................... 5-3Monoisotopic Precursor Selection ........................................... 5-4

Use Non-Peptide Monoisotopic Recognition....................... 5-4

Chapter 6 Instrument Configuration............................................................... 6-1Starting Instrument Configuration.......................................... 6-2FT Settings Page ..................................................................... 6-3

Active Noise Reduction........................................................ 6-3Automatic Magnetic Field Drift Correction ......................... 6-3IRMPD Configured............................................................. 6-4ECD Configured ................................................................. 6-4ECD Target Gate Current ................................................... 6-4

FT Mass Lists Page.................................................................. 6-5Name................................................................................... 6-5Mass List.............................................................................. 6-5Save...................................................................................... 6-5Save As................................................................................. 6-5Delete .................................................................................. 6-6Import ................................................................................. 6-6Export.................................................................................. 6-6Show Factory Lists ............................................................... 6-6

FT TIC Correction Page......................................................... 6-7FT Full MS.......................................................................... 6-7FT Wide SIM ...................................................................... 6-7FT Full MSn........................................................................ 6-7ECD .................................................................................... 6-7IRMPD................................................................................ 6-7ECD & IRMPD .................................................................. 6-7

Chapter 7 ECD..................................................................................................... 7-1Instrument Configuration for ECD ........................................ 7-2ECD Status Monitor............................................................... 7-3

Heater Current .................................................................... 7-4Heater Resistance ................................................................. 7-4Vacuum Quality .................................................................. 7-4Gate Current........................................................................ 7-5

ECD Cathode Heating during Bakeout .................................. 7-6

Thermo Electron Corporation LTQ FT Ultra Getting Started xi

Contents

ECD Cathode Activation ........................................................ 7-8ECD Calibration and Tuning Solution................................... 7-9FT ECD Calibration Page..................................................... 7-10

Calibrate Heater Current ................................................... 7-10Calibrate Potentials and Timing......................................... 7-11Check Heater Current........................................................ 7-11Check Potentials and Timing............................................. 7-11

FT ECD Dialog Box............................................................. 7-12Define Scan Dialog Box with ECD Option .......................... 7-15

Scan Range of ECD Scans.................................................. 7-17Tuning of ECD Energy and Exposure Time ......................... 7-18Scan Time Settings with ECD Option.................................. 7-18Injection Control Box with ECD Option ............................. 7-19Set Device Page with ECD Option ....................................... 7-20Data Dependent Settings with ECD Option......................... 7-21

Activate ECD for Data Dependent Scans........................... 7-21ECD Settings ..................................................................... 7-21Charge State Dependent ECD Time.................................. 7-22

Chapter 8 IRMPD................................................................................................ 8-1Instrument Configuration for IRMPD Option ....................... 8-2IRMPD Tuning Solution........................................................ 8-3FT IRMPD Dialog Box .......................................................... 8-4Define Scan Dialog Box with IRMPD Option........................ 8-4

Scan Range of IRMPD Scans ............................................... 8-6Tuning of IRMPD Energy and Duration................................ 8-7Scan Time Settings with IRMPD Option ............................... 8-7Injection Control Dialog Box with IRMPD Option ............... 8-8IRMPD Efficiency Test .......................................................... 8-9

Fragmentation Efficiency at the current Settings .................. 8-9Pulse Duration required for Halving the Intensity of the Precursor Ion ....................................................................... 8-9

Data Dependent Settings with IRMPD Option .................... 8-10Activate IRMPD for Data Dependent Scans ...................... 8-10IRMPD Settings ................................................................ 8-10

ECD and IRMPD................................................................. 8-12Instrument Configuration for ECD and IRMPD............... 8-12Define Scan Dialog Box with ECD and IRMPD Option... 8-12Scan Time Settings with ECD and IRMPD Option .......... 8-13Injection Control Dialog Box with ECD and IRMPD Option ............................................................................... 8-14Data Dependent Settings with ECD and IRMPD Option . 8-14

xii LTQ FT Ultra Getting Started Thermo Electron Corporation

Contents

Appendix A Miscellaneous Information...........................................................A-1FT Analyzer Information in Scan Header................................A-2

FT Analyzer Settings ............................................................A-2FT Analyzer Messages ..........................................................A-3

Data Size of FT Raw Files .......................................................A-4

Appendix B Safety Information for Optional Hardware.................................B-1Safety Components for IRMPD..............................................B-2

Safety Interlocks...................................................................B-2Safety Lock for Electronics Cabinet......................................B-3

Safety Instructions for IRMPD ...............................................B-4Laser Safety ..........................................................................B-4General Advice for Safe Operation .......................................B-4Glass Cover ..........................................................................B-4Diode Pointer (Adjusting Laser)...........................................B-5Optical Components............................................................B-5Hazard Labels ......................................................................B-6

Glossary ............................................................................................G-1

Index................................................................................................... I-1

Thermo Electron Corporation LTQ FT Ultra Getting Started xiii

Figures

Tune Plus window ...................................................................................... 2-2Spectrum View page .................................................................................... 2-3Spectrum Display Options dialog box: FT tab ............................................ 2-4Shortcut menu of the Spectrum view .......................................................... 2-5Graph view page ......................................................................................... 2-6Status view: All page .................................................................................... 2-7User Status Display Configuration dialog box ............................................. 2-8Define Scan dialog box .............................................................................. 2-10Scan Time Settings dialog box ................................................................... 2-16Spectrum Averaging dialog box ................................................................. 2-17FT Transfer Ion Optics dialog box ............................................................ 2-18Ion Trap tab of the Injection Control dialog box ...................................... 2-19FT tab of the Injection Control dialog box ................................................ 2-20FT Cryo Monitor dialog box ..................................................................... 2-21Ion Trap tab of the Vacuum dialog box .................................................... 2-21FT tab of the Vacuum dialog box .............................................................. 2-22Recommended settings in the Define Scan dialog box during an automatic tune of the ion trap ........................................................................................... 3-11Ion trap spectrum of the LTQ FT calibration solution, scan range 150–2000, positive ion polarity mode ......................................................................... 3-12Ion trap spectrum of the LTQ FT calibration solution, scan range 150–2000, negative ion mode ..................................................................................... 3-13Automatic page of the Calibrate dialog box ............................................... 3-14Semi-Automatic page of the Calibrate dialog box ...................................... 3-16Check page of the Calibrate dialog box ..................................................... 3-18FT Manual page of the Calibrate dialog box ............................................. 3-20FT Advanced page of the Calibrate dialog box .......................................... 3-22Automatic FT Advanced calibration showing the automatic calibration of the trapping voltage for the positive ion mode ................................................. 3-24Manual FT Advanced calibration, showing the manual calibration of the excite amplitude .................................................................................................. 3-25Manual coarse calibration page of the Diagnostics dialog box .................... 3-27System evaluation page of the Diagnostics dialog box .................................. 4-2Progress of the FT noise test displayed in the Graph View .......................... 4-4Result of the FT stability test displayed in the Graph View ......................... 4-5Toggles page of the Diagnostics dialog box ................................................. 4-7Spectrum view with the “FT detect during excite” toggle switched on ......... 4-8Set device page of the Diagnostics dialog box ............................................ 4-11Display settings page of the Diagnostics dialog box ................................... 4-14

Thermo Electron Corporation LTQ FT Ultra Getting Started xv

Figures

Data Dependent Settings dialog box: Current Scan Event page ................... 5-2Data Dependent Settings dialog box: Scan Widths page ............................. 5-2Data Dependent Settings dialog box: Current Segment page ...................... 5-3Data Dependent Settings dialog box: Charge State page with Advanced Features On .............................................................................................................. 5-4Instrument Configuration dialog box .......................................................... 6-2LTQ FT Ultra Configuration dialog box: FT Settings page ........................ 6-3LTQ FT Ultra Configuration dialog box: FT Mass Lists page ..................... 6-5LTQ FT Ultra Configuration dialog box: FT TIC Correction page ............ 6-7FT Settings page of the LTQ FT Ultra Configuration dialog box ............... 7-2FT ECD page of the Diagnostics dialog box ............................................... 7-3ECD monitor results displayed in the Graph view ...................................... 7-3ECD monitor parameters displayed in the Graph View during a bakeout ... 7-6ECD monitor parameters displayed in the Graph View during an ECD cathode activation .................................................................................................... 7-8FT ECD page of the Calibrate dialog box ................................................. 7-10Toolbar button of the FT ECD dialog box for ECD heater, a: off, b: standby, c: heating or not ready, and d: ready ......................................................... 7-12FT ECD dialog box .................................................................................. 7-13Define Scan dialog box with ECD option ................................................. 7-15Define Scan dialog box, showing a setup for a CID experiment followed by an ECD experiment ....................................................................................... 7-17Scan Activation Parameters dialog box with ECD option .......................... 7-18Scan Time Settings dialog box: FT tab with ECD option ......................... 7-19Injection Control dialog box: FT tab with ECD option ............................ 7-19Set device page of the Diagnostics dialog box with ECD option ................ 7-20Activation page of the Data Dependent Settings dialog box with ECD option ....................................................................................................... 7-21FT ECD/IRMPD page of the Data Dependent Settings dialog box with ECD option ....................................................................................................... 7-22Charge State page of the Data Dependent Settings dialog box with ECD option and Advanced Features On ....................................................................... 7-23LTQ FT Ultra Configuration dialog box: FT Settings page ........................ 8-2FT IRMPD dialog box ................................................................................ 8-4Define Scan dialog box with IRMPD option .............................................. 8-4Define Scan dialog box, showing a setup for a CID experiment followed by an IRMPD experiment .................................................................................... 8-6Scan Activation Parameters dialog box with IRMPD option ....................... 8-7Scan Time Settings dialog box: FT tab with IRMPD option ....................... 8-7Injection Control dialog box: FT tab with IRMPD option ......................... 8-8Activation page of the Data Dependent Settings dialog box with IRMPD option ....................................................................................................... 8-10FT ECD/IRMPD page of the Data Dependent Settings dialog box with IRMPD option ....................................................................................................... 8-11LTQ FT Ultra Configuration dialog box: FT Settings page ...................... 8-12Define Scan dialog box with ECD and IRMPD option ............................ 8-13

xvi LTQ FT Ultra Getting Started Thermo Electron Corporation

Figures

Scan Time Settings dialog box: FT tab with ECD and IRMPD option ..... 8-13Injection Control dialog box: FT tab with ECD and IRMPD option ....... 8-14Activation page of the Data Dependent Settings dialog box with ECD and IRMPD option ......................................................................................... 8-15FT ECD/IRMPD page of the Data Dependent Settings dialog box with ECD and IRMPD option ................................................................................... 8-16Safety interlock at support frame (side panel removed) ................................B-2Mirror unit for IRMPD, showing the safety interlock and the adjusting knobs ..........................................................................................................B-3Safety lock for electronics cabinet ................................................................B-3

Thermo Electron Corporation LTQ FT Ultra Getting Started xvii

Tables

Relation between pressure and vacuum quality ............................................ 7-4ECD heater current in dependence on ECD status ................................... 7-13Actual settings of manual toggles .................................................................A-2Typical data sizes (per scan) of an FT spectrum ...........................................A-4Safety labels for the optional hardware ........................................................B-7

Thermo Electron Corporation LTQ FT Ultra Getting Started xix

Chapter 1 Introduction

This manual describes only the FT relevant settings and procedures of the LTQ FT Ultra software (Tune plus version 2.2). For ion trap relevant settings and procedures, refer to the Getting Started manual that came with the ion trap.

In addition to this manual, the LTQ FT Ultra Tune Plus Online Help gives information to specific topics. Nevertheless, it is recommended to read this manual entirely.

Thermo Electron Corporation LTQ FT Ultra Getting Started 1-1

Chapter 2 Tune Plus Window

This chapter provides LTQ FT Ultra specific information about the Tune Plus window. It contains the following topics:

• “Preliminary Remarks” on page 2-2

• “View Menu” on page 2-3

• “Control Menu” on page 2-9

• “Scan Mode Menu” on page 2-10

• “Display Menu” on page 2-17

• “Setup Menu” on page 2-18

• “Tune Methods” on page 2-23


Tune Plus WindowPreliminary Remarks

Preliminary Remarks The Tune Plus window shows the schematic view of the LTQ FT Ultra and the instrument name. See Figure 2-1. Below the schematic view, an additional Vacuum button allows checking the pressure values for the ICR cell chamber. An FT Cryo Monitor button allows checking the current cryogen fill levels.

Note The Tune Plus window needs a minimum screen resolution of 1152×864 to be fully displayed.

To access the functions of the Tune Plus window, use the menu commands, toolbar buttons, and display views. The FT relevant changes or additions of the menu commands, toolbar buttons, and display views are explained in the following chapters.

Figure 2-1. Tune Plus window

2-2 LTQ FT Ultra Getting Started Thermo Electron Corporation

Tune Plus WindowView Menu

View Menu This section describes those elements of the View menu that are different from the ion trap version of the Tune Plus window.

Spectrum View The Spectrum view displays real-time ion trap or FT mass spectra depending on the analyzer type selected in the Define Scan dialog box. See Figure 2-2.

The Spectrum view page has a shortcut menu that is displayed when you right-click anywhere on the page. To open the Spectrum Display Options dialog box, choose Display Options. The dialog box has two tabs, Ion Trap, and FT.

Figure 2-2. Spectrum View page



On the FT tab, you can determine the number of decimals shown on value tables. See Figure 2-3. To change the number of decimals, click on the arrows in the spin box to increment [up arrow] or decrement [down arrow] the value. You can set the number of decimals to any value from 0 to 5. Alternatively, you can enter a value in the spin box text field. The LTQ FT Ultra changes the number of decimals when you click on Apply or OK.

You can decide whether or not to show the resolution and/or the charge state of peaks in the FT spectrum by clearing or checking the corresponding check boxes.

Another check box allows you to show additional analyzer information for FTMS scans. This information will be displayed above the spectrum graph if the box is checked. Refer to “FT Analyzer Messages” on page A-3 for a list of items that may be displayed as analyzer information.

If the FT analyzer is used, it is possible to display different views in the Spectrum view.

FT Mass Spectrum View Without any action, the usual FT spectra are shown as mass spectra in the Spectrum view.

FT Frequency Spectrum View If the toggle FT view frequency on the Toggles page of the Diagnostics dialog box is enabled, the FT spectra are shown as frequency spectra. In this frequency mode, the labels of the x-coordinate in the Spectrum view correspond to kHz although the x-coordinate is still misleadingly labeled with m/z. To return to the FT mass spectrum view, you have to disable the toggle FT view frequency on the Toggles page of the Diagnostics dialog box.

Figure 2-3. Spectrum Display Options dialog box: FT tab



FT Transient View To display the transient in the Spectrum view, do the following:

1. Enable the toggle FT include transients on the Toggles page of the Diagnostics dialog box.

2. Choose Show FT Transient in the shortcut menu of the Spectrum view that is displayed when you right-click anywhere on the page. See Figure 2-4.

3. Ensure that profile (instead of centroid) is chosen as data format.

FT Excite Signal View To display the excite signal in the Spectrum view, do the following:

1. Enable the toggle FT detect during excite on the Toggles page of the Diagnostics dialog box.

2. Choose Show FT Transient in the shortcut menu of the Spectrum view that is displayed when you right-click anywhere on the page. See Figure 2-4.

3. Ensure that profile (instead of centroid) is chosen as data format.

Figure 2-4. Shortcut menu of the Spectrum view



Graph View The Graph view displays, in a variety of traces, real-time data generated during calibration, tuning, and diagnostic tests. For example, Figure 2-5 shows the progress of the FT mass calibration.

Status View The Status view displays real-time status information for the LTQ FT Ultra. See Figure 2-6 on page 2-7. The Status view has two pages, the All page and the User page:

• The Status view - All page displays the real-time status information for about 80 parameters of the LTQ FT Ultra. You can scroll through the list to observe the status of the parameters.

Figure 2-5. Graph view page



• The Status view - User page displays real-time status information for LTQ FT Ultra parameters that you have selected in the User Status Display Configuration dialog box. (See page 2-8.)

Figure 2-6. Status view: All page



User Status DisplayConfiguration Dialog Box

Figure 2-7 shows the User Status Display Configuration dialog box. Use the following procedure to configure the User page:

1. Choose View > Display Status View.

2. Click on the User tab. Right-click on the User page to display the shortcut menu.

3. Choose Configure.

Figure 2-7. User Status Display Configuration dialog box


Tune Plus WindowControl Menu

Control Menu This section describes the elements of the Control menu that are different from the ion trap.

Advanced CalibrationFeatures

With the Advanced Calibration Features command, you display the additional calibration page FT Advanced in the Calibrate dialog box.

This command is a toggle. A check mark () appears next to the command when it is activated.

Usually it is not necessary to use the FT Advanced calibration page, which is provided for advanced users only. An incorrect usage of the FT Advanced page may harm the functionality of the LTQ FT Ultra. Thus, it is recommended to deactivate the Advanced Calibration Features. For further details about the FT Advanced calibration page, please refer to the topic “FT Advanced Calibration Page” on page 3-22.


Tune Plus WindowScan Mode Menu

Scan Mode Menu This section describes the elements of the Scan Mode menu that are different from the ion trap.

Define Scan The Define Scan dialog box allows you to define a scan in various ways depending on the scan mode and scan type combination. Also, the Define Scan dialog box allows you to choose the ion trap or the FT cell as analyzer. Figure 2-8 shows the Define Scan dialog box showing the Advanced Scan features. The Advanced Scan features can be activated in the Scan Mode menu of Tune plus.

Scan Description The Scan Description group box contains the following elements:

Figure 2-8. Define Scan dialog box

Analyzer At the top, the Analyzer combo box allows you to select the analyzer type used during the currently selected scan event. The options are as follows:

• FTMS selects the FTICR detector.

• Ion Trap selects the ion trap detector.



Mass Range If you choose Ion Trap as analyzer, you can select the following mass ranges:

• Low: 15–200

• Normal: 50–2000

• High: 100–4000

When you have selected the entry FTMS in the Analyzer combo box, this combo box has the entries Normal and High corresponding to these scan ranges:

• Normal: 50–2000

• High: 100–4000Scan Rate / Resolution When you have selected the entry ion trap in the

Analyzer combo box, this combo box allows you to set the scan rate (Normal, Enhanced, Turbo, Zoom, UltraZoom).

When you have selected the entry FTMS in the Analyzer combo box, this combo box allows you to set the resolution of the FT mass spectra. The mass resolution is selectable between several options. Available resolutions are 12500, 25000, 50000, 100000, 200000, 400000, 750000, and 1000000.

The resolution setting of 1000000 is not available if the first mass of the scan range is below m/z 98.

The default resolution setting is 100000, but for many applications a resolution setting of 50000 is sufficient.

The mass resolution in FTMS is proportional to 1/(m/z). The value selected in the Define Scan Box refers to m/z 400. Thus, at a resolution setting of 100000 the mass resolution at m/z 200 is actually 200000 and the mass resolution at m/z 800 is actually 50000.



With increasing resolution, the detect time and thus the scan time is increased. At a selected resolution of 100000, the scan speed is about 1 scan per second. At a resolution setting of 50000, the scan speed is about 2 scans per second.

Scan Type Full:Allows you to select a full scan type. To select the full scan type, select the Scan Type: Full option button. You can perform a full scan with any of the scan modes: MS, MS/MS, or MSn. Usage of the scan type Full MS using the FTMS analyzer is analogous to the ion trap analyzer.

While there is only one SIM (selected ion monitoring) scan type for the ion trap analyzer, two different SIM modes can be selected if FTMS is chosen as analyzer:

• Narrow SIM:SIM scan type that is only available if FTMS is chosen as analyzer. To define a Narrow SIM experiment, clear all parent masses from the MSn Settings table and select Scan Type: Narrow SIM. The scan range width is limited to 30. In a Narrow SIM scan, the electrodes of the FT transfer optics are set to low potentials to increase the detection efficiency for ions within a small m/z range. Only one scan range is available for Narrow SIM.

• Wide SIM:SIM scan type that is only available if FTMS is chosen as analyzer. To define a Wide SIM experiment, clear all parent masses from the MSn Settings table and select Scan Type: Wide SIM. The scan range width has to be within 30–600. In a Wide SIM scan, the electrodes of the FT transfer optics are set to the same potentials as in full scans to allow detection of ions over a wider m/z range. Only one scan range is available for Wide SIM.



Scan Time The Scan Time group box contains the following elements:

SRM:Allows you to select a selected reaction monitoring (SRM) scan type. To define an SRM experiment, enter one parent mass in the MSn Settings table and select Scan Type: SRM. Only one scan range is available for SRM if FTMS is chosen as analyzer.

CRM:Allows you to select a consecutive reaction monitoring (CRM) scan type. To define a CRM experiment, enter two or more parent masses in the MSn Settings table and select Scan Type: CRM. Only one scan range is available for CRM if FTMS is chosen as analyzer.

Microscans The number of microscans determines how many spectra are averaged in one analytical scan. If the FT is chosen as the analyzer, transients are averaged in one analytical scan.

The number of microscans can be set individually for FT MS, Ion Trap MS, FT SIM, Ion Trap SIM, FT MSn, Ion Trap MSn, and Ion Trap Zoom.

Max Inject Time The inject time is automatically controlled by the automatic gain control (AGC). The entry in this spin box limits the inject time to a maximum value. To ensure the high mass accuracy of the LTQ FT Ultra, the maximum inject time should not be reached. Otherwise, the number of ions does not correspond to the AGC target value. In particular during (manual) mass calibration, it is absolutely necessary that the maximum inject time is not reached.

The maximum inject time can be set individually for FT MS, Ion Trap MS, FT Narrow SIM, FT Wide SIM, FT SIM, Ion Trap SIM, FT MSn, Ion Trap MSn, and Ion Trap Zoom.



MSn Settings If ECD and/or IRMPD is configured in the Instrument Configuration, it is possible to choose ECD and/or IRMPD as last event of an MSn experiment. For further details, please refer to Chapter 7: “ECD” and Chapter 8: “IRMPD”.

Note If the maximum inject time is reached the number of ions may not correspond to the current AGC target value. This may harm the mass accuracy of FT spectra. Thus as a rule for good mass accuracy, the maximum inject time should be chosen as high as reasonable.

Wide Scan Range (WSR)

If FTMS is chosen as analyzer, it is possible to enable the Wide Scan Range (WSR) mode for FT Full MS and FT MSn scan types. If the WSR mode is enabled, transients of scans acquired at different transfer settings are averaged to allow larger scan ranges to be covered. The number of averaged transients is automatically determined in dependence on the scan range, scan type, and target value. Since more than one transients are acquired, the scan time is increased if the WSR mode is enabled. If n transients are averaged for the WSR mode of a given scan range at one microscan, and m is set as number of microscans, m × n transients will be averaged. The selected WSR mode (enabled or disabled) is saved as tune parameter, independently for FT full and FT MSn scans.

Scan Time Settings Overview

By pressing the All… button of the Define Scan dialog box, the Scan Time Settings dialog box is displayed. It allows displaying and setting all scan time settings for all scan types at the same time for both the ion trap and the FT analyzer. For further details, refer to topic “Scan Time Settings” on page 2-15.



Scan Ranges Different transfer-, excitation- and mass calibration parameters are used for three different scan ranges defined by the first mass and for the ion polarity mode. The resulting six calibration ranges are shortened in this manual and in the display messages of Tune plus by the following terms

For example, different transfer-, excitation- and mass calibration parameters are used for the scan ranges 90–900 and 110–900, whereas the same transfer-, excitation- and calibration parameters are used for the scan ranges 200–2000 and 1000–2000, if the same ion polarity mode is chosen.

Scan Ranges for Full Scans In principle, for FT scans it is possible to use any scan range within 50–2000 (normal mass range) or within 100–4000 (high mass range). However, it does not make sense to use a scan range that extends over more than one decade, i.e. last mass = 10 × first mass, for example 190–1900 or 70–700.

Scan Ranges forSIM/SRM/CRM Scans

For ion trap SIM, SRM, or CRM scans, the mass window around the center mass is limited to 100. For FT Narrow SIM scans, the mass window around the center mass is limited to 30. For FT Wide SIM scans, the mass window has to be within 30–600.For FT SRM and FT CRM scans, the mass window around the center mass is limited to 600.

Scan Ranges for ECD/IRMPD If ECD and/or IRMPD is set as last event in the Define Scan dialog box (only possible if ECD and/or IRMPD is configured in the Instrument Configuration), the scan ranges are limited by other rules. For further details, please refer to Chapter 7: “ECD” and Chapter 8: “IRMPD”.

Scan Time Settings The Scan Time Settings dialog box displays the current scan time settings for all scan types of both the ion trap and the FT analyzer. See Figure 2-9 on page 2-16. In addition to the number of microscans and the maximum

fm > 50 (+) for 50 ≤ first mass < 98 positive polarity mode fm > 98 (+) for 98 ≤ first mass < 190 positive polarity modefm > 190 (+)for 190 ≤ first mass positive polarity modefm > 50 (-) for 50 ≤ first mass < 98 negative polarity modefm > 98 (-) for 98 ≤ first mass < 190 negative polarity modefm > 190 (-) for 190 ≤ first mass negative polarity mode



inject time, it is possible to enable or disable the Wide Scan Range (WSR) mode for FT Full MS and FT MSn scans. For further details about the wide scan range mode, refer to topic “Define Scan” on page 2-10.

If ECD and/or IRMPD is configured in the Instrument Configuration, additional scan time settings for ECD and/or IRMPD are displayed in this dialog box. Refer to Chapter 7: “ECD” and Chapter 8: “IRMPD” for further details.

The Scan Time Settings dialog box can also be called from the Define Scan dialog box by pressing the button All… See page 2-14.

Centroid/Profile With this pair of buttons, you switch between the Centroid and the Profile format.

With the toggle “FT profile mode” under Diagnostics > Diagnostics > Tools > Toggles, you can select whether the FT profile mode corresponds to a Full Profile format or to a Reduced Profile format. It is recommended to use the Reduced Profile format for data acquisition since the data size of the raw file is significantly decreased by using the Reduced Profile. For further information, see also topic “Data Size of FT Raw Files” on page A-4.

Positive/Negative With this pair of buttons, you can toggle between positive ion and negative ion polarity. Different FT transfer, excite and mass calibration parameters are used for the different polarities.

Figure 2-9. Scan Time Settings dialog box


Tune Plus WindowDisplay Menu

Display Menu This section describes the elements of the Display menu that are different from the ion trap.

Spectrum Averaging With this toggle, spectrum averaging can be switched on or off. If spectrum averaging is enabled the displayed spectrum is the moving average of several spectra before. Averaging FT scans is actually an averaging of transient.

Use this functionality in analogy to ion trap scans:

1. In the Tune Plus window, choose Display > Spectrum Averaging > Settings… to display the Spectrum Averaging dialog box. (See Figure 2-10.)

2. Enter the number of transients to average into the spin box.

3. Click on OK to save your changes and close the dialog box.

If the FT analyzer is chosen it is also possible to display the excite signal, a frequency spectrum, or transients by using the corresponding toggles on the Toggles page of the Diagnostics dialog box. (See topic “Toggles” on page 4-7.)

Figure 2-10. Spectrum Averaging dialog box


Tune Plus WindowSetup Menu

Setup Menu This section describes the elements of the Setup menu that are different from the ion trap.

FT Ion Optics The FT transfer parameters are only allowed to be changed by an FT ion transmission calibration, which is usually only necessary when the hardware of the system is modified somehow. Therefore, this message box only displays the corresponding FT readback values. See Figure 2-11.

To open this message box:

• From the Tune Plus window, choose Setup > FT Ion Optics, or

• click on in the Instrument Setup toolbar.

FT Injection Control The Injection Control dialog box allows you to set the automatic gain control (AGC) target values. In addition, the Injection Control dialog box allows you to enable or disable the injection waveforms.

To open this dialog box:

• From the Tune Plus window, choose Setup > FT Injection Control, or

Figure 2-11. FT Transfer Ion Optics dialog box



• Click on in the Instrument Setup toolbar.

The Injection Control dialog box has two tabs to enable an independent selection of target values for Ion Trap and FT scans.

Ion Trap We recommend the following target values for the ion trap:

FT For FT measurements, the Full MS Target, the Narrow SIM Target, the Wide SIM Target, and the MSn Target can be set. See Figure 2-13 on page 2-20.

Recommended target values for the FT analyzer, especially in terms of good mass accuracy results:

Full MS Target: 3e+04SIM Target: 1e+04MSn Target: 1e+04Zoom Target: 3000.0

Figure 2-12. Ion Trap tab of the Injection Control dialog box

Full MS Target: 5e+05Narrow SIM Target: 5e+04Wide SIM Target: 5e+05MSn Target: 2e+05



If ECD and/or IRMPD are configured in the Instrument Configuration, additional target values can be set in the FT Injection Control Box. For further details, refer to Chapter 7: “ECD” and/or Chapter 8: “IRMPD”.

Enable Full Scan InjectionWaveforms

You can enable or disable the injection waveforms independently for ion trap and FT scans.

If the injection waveforms are enabled, a filter on the ions injected into the ion trap is applied. The ions above and below the selected ion or ion range selected are rejected. This option is often useful if the ion trap is being filled with ions of greater or lesser mass than the ion mass or ion mass range of interest. For example, this option can be used to remove high mass ions that are not of interest and ensures that more target ions can enter the trap before the trap is full.

Note The FT injection waveforms option only applies to full scan MS scans performed with the FT-ICR mass analyzer. In FT Narrow SIM, FT Wide SIM, and FT MS^n scans, the injection waveforms are automatically enabled.

FT Cryo Monitor The FT Cryo Monitor dialog box displays the cryogen fill levels of the superconducting magnet as a percentage of the maximum fill level. See Figure 2-14 on page 2-21. You can also monitor the cryogen fill levels on the front of the instrument.

Note In case of doubt the display on the front of the system is relevant.

Figure 2-13. FT tab of the Injection Control dialog box




• From the Tune Plus window, choose Setup > FT Cryo Monitor… or

• Click on in the Instrument Setup tabular.

Note The cryogen fill levels are automatically saved in the logfile system_monitor.log, which is located in C:\Xcalibur\system\ltq\logs.

FT Vacuum The Vacuum dialog box allows you to monitor the vacuum system parameters. The Vacuum dialog box has two tabs to enable an independent selection of displaying the vacuum data of the ion trap (See Figure 2-15.) or the FT part. (See Figure 2-16 on page 2-22.)

Figure 2-14. FT Cryo Monitor dialog box

Figure 2-15. Ion Trap tab of the Vacuum dialog box




• From the Tune Plus window, choose Setup > Vacuum…, or

• Click on in the Instrument Setup tabular.

FT ECD The command FT ECD is only part of the Setup menu if ECD is configured in the Instrument Configuration. For further details, refer to Chapter 7: “ECD”.

FT IRMPD The command FT IRMPD is only part of the Setup menu if IRMPD is configured in the Instrument Configuration. For further details, refer to Chapter 8: “IRMPD”.

Figure 2-16. FT tab of the Vacuum dialog box


Tune Plus WindowTune Methods

Tune Methods Several parameters, like the ion source parameters, ion trap optics parameters, AGC target values etc., are stored in tune methods. This topic points out for which parameters a differentiation between ion trap and FT is made. The Tune Plus title bar displays the name of the current Tune Method. If you are not currently editing a preexisting Tune Method, the title bar displays the word Untitled.

Parameters withDifferentiation between

Ion Trap and FT Scans

A differentiation between Ion Trap and FT scans is made for the tune parameters listed in the following topics.

AGC Target Values They can be set and saved independently for these experimental modes (No differentiation is made between positive and negative ion polarity mode.):

• Ion Trap Full MS Target

• Ion Trap SIM Target

• Ion Trap MSn Target

• Ion Trap Zoom Target

• FT Narrow SIM Target

• FT Wide SIM Target

• FT MSn Target

Microscans and the MaximumInject Time

They can be set and saved independently for these experimental modes:

• Ion Trap Full MS, positive ion mode

• Ion Trap SIM, positive ion mode

• Ion Trap MSn, positive ion mode

• Ion Trap Zoom, positive ion mode

• FT Full MS, positive ion mode

• FT Narrow SIM, positive ion mode

• FT Wide SIM, positive ion mode



• FT MSn, positive ion mode

• Ion Trap Full MS, negative ion mode

• Ion Trap SIM, negative ion mode

• Ion Trap MSn, negative ion mode

• Ion Trap Zoom, negative ion mode

• FT Full MS, negative ion mode

• FT Narrow SIM, negative ion mode

• FT Wide SIM, negative ion mode

• FT MSn, negative ion mode

Inject Waveform Flags The flag whether the inject waveform is enabled or disabled can be set and saved independently for

• Ion trap scans, and

• FT full scans.

Wide Scan Range Mode The flag whether the wide scan range mode is enabled or disabled for FT Full MS and FT MSn scans can be set and saved independently for these scan types.

Parameters withoutDifferentiation between

Ion Trap and FT Scans

No differentiation between Ion Trap and FT scans is made for all ESI parameters, and for all ion source and ion optics parameters.

Parameters not saved in aTunefile

All parameters that can be set in an instrument method are not saved in the tune method. Thus, the following parameters are not saved in a tune method:

• Analyzer (Ion Trap or FTMS)

• Mass Range (Low, Normal, or High)

• Scan Rate



• Resolution

• Scan Type (Full, SIM, SRM, CRM)

• Scan Range

• Polarity* (positive or negative)

• Data type* (centroid or profile)

* Only the data format (centroid or profile) and the ion polarity, which are set after a new start of TunePlus, are saved in a tunefile.


Chapter 3 Calibrating the LTQ FT Ultra for FT Measurements

This chapter provides procedures to calibrate your LTQ FT Ultra for FTmeasurements. It contains the following topics:

• “Preliminary Remarks” on page 3-2

• “Calibration Files and their Backups” on page 3-4

• “Calibration Solutions” on page 3-5

• “Calibration and Tuning of the Ion Trap” on page 3-10

• “Automatic Calibration Page” on page 3-14

• “Semi-Automatic Calibration Page” on page 3-16

• “Check Calibration Page” on page 3-18

• “FT Manual Calibration Page” on page 3-20

• “FT Advanced Calibration Page” on page 3-22

• “Manual Coarse Two-Point Mass Calibration” on page 3-27


Calibrating the LTQ FT Ultra for FT MeasurementsPreliminary Remarks

Preliminary Remarks There are no tune procedures for the FT part since a change of the transfer or excitation settings would potentially require a new mass calibration. Thus, all transfer and excitation parameters are treated as calibration parameters and determined in calibration procedures.

Different sets of transfer, excitation and mass calibration parameters are used for six different calibration ranges depending on the first mass and the ion polarity mode. These ranges are:

In the automatic calibration, the FT transmission calibration and the FTmass calibration are automatically performed for all six calibration ranges. In the semi-automatic calibration, it is possible to decide whether the transmission and/or mass calibration are performed only for the positive ion mode (three calibration ranges), only for the negative ion mode (the other three calibration ranges) or for both polarities (six calibration ranges). See topics “Automatic Calibration Page” on page 3-14 and “Semi-Automatic Calibration Page” on page 3-16 for further details.

On the FT Manual calibration page, you can select your own calibration masses for FT ion transmission and FT mass calibration. Note that a manual FT calibration is only performed for that calibration range corresponding to the first mass and ion polarity specified during the calibration. See topic “FT Manual Calibration Page” on page 3-20 for further details. The FT Manual page is only displayed if the Advanced Calibration Features are activated in the Control menu. See topic “Advanced Calibration Features” on page 2-9 for instructions.

Apart from the FT transmission and FT mass calibration parameters, further calibration parameters are saved in the calibration file. Since these additional calibration parameters should usually not be changed unless the hardware is modified, the corresponding calibration procedures are not part of the Automatic page or Semi-Automatic calibration page. They are part of the FT Advanced calibration page and should – as the name suggests – only performed by advanced users. See topic “FT Advanced Calibration Page” on page 3-22 for further details. The FT Advanced page is only displayed if the Advanced Calibration Features are activated in the Control menu.

fm > 50 (+) for 50 ≤ first mass < 98 positive polarity mode,fm > 98 (+) for 98 ≤ first mass < 190 positive polarity mode,fm > 190 (+)for 190 ≤ first mass positive polarity mode,fm > 50 (-) for 50 ≤ first mass < 98 negative polarity mode,fm > 98 (-) for 98 ≤ first mass < 190 negative polarity mode,fm > 190 (-) for 190 ≤ first mass negative polarity mode.


Calibrating the LTQ FT Ultra for FT MeasurementsPreliminary Remarks

Note It is recommended to use the semi-automatic calibration.

If ECD is configured in the Instrument Configuration, the ECD calibration page is also part of the Calibrate dialog box. The ECD calibrations are discussed in Chapter 7: “ECD”.


Calibrating the LTQ FT Ultra for FT MeasurementsCalibration Files and their Backups

Calibration Files andtheir Backups

After a successful or partly successful calibration, the ion trap and FT calibration parameters are saved automatically. All ion trap and FT calibration parameters are stored in the calibration file master.LTQCal, which is located in C:\Xcalibur\system\ltq\msx.

It is possible to create a backup of the current calibration file manually or by choosing File > Backup Current Calibration in the Tune Plus window. The Backup Current Calibration and Restore Backup Calibration items work by copying the master.LTQCal to user.LTQCal and vice versa.

If already a backup calibration user.LTQCal was generated before, the old user.LTQCal will be backed up to a file named userXYZ.LTQCal. If you perform backup calibrations at regular intervals, then a history of your calibration files is generated in C:\Xcalibur\system\ltq\msx.

Upon Restore Backup Calibration, the calibration values saved in user.LTQCal are automatically downloaded to the instrument. Therefore, we recommend generating a current backup after a successful calibration.

We recommend using the Restore Backup Calibration command instead of the Restore Factory Calibration command since the backup calibration file is newer than the factory calibration file.


Calibrating the LTQ FT Ultra for FT MeasurementsCalibration Solutions

Calibration Solutions This section provides information about preparing the calibration solutions for the LTQ FT Ultra.

LTQ Calibration Solution The LTQ calibration solution consists of caffeine, MRFA, and Ultramark 1621 in an acetonitrile:methanol:water solution containing 1% acetic acid.

Note Vials of caffeine, MRFA, and Ultramark 1621 are included in the API accessory kit. To order more of these compounds, write or call:

Sigma Chemical CompanyP. O. Box 14508St. Louis, Missouri, USA 63178-9916Phone (800) 325-3010 (in the USA or Canada)

(314) 771-3750 (outside the USA or Canada)

Caution AVOID EXPOSURE TO POTENTIALLY HARMFUL MATERIALS.

Always wear protective gloves and safety glasses when you handle solvents or corrosives. Also contain waste streams and use proper ventilation. Refer to your supplier's Material Safety Data Sheet (MSDS) for proper handling of a particular solvent.

To prepare the LTQ calibration solution, use the following procedure:

1. Stock Solution: MRFA

Prepare a 1.5 mL stock solution of 166.7 pmol/µL MRFA in 50:50 methanol:water as follows:

a. Obtain the vial of L-methionyl-arginyl-phenylalanyl-alanine acetate × H2O (MRFA) in your accessory kit. In this form, the MRFA sample has an average molecular weight of 607.7 u. Carefully weigh 3.0 mg of the MRFA sample.

b. Dissolve the MRFA sample in a total volume of 1.0 mL of 50:50 methanol:water. Mix the solution (5.0 nmol/µL) thoroughly.

c. Transfer 50 µL of the 5 nmol/µL solution into a clean polypropylene tube.

d. Add 1.45 mL of 50:50 methanol:water to the tube. Mix this solution (166.7 pmol/µL) thoroughly.



e. Label the tube MRFA stock solution and store it in a refrigerator until it is needed.

2. Ultramark 1621 stock solution

Prepare a 10 mL stock solution of 0.1% Ultramark 1621 in acetonitrile as follows:

a. Obtain the vial of Ultramark 1621 in your accessory kit.

b. Using a syringe, measure out 10 µL of Ultramark 1621, and dissolve it in 10 mL of acetonitrile. Mix the solution thoroughly.

c. Label the vial Ultramark 1621 stock solution and store it in a refrigerator until it is needed.

3. LTQ calibration solution

Prepare 10 mL of the LTQ calibration solution as follows:

a. Obtain the 1 mg/mL stock solution of caffeine in 100% methanol that is provided in your accessory kit.

b. Pipet 200 µL of the caffeine stock solution into a clean, dry 10 mL volumetric flask.

c. Pipet 100 µL of the MRFA stock solution into the flask.

d. Pipet 100 µL of the Ultramark 1621 stock solution into the flask.

e. Pipet 100 µL of glacial acetic acid into the flask.

Note Use only glass pipets or stainless steel syringes when measuring glacial acetic acid. Using plastic pipet tips causes contamination of acid stock solutions that can introduce contaminants into the calibration solution.

f. Pipet 5 mL of acetonitrile into the flask.

g. Bring the volume of the solution up to the 10 mL-mark on the flask with 50:50 methanol:water.

h. Mix the calibration solution thoroughly.

i. Transfer the solution to a clean, dry vial.

j. Label the vial LTQ Calibration Solution and store it in a refrigerator until it is needed.



LTQ FT CalibrationSolution

In contrast to the ion trap calibration, the FT calibration of the LTQ FT Ultra requires both positive and negative ions. The LTQ ESI calibration solution does not provide applicable negative ions below m/z 1000. Thus, sodium dodecyl sulfate (gives anion at m/z 265) and sodium taurocholate (gives anion at m/z 514) is added to the LTQ ESI calibration solution to generate the LTQ FT calibration solution. In addition, the ratio of MRFA is increased versus the LTQ calibration solution.

Note Vials of sodium dodecyl sulfate and sodium taurocholate are shipped with the instrument. Refer to the Material Safety Data Sheet (MSDS) for proper handling of these substances. To order more of these compounds, contact Sigma-Aldrich. The product number of sodium dodecyl sulfate is L4509-10G, the product number of sodium taurocholate is T4009-250MG. Note that sodium dodecyl sulfate and sodium taurocholate are not included in the standard LTQ API accessory kit.

Caution AVOID EXPOSURE TO POTENTIALLY HARMFUL MATERIALS.

Always wear protective gloves and safety glasses when you use solvents or corrosives. Also, contain waste streams, and use proper ventilation. Refer to your supplier's Material Safety Data Sheet (MSDS) for proper handling of a particular solvent.

To prepare the LTQ FT calibration solution, use the following procedure:

1. Stock Solution: Sodium Dodecyl Sulfate

a. Obtain the vial of sodium dodecyl sulfate. In this form the sample has an average molecular weight of 288.4 u.

b. Prepare the stock solution of sodium dodecyl sulfate by dissolving 2.88 mg in 10 mL of 50:50 methanol:water.

c. Mix the solution (1.0 nmol/µL) thoroughly.

d. Label the vial Sodium Dodecyl Sulfate stock solution (1 nmol/µL).

2. Stock Solution: Sodium Taurocholate

a. Obtain the vial of sodium taurocholate. In this form the sample has an average molecular weight of 537.7 u.

b. Prepare the stock solution of sodium dodecyl sulfate by dissolving 5.38 mg in 10 mL of 50:50 methanol:water.




d. Label the vial Sodium Taurocholate stock solution (1 nmol/µL).

3. Stock Solution: MRFA

To prepare the MRFA stock solution (5.0 nmol/µL or 166.7 pmol/µL in 50:50 methanol:water), refer to step 1 of topic ”LTQ Calibration Solution” above.

4. LTQ FT Calibration Solution

Prepare 10 mL of the LTQ FT calibration solution, as follows:

a. Pipet 10 mL of the standard LTQ ESI calibration solution into a vial.

b. Add 100 µL of the Sodium Dodecyl Sulfate stock solution.

c. Add 100 µL of the Sodium Taurocholate stock solution.

d. Add 6.6 µL of the 5.0 nmol/µL MRFA stock solution or 200 µL of the 166.7 pmol/µL MRFA stock solution, respectively.

e. Mix the solution thoroughly.

f. Label the vial LTQ FT calibration solution and store it in a refrigerator until it is needed.

Applicable CalibrationSolutions for Automatic

Calibration

In an automatic calibration of the LTQ FT Ultra, both the ion trap and the FT calibrations (positive and negative ion mode) are performed. Thus for an automatic calibration, the LTQ FT calibration solution has to be used.

Applicable CalibrationSolutions for

Semi-AutomaticCalibration

For the ion trap calibrations, the LTQ ESI calibration solution or the LTQ FT calibration solution can be used. For the FT calibrations of the positive ion mode, the LTQ ESI calibration solution or the LTQ FT calibration solution can be used. For the FT calibrations of the negative ion mode, the LTQ FT calibration solution has to be used. Thus, for a complete FT calibration the LTQ FT calibration has to be used, too.



Applicable CalibrationSolutions for FT Manual

Calibration

Since the FT Manual Calibration page allows using your own calibration masses, there is in principle no limitation of the calibration solution here. However, you should take care that the calibration masses are uniformly distributed among the used scan range.

Calibration Solution forECD and/or IRMPD

If ECD and/or IRMPD is installed at your instrument, refer to Chapter 7: “ECD” and/or Chapter 8: “IRMPD” for further details about the extra calibration solution for ECD and/or IRMPD.


Calibrating the LTQ FT Ultra for FT MeasurementsCalibration and Tuning of the Ion Trap

Calibration andTuning of the Ion Trap

This chapter describes the calibration and tuning of the ion trap for FTmeasurements. It contains the following topics:

• “Calibration of the Ion Trap” on page 3-10

• “Tuning the Ion Trap for Positive Ion Mode” on page 3-10

• “Tuning the Ion Trap for Negative Ion Mode” on page 3-12

Calibration of the Ion Trap To perform an FT calibration, the ion trap has to be successfully calibrated before. However, it is not imperatively necessary to perform the ion trap mass and resolution calibrations for Zoom Scan and UltraZoom Types. It is very important that the electron multiplier gain is correctly calibrated since the AGC prescan is performed in the ion trap. Thus, the electron multiplier gain calibration should be checked before an FT calibration is performed. Note that it is not necessary to use the ESI standard solution to perform a multiplier gain calibration. It is sufficient to use an MRFA solution (for example 5×10-6 M in 100% methanol, 1% acetic acid).

For the ion trap calibrations, the LTQ ESI calibration solution or the LTQ FT calibration solution can be used.

Tuning the Ion Trap forPositive Ion Mode

For the positive ion mode, it is recommended to perform an automatic tune of m/z 524 at a Full MS Target of 1e4–3e4. Use the LTQ ESI calibration solution or the LTQ FT calibration solution with the following settings in the Define Scan dialog box. See Figure 3-1 on page 3-11.



After the automatic tune, a manual adjustment of the tube lens should be used to get an ion trap spectrum in the scan range 150–2000. At m/z 195 is the base peak (100%), and the highest Ultramark peaks are at about 30–50%, as shown in Figure 3-2 on page 3-12. The inject time should be less than 1 ms (if a Full MS target of 1e4 is used). Do not forget to save the tune method after a successful tuning.

Figure 3-1. Recommended settings in the Define Scan dialog box during an automatic tune of the ion trap



Tuning the Ion Trap forNegative Ion Mode

For the negative ion mode, it is recommended to perform an automatic tune of m/z 514 at a Full MS Target of 1e4–3e4. Use the LTQ FT calibration solution with the settings shown in Figure 3-1 on page 3-11.

After the automatic tune, a manual adjustment of the tube lens should be used to get an ion trap spectrum in the scan range 150–2000. At m/z 265 is the base peak (100%), and the highest Ultramark adduct ion peaks are at about 80%, as shown in Figure 3-3 on page 3-13.

If the tube lens is set to a very high negative voltage, in-source fragmentation of the Ultramark adduct ions may occur. Thus, if you observe rather ions at m/z 906, 1006, … than ions at m/z 1280, 1380, … the tube lens setting has to be decreased. The inject time should be less than 1 ms (if a target of 1e4 is used). Do not forget to save the tune method after a successful tuning.

Figure 3-2. Ion trap spectrum of the LTQ FT calibration solution, scan range 150–2000, positive ion polarity mode

#4532 IT: 0.254 ST: 0.50 uS: 3 NL: 5.18E5

F: ITMS + p ESI Full ms [ 150.00-2000.00]

200 400 600 800 1000 1200 1400 1600 1800 2000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

RelativeIntensity

195

1422

1522

1322 1622

17221222

524

1122 1822259

1022 1348663521 857 1183339 1922549 685345 1613 1639725 904 180214901226982 1960



Figure 3-3. Ion trap spectrum of the LTQ FT calibration solution, scan range 150–2000, negative ion mode

#3906 IT: 0.426 ST: 0.83 uS: 5 NL: 8.25E5

F: ITMS - p ESI Full ms [ 150.00-2000.00]

200 400 600 800 1000 1200 1400 1600 1800 2000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

RelativeIntensity

265.2

514.4

1579.6

1679.51479.7

1779.51379.7

1879.51279.8

1206.11106.11306.0

1006.11785.5

1885.51406.0

1179.8 1505.8906.1

596.4210.9 806.1 1079.9379.9 678.3470.1 994.1


Calibrating the LTQ FT Ultra for FT MeasurementsAutomatic Calibration Page

Automatic CalibrationPage

The Automatic page of the Calibrate dialog box (see Figure 3-4) allows you to perform an automatic calibration of all the calibration parameters including all ion trap calibrations and all FT calibrations.

In addition to the ion trap calibrations, the automatic calibration procedure calibrates the following:

• FT Multipole 1 and 2 RF Frequency

• FT Multipole 3 RF Frequency

• FT Ion Transfer

• FT Mass

Figure 3-4. Automatic page of the Calibrate dialog box


Calibrating the LTQ FT Ultra for FT MeasurementsAutomatic Calibration Page

In an automatic calibration, these four FT calibration procedures are performed automatically one after another subsequent to the ion trap calibration. The FT multipole RF calibration parameters are the same for all six calibration ranges and thus these calibrations are performed only once.

The FT ion transmission calibration and the FT mass calibration are performed for all six calibration ranges, i.e. fm > 50 (+), fm > 98 (+), fm > 190 (+), fm > 50 (-), fm > 98 (-), and fm > 190 (-).

To perform an automatic calibration, the LTQ FT calibration solution has to be used.

The calibration masses and all experimental parameters like target values, scan ranges, resolution settings, etc. are set automatically and cannot be influenced by the user.

The current mass calibration has to be good enough to ensure the identification of the calibration masses. If the mass calibration is off by more than 100 ppm, you have to perform a manual coarse two-point calibration, which can be performed on the Mass Calibration Page in the Diagnostics dialog box. (Refer to topic “Manual Coarse Two-Point Mass Calibration” on page 3-27 for further details.)

Note Usually you do not need to perform a complete ion trap calibration or an FT ion transmission calibration unless the hardware is modified in some way. However, it is necessary to repeat the electron multiplier calibration and the FT mass calibration on a regular basis. Thus, in the most cases it is not recommended to perform an automatic calibration of the LTQ FT Ultra since all calibrations are performed, which takes about 1 hour. To run only a multiplier gain calibration or an FT mass calibration (which takes only some minutes), it is recommended to use the semiautomatic calibration.


Calibrating the LTQ FT Ultra for FT MeasurementsSemi-Automatic Calibration Page

Semi-AutomaticCalibration Page

The Semi-Automatic page of the Calibrate dialog box (see Figure 3-5) allows you to select specific calibration parameters to calibrate, for example only the ion trap calibrations or only the FT calibrations. For FT calibrations, it is also possible to differentiate between positive and negative ion mode.

To calibrate one or more selected parameters, clear the Select All check box to make the individual calibration parameters available. Select the parameter(s) you want to calibrate, then click on Start.

Figure 3-5. Semi-Automatic page of the Calibrate dialog box


Calibrating the LTQ FT Ultra for FT MeasurementsSemi-Automatic Calibration Page

For example:

• To run a complete automatic calibration (ion trap and FT), tick the Select All check box. Then, click on Start. This is analogous to the automatic calibration. As already described before, it is not recommended to perform an automatic calibration of the LTQ FT Ultra if not really necessary since all calibrations are performed, which takes about 1 hour.

• To run an automatic calibration of the ion trap, tick the Select All - Ion Trap check box. Then, click on Start.

• To run an automatic calibration of the FT part, tick the Select All - FT check box. Then, click on Start.

• To run a FT mass calibration, tick the Mass Calibration check box. Then, click on Start.

In a semi-automatic calibration, the selected FT calibration procedure(s) are performed automatically one after another. If the FT ion transmission calibration and/or the FT mass calibration are selected for the positive ion mode, the calibrations are only performed for the calibration ranges fm > 50 (+), fm > 98 (+), and fm > 190 (+). If the FT ion transmission calibration and/or the FT mass calibration are selected for the negative ion mode, the calibrations are only performed for the calibration ranges fm > 50 (-), fm > 98 (-), and fm > 190 (-). If the calibrations are selected for both polarity modes, all six calibration ranges are calibrated.

All calibrations apart from the FT ion transmission and mass calibration for the negative ion mode can be performed with the LTQ ESI calibration solution or with the LTQ FT calibration solution. To run the FT ion transmission and/or mass calibration for the negative ion mode, the LTQ FT calibration solution has to be used.

The calibration masses and all experimental parameters like target values, scan ranges, resolution settings, inject waveforms etc. are set automatically and cannot be influenced by the user. Sole exception: the number of microscans used during a transmission calibration can be set by the user in the Define Scan dialog box (limited to three).

The current mass calibration has to be good enough to ensure the identification of the calibration masses. If the mass calibration is off by more than 100 ppm, you have to perform a manual coarse two-point calibration, which can be performed on the Mass Calibration Page in the Diagnostics dialog box. (Refer to topic “Manual Coarse Two-Point Mass Calibration” on page 3-27 for further details.)


Calibrating the LTQ FT Ultra for FT MeasurementsCheck Calibration Page

Check CalibrationPage

The Check page of the Calibrate dialog box allows you to check automatically several calibration settings. See Figure 3-6. All calibration checks apart from those for the FT negative ion mode can be performed with the LTQ FT calibration solution or the LTQ ESI calibration solution. To check the FT ion transmission and FT mass calibration in the negative ion mode, the LTQ FT calibration solution has to be used. The calibration masses and all experimental parameters like target values, scan ranges, resolution settings, etc. are set automatically.

At the conclusion of the check procedure, the ion trap displays a message that indicates whether the parameter(s) are calibrated properly or not.

Figure 3-6. Check page of the Calibrate dialog box


Calibrating the LTQ FT Ultra for FT MeasurementsCheck Calibration Page

Using the Check page of the Calibrate dialog box, you can select the following parameters:

Select All This check box allows you to specify whether or not to check all of the calibration parameters. To check all calibration parameters, select the Select All check box. In this case, all ion trap calibration parameters and all FT calibration parameters are checked. You can also check each calibration parameter individually. To make the individual calibration parameters available, clear the Select All check box.

Select All Ion Trap This check box allows you to specify whether or not to check the calibration of the linear ion trap parameters only.

Select All-FT This check box allows you to specify whether or not to check the calibration of the FT-ICR ion transfer optics and mass analyzer only.

Multipole 1 and 2 RF Frequency

This check box allows you to specify whether or not to check the frequency of the RF voltage of the first and second multipole in the FT transfer ion optics.

Multipole 3 RF Frequency

This check box allows you to specify whether or not to check the frequency of the RF voltage of the third multipole in the FT transfer ion optics.

Positive Ion Mode This check box allows you to specify whether or not to check the FT ion transmission calibration and FTmass calibration of the positive ion mode.

Negative Ion Mode This check box allows you to specify whether or not to check the FT ion transmission calibration and FTmass calibration of the negative ion mode.

Ion Transmission This check box allows you to specify whether or not to check the FT ion transmission calibration. The ion transmission from the ion trap to the FT-ICR cell is checked by means of the calibration masses in SIM experiments at different AGC target values. The FT ion transmission calibration can be checked for the positive and negative ion mode independently.

Mass Calibration This check box allows you to specify whether or not to check the mass calibration of the FT-ICR mass analyzer. In this check, the current mass calibration is checked, i.e it is a check of the external mass calibration. The FT mass calibration can be checked for the positive and negative ion mode independently.


Calibrating the LTQ FT Ultra for FT MeasurementsFT Manual Calibration Page

The Last Check Date readback column gives the date of the last successful check for each item. If a check is performed that fails, the last successful check date still appears in the Last Check Date readback column. The last successful check continues to be in effect in the instrument. However, the result column will show a red x mark indicating that the current attempt check has failed or was aborted.

FT Manual CalibrationPage

The FT Manual page of the Calibrate dialog box allows you to perform or to check an FT transmission calibration and/or an FT mass calibration with user-defined calibration masses. See Figure 3-7.

All experimental parameters like scan ranges, target values, resolution settings, etc. are set automatically during a manual FT calibration.

Note Only the three calibration ranges of the current ion polarity mode are calibrated during a manual calibration.

Figure 3-7. FT Manual page of the Calibrate dialog box


Calibrating the LTQ FT Ultra for FT MeasurementsFT Manual Calibration Page

Note To allow to use different reference masses for a manual FT transmission and the succeeding FT mass calibration, a manual FT transmission calibration is not automatically followed by an FT mass calibration. Nevertheless, it is implicitly necessary to perform a new FTmass calibration after a new transmission calibration.

Using the FT Manual calibration page, it is also possible to use calibration masses with m/z >2000. If you want to use calibration masses beyond m/z 2000, you have to select the High mass range on the Define Scan dialog box, which allows to use a scan range within the range 200–4000. (Refer to topic “Scan Mode Menu” on page 2-10 for further details.)

Note There are no separate calibration parameters for the mass range beyond m/z 2000. For example, the same calibration parameters are used for the scan ranges 190–1900, 300–3000, and 2000–4000, i.e. the parameters from the calibration range fm >190(+) or fm >190(-).

By means of the toggle “FT manual calibration in diagnostic mode”, the functionality of the FT Manual page can be changed for diagnostic purposes. Refer to “FT Manual Calibration in Diagnostic Mode” on page 4-9 for further details.

Mass List Group Box The calibration masses for the manual calibration can be defined in the corresponding mass list on the FT Manual page of the Calibrate dialog box. Mass lists can be imported and exported by means of the Instrument Configuration page, see further details in Chapter 6: “Instrument Configuration”.

Note Ensure that you use calibration masses of sufficient accuracy (sub ppm).

Name This list box lists the names of the factory supplied and user created mass lists. To delete a user created entry in the Name list, select the entry and click on Delete.

Mass List This table lists the mass-to-charge ratios of the ions that you are using to calibrate the FT-ICR mass analyzer. You can select an existing mass list in the Name list box, or you can create or modify a mass list by clicking on and editing the entries in the Mass List table.

Save Click on Save to save the mass list with the name that is selected in the Name list box.


Calibrating the LTQ FT Ultra for FT MeasurementsFT Advanced Calibration Page

Save As Click on Save As a to save the mass list with a new name.

Delete Click on Delete to delete the mass list that is selected in the Name list box.

Factory-Supplied Mass Lists There are also two factory supplied mass lists, calmix_positive (factory) and calmix_negative (factory). They contain the exact masses of all main ion peaks, which should appear if the LTQ FT calibration solution is used in positive or negative ion mode, respectively.

FT AdvancedCalibration Page

The FT Advanced calibration page is only displayed if the Advanced Calibration Features are activated in the Control menu. See Figure 3-8.

Figure 3-8. FT Advanced page of the Calibrate dialog box



The FT transmission and FT mass calibration parameters are determined in the corresponding calibration procedures on the Automatic page or Semi-Automatic page of the Calibrate dialog box. In addition, further calibration parameters are saved in the calibration file: the excitation amplitudes, the trapping voltages, and the DC offset voltages of the excite and detect electrodes. The optimum excitation amplitudes, trapping voltages, and DC offset voltages are set in the factory. They should not be changed unless the hardware is modified.

Caution It is not recommended to perform a new calibration of the excitation amplitudes, the trapping voltages, and the DC offset voltages unless the hardware is modified. The excitation amplitude, the trapping voltages, and the DC offset voltages are very critical parameters. An adverse excitation amplitude, trapping voltage, or DC offset voltage may harm the functionality of the LTQ FT Ultra. The FT mass calibration has to be performed after changing one of the advanced calibration parameters.

Note Before starting calibration procedures or performing manual changes on this page, it is mandatory to click on the Refresh button to ensure that the actual values are listed. Otherwise, false new values may be set accidentally.

What to calibrate Here, you can decide whether the excite amplitude, the trapping voltages, or the DC offset voltages are calibrated.

How to calibrate Here you can decide whether the calibration of the excite amplitude, the trapping voltages, or the DC offset voltages are performed automatically or manually.

Automatic If an automatic calibration is selected, all settings like scan range, target and resolution settings are set automatically. For an automatic calibration of the positive ion mode, the LTQ ESI calibration solution or the LTQ FT calibration solution can be used. For the automatic FT advanced calibrations of the negative ion mode, the LTQ FT calibration solution has to be used. Thus, for a complete automatic advanced calibration the LTQ FT calibration has to be used.

The excitation amplitude is independently set for all six calibration ranges, i.e. fm>190(+), fm>98(+), …, fm>50(-). A new calibration of the excite amplitude is only performed for those calibration ranges, which are checked in the Option field.



The trapping voltage is independently set for the positive and the negative ion mode. A new calibration of the trapping voltage is only performed for those polarity modes which are checked in the Option field.

The set of four DC offset voltages of the excite and detect electrodes (E1, E2,D1, and D2) are independently set for the positive and the negative ionmode. A new calibration of these offset voltages is only performed for thosepolarity modes which are checked in the Option field.

At the completion of the automatic calibration, the new calibration parameter(s) of all selected calibration ranges or polarity modes are entered into the corresponding row of the New Value column. To actually set the new values, press the Apply button. To save the new calibration parameter(s) in the calibration file, press the Save button.

Figure 3-9. Automatic FT Advanced calibration showing the automatic calibration of the trapping voltage for the positive ion mode



Manual If a manual calibration is selected, all settings like scan range, AGC target value, and resolution settings are set by the user. The wide scan range mode is switched off automatically. Only the advanced calibration parameters (excite amplitude, trapping voltage, set of four DC offset voltages) of the current calibration range or polarity mode are calibrated.

In addition, it is possible to use your own calibration masses. The calibration masses for the manual calibration can be defined in the corresponding mass list analogous to the mass lists on the FT Manual page of the Calibrate dialog box. (See topic “FT Manual Calibration Page” on page 3-20.) It is possible to import or export mass lists by means of the Instrument Configuration page, see Chapter 6: “Instrument Configuration” for further details.

Figure 3-10. Manual FT Advanced calibration, showing the manual calibration of the excite amplitude



At the completion of the manual calibration, the new calibration parameter of the selected calibration range or polarity mode is entered into the corresponding row of the New Value column. To actually set the new values, press the Apply button. To save the new calibration parameter(s) in the calibration file, press the Save button.

Refresh By means of the Refresh button, the displayed values in the column Current Value are updated to their actual values.

Apply By means of the Apply button, the values displayed in the column New Value are actually set. The new values can be the result suggested by an automatic or manual calibration or can be set manually. As long as the Save button is not pressed or no other automatic or semi-automatic calibration is successfully completed, the new values are not saved in the calibration file.

Save By means of the Save button, all current settings are saved in the calibration file.


Calibrating the LTQ FT Ultra for FT MeasurementsManual Coarse Two-Point Mass Calibration

Manual CoarseTwo-Point Mass

Calibration

A manual coarse two-point FT mass calibration is necessary if the current calibration is off by more than 100 ppm, since otherwise the normal calibration procedure cannot identify the correct peaks. Usually it is not necessary to perform a manual coarse calibration once the LTQ FT Ultra is mass calibrated during its installation. A miscalibration of more than 100 ppm should only occur if the hardware is modified in some way, especially if the magnet is recharged.

The procedure is analogous to the manual coarse mass calibration of the ion trap on the Mass Calibration Page of the Diagnostics dialog box. The manual coarse FT mass calibration becomes only effective for the current calibration range, which depends on the first mass currently set in the Define Scan dialog box and on the ion polarity mode. However, it is possible to carry over the new mass calibration parameters of this calibration range to the other five calibration ranges.

A manual coarse calibration can be performed with any sample solution. If the standard calibration solutions are used, it is recommended to use the default mass lists for FTMS, i.e. the exact masses 524.265 and 1421.978.

Figure 3-11. Manual coarse calibration page of the Diagnostics dialog box


Calibrating the LTQ FT Ultra for FT MeasurementsManual Coarse Two-Point Mass Calibration

Use the following procedure to perform a two-point FT mass calibration from the Tune Plus window:

1. Choose Diagnostics > Diagnostics to display the Diagnostics dialog box.

2. Click on Tools and select Mass calibration from the list. See Figure 3-11 on page 3-27.

3. Identify unequivocally two known peaks in the FT spectrum. To identify ion signals in a FT spectrum it may help to perform MS/MS experiments of known ions using no collision energy. Note that in an MS/MS experiment the first mass of the scan range is automatically calculated and may be changed to another calibration range. In this case you may have to increase the first mass manually to get into the wanted calibration range.

4. Enter both the expected masses and the observed masses using their accurate masses with two digits.

5. Afterwards, click on Execute corresponding to Calibrate current scan type (Execute button on the top).

6. If you want to carry over the new mass calibration parameters of the current calibration range to the other five calibration ranges, click on the Execute button corresponding to Estimate other calibration modes using current full scan (Execute button at the bottom).

Note After a manual coarse FT mass calibration, you have to perform a regular FT mass calibration (automatic or manual). This is necessary because the manual coarse mass calibration may be off by several ppm and is only performed for one of the six calibration ranges.

Caution Do not perform accidentally a two-point calibration of an FT spectrum if the ion trap is chosen as analyzer - or vice versa.


Chapter 4 Performing Diagnostics/Checks

This chapter describes several diagnostic procedures for the LTQ FT Ultra. It contains the following topics:

• “System Evaluation Procedures” on page 4-2

• “Toggles” on page 4-7

• “Set Device” on page 4-11

• “Display Settings” on page 4-14


Performing Diagnostics/ChecksSystem Evaluation Procedures

System EvaluationProcedures

The System Evaluation page in the Diagnostics dialog box allows you to evaluate system performance. See Figure 4-1.

Besides several ion trap relevant system evaluation procedures, you can perform the following FT system evaluation procedures:

• “FT Sensitivity Test” on page 4-3

• “FT Noise Test” on page 4-3

• “FT Isolation Test” on page 4-4

• “FT Dynamic Range Test” on page 4-4

• “FT Stability Test” on page 4-4

• “FT Transfer Optics Test” on page 4-5

• “FT High Mass Range Target Compensation” on page 4-6

If IRMPD is configured at your instrument, an additional system evaluation tool, i.e. the FT IRMPD efficiency test, is available. Refer to Chapter 8: “IRMPD” for further details.

Figure 4-1. System evaluation page of the Diagnostics dialog box



FT Sensitivity Test The FT sensitivity test is only applicable for an infusion experiment with Reserpine. The test assumes that a Reserpine solution of 5×10-9 M (100% methanol, 1% acetic acid) is used. The following test are performed one after another:

1. SIM of m/z 609.28 using the ion trap as analyzer and an AGC target of 2e+03.

2. SIM of m/z 609.28 using the FT cell as analyzer and an AGC target of 5e+03.

3. SIM of m/z 609.28 using the FT cell as analyzer and an AGC target of 5e+04.

4. MS/MS of m/z 609.28 using the FT cell and an AGC target of 5e+04.

The test fails

• if the inject time which is necessary to reach the selected AGC target value is too high,

• if the ratio of the reserpine signal to the overall signal inside the SIM window is too low,

• if the transmission from the ion trap to the FT cell is too low, or

• if the intensity of the product ions of reserpine is too low.

FT Noise Test This test determines resistant noise peaks in the selected scan range. In this test ions are “switched off ” automatically. At the conclusion of the FT noise test, a list of resistant noise peaks is displayed in the Testing text box sorted by both frequency and intensity.



FT Isolation Test This test is only applicable for an infusion experiment with a solution containing MRFA, for example the standard LTQ calibration solution or a MRFA alone solution (for example 5×10-6 M in 100% methanol/water, 1% acetic acid). This test is analogous to the “Check of the ion isolation waveform” on the Check page of the Calibrate dialog in which the isolation of m/z 525.3 is performed at a target of 2000 and analyzed by the ion trap. In contrast to this, the FT isolation test is performed at higher targets and uses the FT analyzer. Thus this test determines the maximum AGC target value that allows performing a unit isolation of m/z 525.3 at the presence of m/z 524.3 and 526.3.

FT Dynamic Range Test This test is only applicable for an infusion experiment with a solution containing MRFA, for example the standard LTQ calibration solution or a MRFA alone solution (for example 5×10-6 M in 100% methanol/water, 1% acetic acid). This test determines the signal-to-noise ratio of an isolated MRFA signal.

FT Stability Test This test is applicable for an infusion experiment with any sample solution. This test procedure checks the stability of the FT TIC (total ion current) detected in the selected scan range by means of 600 scans. In principle, the test can be performed at any experimental conditions but it is recommended

Figure 4-2. Progress of the FT noise test displayed in the Graph View

FT detect resident noise test (check 18.000000)

50 100 150 200 250 300 350 400 450 500 550 600 650 700 750

Frequency [kHz]

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

Intensit

y/sigma



to perform this test in Full scan mode using one microscan, a resolution setting of 50000 and a FT Full MS Target of 5e+05 or 1e+06. At the conclusion of the FT stability test, the AGC stability and the corresponding signal variation is displayed.

FT Transfer Optics Test This test is applicable for an infusion experiment with any sample solution. However, it is recommended to use the LTQ calibration solution with a high AGC target (1e6) and a low-resolution setting (25000). This test procedure checks whether all FT transfer optic electrodes are obviously connected. During this test, the FT multipole and intermultipole lens DC offsets and the multipole RF amplitudes are consecutively set to an absurd value. This should result in a significant drop of the signal in the FTmass spectrum. A missing signal drop should provide an indication that the corresponding FT transfer optic electrode is not connected correctly.

Note If no ions at all can be observed, this test cannot help for error diagnostics.

Figure 4-3. Result of the FT stability test displayed in the Graph View

FT stability test (600 scans)

50 100 150 200 250 300 350 400 450 500 550 600 650 700

Scan

0

5000000

10000000

15000000

20000000

25000000

30000000

35000000

40000000

45000000

50000000

55000000

60000000

65000000

70000000

75000000

80000000

85000000

90000000

95000000

TIC



FT High Mass RangeTarget Compensation

This procedure determines an AGC target compensation factor, which ensures that the FT mass calibration is still valid if the instrument is set into the high mass range mode. The resulting compensation factor will be saved in the calibration file. Usually, it is sufficient to run this procedure once. It is not necessary to repeat this procedure on a regular basis. It is recommended to use the LTQ calibration solution for this test. However, you can also use any other solution that gives reasonable ion signals at 1000 < m/z < 2000.


Performing Diagnostics/ChecksToggles

Toggles The Toggles page in the Diagnostics dialog box allows you to toggle [change] a subsystem from one state to another state. See Figure 4-4.

To display this page:

From the Tune Plus window, choose Diagnostics > Diagnostics > Tools > Toggles.

Caution All toggles should only be used for diagnostic purposes. The functionality of the LTQ FT Ultra may be harmed if a toggle is switched to a status which differs from its default value.

If one of the FT toggles is (accidentally) different from its default value during data acquisition, the FT Analyzer Settings of the Scan Header of a raw file contains a reference to this. See Appendix A: “Miscellaneous Information” for further details.

Note The status of a toggle is not saved in the tune method and is set back to its default value after an instrument reset.

Figure 4-4. Toggles page of the Diagnostics dialog box



FT Apodization With this toggle, the apodization can be switched on or off. The default setting is On.

FT Detect during Excite If the FT Detect During Excite toggle is switched on, the FT excitation and detection are performed simultaneously. Without any other action, the resulting FT mass spectra are displayed in the Spectrum view. The excite signal itself can be displayed (as a transient) in the Spectrum view by choosing Show FT Transient in the shortcut menu of the Spectrum view. The menu is displayed when you right-click anywhere on that page (see topic “Spectrum View” on page 2-3 for further details). Note that a transient view is only possible if profile (instead of centroid) is chosen as data format. If the excite amplifier and the FT cell plates are connected correctly, the excite signal should like shown in Figure 4-5. The default setting of the FT Detect During Excite toggle is Off.

Figure 4-5. Spectrum view with the “FT detect during excite” toggle switched on



FT Excite Amplifier This toggle can be used to switch on or off the excite amplifier. If the excite amplifier is switched on, the blue diagnostic LED of the excite amplifier board labeled RF-ON should be on (indicating that the RF Input is switched on). See the LTQ FT Ultra Hardware Manual for further details. The default setting is On.

FT Include Transients If this toggle is on, it is possible to display transients in the Spectrum view by choosing Show FT Transient in the shortcut menu of the Spectrum view. The menu is displayed when you right-click anywhere on that page. See topic “Spectrum View” on page 2-3 for further details. Note that a transient view is only possible if profile (instead of centroid) is chosen as data format. The default setting is Off.

Note It is not possible to acquire transients into an Xcalibur raw file.

FT Manual Calibration inDiagnostic Mode

If this toggle is on, the functionality of the FT Manual calibration page is changed. If the FT Manual page is set into its diagnostic mode, only one of the six calibration ranges is calibrated during a manual calibration. The LTQ FT Ultra only calibrates the range corresponding to the first mass and ion polarity that you specify. In addition, the scan range and target values used during the calibration can be chosen by the user. The default setting of this toggle is Off.

FT Multipole 1 and 2 RF This toggle can be used to switch on or off the multipole 1 and 2 RF. The default setting is On. If the FT multipole 1 RF is switched on, the corresponding blue diagnostic LED of the ion optic supply board should be on (indicating that the RF1 generator is switched on). See the LTQ FT Ultra Hardware Manual for further details.

FT Multipole 3 RF This toggle can be used to switch on or off the multipole 3 RF. The default setting is On. If the FT multipole 3 RF is switched on, the corresponding blue diagnostic LED of the ion optic supply board should be on (indicating that the RF2 generator is switched on). See the LTQ FT Ultra Hardware Manual for further details.

FT Profile Mode With this toggle you can select whether the FT profile mode corresponds to a Full Profile format or to a Reduced Profile format. It is recommended to use the Reduced Profile Mode for data acquisition since the data size of the raw file is significantly decreased by using the Reduced Profile. The default setting is Reduced. For further information, see also topic “Data Size of FT Raw Files” on page A-4.



FT SIM and MSn InjectionWaveforms

Usually, for FT SIM and FT MSn scans, the injection waveforms are automatically enabled. It is not possible to change this setting in the Injection Control dialog box. By means of this toggle, it is possible to disable or enable the injection waveforms manually for diagnostic purposes. The default setting is On.

FT View Frequency If this toggle is switched on, the FT spectrum is shown as a frequency spectrum. If the system is on and the FT is chosen as analyzer, the frequency spectrum is displayed in the Spectrum view. The default setting is Off.

Note that the x-coordinate is misleadingly labeled with m/z instead of kHz.

This toggle is for diagnostic purposes only. Therefore, it is not possible to acquire frequency spectra.

FT Zero Offset If this toggle is switched on an offset is added to the spectrum. This enables to view the full noise band. The default setting is Off, if the Reduced Profile format is used. The default setting is On, if the Full Profile format is used.


Performing Diagnostics/ChecksSet Device

Set Device The Set Device page of the Diagnostics dialog box (See Figure 4-6.) allows you to select devices or experimental parameters from the list and to set the value for that device or parameter. Do the following:

1. Select the device or parameter you would like to set from the Device list box.

2. Enter the device parameter's value in the text box below the Device list box.

3. Click on Set to apply the change to the device value or parameter.

Note The value in the text box below the Device list box, which is displayed after the call of this page, does not correspond to the actual value.

There are several FT relevant parameters, which may be changed from this page. If ECD is configured in the Instrument Configuration, additional parameter can be set on this page. Please refer to topic “Set Device Page

Figure 4-6. Set device page of the Diagnostics dialog box



with ECD Option” on page 7-20 for further details. Note that after an instrument reset the manual settings are overwritten again with the corresponding calibration parameters.

Caution Changing the instrument settings can harm the functionality of the LTQ FT Ultra, especially if followed by saving the calibration parameters (manually or at conclusion of a calibration procedure). Thus, this option should only be used by very advanced users.

Setting new FT TransferIon Optics Parameters

There are six different sets of FT transfer ion optics parameters, which are saved in the calibration file. An overview of these FT transfer ion optics parameters can be displayed by using the tool “Display FT calibration settings” on the Display settings page of the Diagnostics dialog box. The currently used FT transfer ion optics parameters, i.e. the corresponding parameters of the current calibration range, are displayed in the FT transfer ion optics dialog box.

Usually the FT transfer ion optics parameters are determined in the corresponding FT transmission calibrations of the calibration dialog box and should not be changed manually. Nevertheless, for diagnostic purposes it is possible to change the FT transfer ion optics parameters on this page. These parameters are as follows:

• FT Multipole 1 Offset

• FT Intermultipole Lens 1


• FT Intermultipole Lens 2


• FT Multipole 1 and 2 Amplitude

• FT Multipole 3 Amplitude

Note that only the FT transfer ion optics parameter of the current calibration range is changed. We recommend writing down the current value before changing the value on this page. Thus, you can go back to the original value.

If one of the FT transfer ion optics parameter is set but not saved into the calibration file (manually or automatically at the conclusion of a successful ion trap or FT calibration procedure), the old value of this parameter is used



again after a reset of the instrument. If you actually want to save the manually set value into the calibration file, please use the Save button on the FT Advanced page of the calibration dialog box.

Caution If one of the FT transfer ion optics parameters is changed, a new FT mass calibration of the corresponding calibration range has to be performed.

Note If you have changed an FT transfer ion optics setting and run a successful ion trap or FT calibration procedure afterwards, the new value will be saved automatically in the calibration file, too.

Setting a newFT Transfer Time

The transfer times are automatically calculated as a function of the scan range. Therefore, usually it does not make sense to change the transfer time manually. Nevertheless, for diagnostic purposes it is possible to change the transfer time manually on this page.

The current transfer time, i.e. the transfer time of the current scan range can be displayed by using the “Display FT instrument settings” tool on the Display settings page of the Diagnostics dialog box.

Note that only the transfer time of the current scan range is changed and that is only valid until the scan settings (scan range, resolution, etc.) are not changed. The transfer time is saved neither in the tune method nor in the calibration file.


Performing Diagnostics/ChecksDisplay Settings

Display Settings The Display Settings page in the Diagnostics dialog box allows you to select a variety of instrument settings for display. Do the following:

1. Select the instrument settings you want to display,

2. Click on Start,

3. The LTQ FT Ultra MS detector now displays the requested instrument settings in the Testing text box.

The following FT relevant instrument settings can be displayed:

• FT calibration settings

• FT diagnostics

• FT instrument settings

Figure 4-7. Display settings page of the Diagnostics dialog box


Performing Diagnostics/ChecksDisplay Settings

Display FT CalibrationSettings

Displays all FT relevant calibration parameters in the diagnostics text box.

Display FT Diagnostics Displays current diagnostic readback values of the FT instrument control board, the FT ion optic supply board, and the FT excite amplifier in the diagnostics text box.

Display FT InstrumentSettings

Displays the current values of those FT instrument settings

• that depend on the scan range and ion polarity mode, and

• that can be changed manually on the Set Device page of the Diagnostics dialog box or the FT Advanced page of the Calibrate dialog box.


Chapter 5 Data Dependent Settings

In this chapter, the FT relevant topics of the data dependent settings in the Instrument Setup are discussed. All ECD and/or IRMPD relevant topics of the data dependent settings are discussed in Chapter 7: “ECD” and/or Chapter 8: “IRMPD”.

This chapter contains the following topics:

• “Data Dependent FT Narrow SIM Scans” on page 5-2

• “Preview Mode” on page 5-3

• “Monoisotopic Precursor Selection” on page 5-4


Data Dependent SettingsData Dependent FT Narrow SIM Scans

Data Dependent FTNarrow SIM Scans

A data dependent FT narrow SIM scan is performed around the center mass determined in a previous reference scan event if the check box “Same MS order as referenced scan event” is selected on the Current Scan Event page of the Data Dependent Settings dialog box as shown in Figure 5-1.

The scan width of the data dependent FT narrow SIM scan can be selected on the Scan widths page of the Data Dependent Settings dialog box (Figure 5-2).

Figure 5-1. Data Dependent Settings dialog box: Current Scan Event page

Figure 5-2. Data Dependent Settings dialog box: Scan Widths page


Data Dependent SettingsPreview Mode

Preview Mode Figure 5-3 shows the Current Segment page of the Data Dependent Settings dialog box.

If the preview mode for FTMS master scans is enabled on the Current Segment page, the data dependent decision is made on the basis of the FTmaster scan with lower resolution to increase the duty cycle. The resolution of the FT master scan itself is not changed. Since usually the high resolution is not necessary to make the data dependent decision, it is recommended to enable the preview mode.

Figure 5-3. Data Dependent Settings dialog box: Current Segment page


Data Dependent SettingsMonoisotopic Precursor Selection

MonoisotopicPrecursor Selection

Figure 5-4 shows the Charge State page of the Data Dependent Settings dialog box.

If the monoisotopic precursor selection is enabled on the Charge State page, the data dependent scan is only performed for one molecular ion of the corresponding overall 13C isotopic distribution if Dynamic Exclusion is enabled.

Note This check is only available on the Charge State page if the Advanced features are turned on in the LTQ FT Ultra menu of the Instrument Setup.

Use Non-PeptideMonoisotopic Recognition

This check box is only available if monoisotopic precursor selection is active. If monoisotopic precursor selection is active and this box is not checked, precursor ions in FT master scans must match peptide-type isotopic distribution in order to identify the monoisotopic peak. If this box is checked, monoisotopic peaks will also be identified for small molecules and precursor ions with non-peptide-type isotopic distributions.

Figure 5-4. Data Dependent Settings dialog box: Charge State page with Advanced Features On


Chapter 6 Instrument Configuration

This chapter gives instructions about configuring your instrument. It contains the following topics:

• “Starting Instrument Configuration” on page 6-2

• “FT Settings Page” on page 6-3

• “FT Mass Lists Page” on page 6-5


Instrument ConfigurationStarting Instrument Configuration

Starting InstrumentConfiguration

From the Instrument Configuration dialog box, click on the LTQ FT MS button in the Configured Devices group box. See Figure 6-1. Then, click on the Configure button to open the LTQ FT Ultra Configuration dialog box.

The LTQ FT Ultra Configuration dialog box allows you to enter LTQ FT Ultra configuration information by using several pages, including the FT Settings page and the FT Manual Calibration page. The elements of the pages are described in the following topics.

Figure 6-1. Instrument Configuration dialog box


Instrument ConfigurationFT Settings Page

FT Settings Page The FT Settings page of the LTQ FT Ultra Configuration dialog box allows you to specify that you have an infrared multiphoton dissociation (IRMPD) source or an electron capture dissociation (ECD) source installed on your LTQ FT Ultra. See Figure 6-2. You can also turn on/off Active Noise Reduction and turn on/off Automatic Magnetic Field Drift Correction.

Using the FT Settings page of the LTQ FT Ultra Configuration dialog box, you can select the parameters described in the following topics.

Note Configuration changes will become effective when you reboot your instrument.

Active Noise Reduction Select this check box to turn on Active Noise Reduction. If the Active Noise Reduction is turned on, an advanced algorithm is used to decrease the statistic noise in an FT spectrum without increasing the noise threshold. If the Active Noise Reduction is turned on the data size of acquired raw-files is significantly decreased.

For further information, see also topic “Data Size of FT Raw Files” on page A-4.

Automatic Magnetic FieldDrift Correction

Select this check box to turn on the automatic Magnetic Field Drift Correction. If the automatic magnetic field drift correction is enabled, the LTQ FT Ultra automatically corrects the mass calibration parameter A depending on the time elapsed after the last mass calibration.

Figure 6-2. LTQ FT Ultra Configuration dialog box: FT Settings page


Instrument ConfigurationFT Settings Page

Note that even when the automatic drift correction is enabled here, the automatic correction is only active if enough mass calibrations with the standard calibration solution were performed over a longer period on a regular basis before. The information whether the automatic drift correction is applicable or not is given at the conclusion of FT mass calibrations.

IRMPD Configured Select this check box if you have an infrared multiphoton dissociation (IRMPD) source installed. This will activate the FT IRMPD dialog box in Tune Plus.

ECD Configured Select this check box if you have an electron capture dissociation (ECD) source installed. This will activate the FT ECD dialog box in Tune Plus.

ECD Target Gate Current This text box allows you to set the electron current that is used in ECD. The range is 0.001 to 1.000 mA. The default is 0.03 mA.


Instrument ConfigurationFT Mass Lists Page

FT Mass Lists Page The FT Mass Lists page of the LTQ FT Ultra Configuration dialog box allows you to manipulate the mass lists that are displayed in the FT Manual and FT Advanced Calibration page of the Calibration dialog box in Tune Plus. See Figure 6-3. You can also import or export a mass list as a text file.

Using the FT Mass Lists page of the LTQ FT Ultra Configuration dialog box, you can select the following parameters:

Name This list box lists the names of the factory supplied and user created mass lists.

Mass List This table lists the mass-to-charge ratios of the ions that you are using to calibrate the FT-ICR mass analyzer. You can select an existing mass list in the Name list box, or you can create or modify a mass list by clicking on and editing the entries in the Mass List table.

Note Ensure that you use calibration masses of sufficient accuracy (sub ppm).

Save Click on Save to save the mass list with the name that is selected in the Name list box.

Save As Click on Save As a to save the mass list with a new name.

Figure 6-3. LTQ FT Ultra Configuration dialog box: FT Mass Lists page


Instrument ConfigurationFT Mass Lists Page

Delete Click on Delete to delete the mass list that is selected in the Name list box.

Import Click on Import to import a mass list that is a text file.

Export Click on Export to export a mass list to a text file.

Show Factory Lists Select this check box if you want to show the factory calibration mass lists in Tune Plus.


Instrument ConfigurationFT TIC Correction Page

FT TIC CorrectionPage

If the TIC correction is enabled, the mass calibration parameter are not calculated depending on the AGC target value but depending on the actual total ion current (TIC) acquired by the FT analyzer. With the check boxes on this page (See Figure 6-4.) it is possible to enable or disable the TIC correction for different scan types independently. The default setting is off for all scan types.

It is not possible to enable the TIC correction for Narrow SIM scans.

FT Full MS Select this check box to turn on TIC correction for FT Full MS scans.

FT Wide SIM Select this check box to turn on TIC correction for FT Wide SIM scans.

FT Full MSn Select this check box to turn on TIC correction for FT Full MSn scans.

ECD Select this check box to turn on TIC correction for ECD scans.

IRMPD Select this check box to turn on TIC correction for IRMPD scans.

ECD & IRMPD Select this check box to turn on TIC correction for ECD&IRMPD scans.

Figure 6-4. LTQ FT Ultra Configuration dialog box: FT TIC Correction page


Chapter 7 ECD

This chapter describes the changes in the software when the ECD option is installed on your instrument. ECD is not a standard option of the LTQ FT Ultra. This chapter contains only the additional ECD relevant topics. Reading this chapter is only necessary if your instrument is equipped with the ECD option.

It is assumed that the previous chapters of this manual are read and understood.


• “Instrument Configuration for ECD” on page 7-2

• “ECD Status Monitor” on page 7-3

• “ECD Cathode Heating during Bakeout” on page 7-6

• “ECD Cathode Activation” on page 7-8

• “ECD Calibration and Tuning Solution” on page 7-9

• “FT ECD Calibration Page” on page 7-10

• “FT ECD Dialog Box” on page 7-12

• “Define Scan Dialog Box with ECD Option” on page 7-15

• “Tuning of ECD Energy and Exposure Time” on page 7-18

• “Scan Time Settings with ECD Option” on page 7-18

• “Injection Control Box with ECD Option” on page 7-19

• “Set Device Page with ECD Option” on page 7-20

• “Data Dependent Settings with ECD Option” on page 7-21


ECDInstrument Configuration for ECD

InstrumentConfiguration for ECD

If your instrument is equipped with ECD, you have to configure ECD in the LTQ FT Ultra Configuration dialog box to get access to all ECD relevant topics of Tune plus. See further details in Chapter 6: “Instrument Configuration”. It is recommended to use the default value of the target gate current which is 0.030 mA.

Figure 7-1. FT Settings page of the LTQ FT Ultra Configuration dialog box


ECDECD Status Monitor

ECD Status Monitor The ECD status monitor can be started on the Diagnostics/Tools/FT ECD page. See Figure 7-2. If the ECD status monitor is active the heater current, heater resistance, the vacuum quality and the gate current are displayed in the Graph view.

Figure 7-2. FT ECD page of the Diagnostics dialog box

Figure 7-3. ECD monitor results displayed in the Graph view

ECD Monitor (Ready)

0 500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500Time

[min]

Heater Resistance [Ohm]

Gate Current [10uA]

Heater Current [A]

Vacuum Quality

-1

0

1

2

3

4

5

6

7

8

9

10

11

12

13



Heater Current The heater current is given in Ampere (A). There is no direct readback of the heater current. The displayed value is the set point. The heater current in operating mode is typically around 1.0–1.1 A, the maximum value is 1.5 A. During bakeout or activation the heater current is within 1.0 and 1.5 A.

Heater Resistance The heater resistance is calculated by the formula

heater resistance [Ohm] = heater voltage [V] / heater current [A]

whereby the heater voltage is an actual readback and the heater current is the current set value. The heater resistance increases with increasing temperature of the ECD cathode, i.e. with increasing heater current. The typical heater resistance of a hot ECD cathode at operating temperature is around 4–6 Ohm. If the apparent value of the heater resistance is much higher than 5 Ohm, this is a sign that the heater is not contacted. The maximum value of the displayed heater resistance is given by the maximum heater voltage of 10 V. For example, at a heater current of 1 A the apparent heater resistance is 10 V / 1 A = 10 Ohm.

Vacuum Quality The vacuum quality displayed in the Graph View if the ECD monitor is active is given as the negative logarithm of the vacuum reading in mBar, i.e. the vacuum quality increases with decreasing pressure. For example, a pressure of 1E-08 mBar corresponds to a vacuum quality factor of -log(1E-08) = 8, and a pressure of 5E-09 mBar corresponds to a vacuum quality factor of 8.3 approximately. See Table 7-1 below for further examples.

Table 7-1. Relation between pressure and vacuum quality

Pressure [mBar] Vacuum quality

1.00E-06 6.0

5.00E-07 6.3

2.00E-07 6.7

1.00E-07 7.0

5.00E-08 7.3

2.00E-08 7.7

1.00E-08 8.0

5.00E-09 8.3

2.00E-09 8.7

1.00E-09 9.0



Gate Current The gate current is an actual readback value. For a gate current >0 it is necessary that the ECD cathode is emitting ions, and that the ECD gate has an attracting potential for electrons, i.e. more positive than the cathode. Note that the gate current depends on the fact whether the ECD unit is placed inside or outside the magnet. Thus, an activation and especially a ECD gate current calibration should always be performed when the stem is in the magnet. The target gate current can be set in the FT Settings page of the LTQ FT Configuration dialog box; its default is 30 µA. Note that the unit of the gate current displayed in the Graph View is 10 µA. The maximum of the gate current readback is 200 µA.

8.00E-10 9.1

6.00E-10 9.2

4.00E-10 9.4

2.00E-10 9.7

1.00E-10 10.0

Table 7-1. Relation between pressure and vacuum quality, continued

Pressure [mBar] Vacuum quality


ECDECD Cathode Heating during Bakeout

ECD Cathode Heatingduring Bakeout

During a system bakeout of an instrument equipped with an ECD cathode, it is necessary to keep the ECD cathode temperature at or above the tube temperature. Doing so protects the cathode from being poisoned by the surrounding structure or by the gas products during bakeout. The procedure ECD cathode heating during bakeout on the FT ECD page of the Diagnostics/Tools page ensures a controlled heating of the ECD cathode. During heating the cathode, the gate current and the vacuum quality are monitored. This procedure should be started at each system bakeout. Further details of a system bakeout are discussed in the LTQ FT Ultra Hardware Manual. Figure 7-4 shows a typical progress of the ECD cathode heating during bakeout procedure displayed in the Graph View.

In Figure 7-4 one can see the following stages during a 10 hour system bakeout:

Figure 7-4. ECD monitor parameters displayed in the Graph View during a bakeout

ECD Monitor (Plotting STOPPED)

0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900

Time [min]

Heater Resistance [Ohm] Gate Current [10uA] Heater Current [A] Vacuum Quality

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

9.0

minutes 5-85: pressure beyond 1E-06 mBar, ECD heater off.


ECDECD Cathode Heating during Bakeout

minutes 85-240: vacuum is good enough to set ECD heater on standby.

minutes 240-300: vacuum is good enough to increase heater current.minutes 300-600: ECD heater is constantly at ca. 1.2 A.after 600 minutes: instrument bakeout is stopped. ECD heater is kept

on 1.2 A. minutes 600-900: vacuum quality increases, gate current increases.


ECDECD Cathode Activation

ECD CathodeActivation

An activation of the ECD cathode is necessary if the system was vented meanwhile. Thus, after each venting the procedure ECD cathode activation has to be started on the FT ECD page of the Diagnostics/Tools page. See Figure 7-2 on page 7-3. During the activation the cathode is kept at very high temperature while the gate current and the vacuum quality are monitored. The activation of the cathode is achieved by converting the barium oxide in the tungsten matrix into free barium on the surface of the cathode. The rate of activation is a function of tube cleanliness, cathode poisoning, time, and temperature. All systems are different so there is no “standard” activation schedule. Cathode electron emission is the best indicator of activation. The activation procedure takes about 5–6 hours. The activation is automatically followed by a heater current calibration. Refer to “FT ECD Calibration Page” on page 7-10 for further details. Figure 7-5 shows a typical progress of the ECD cathode activation procedure displayed in the Graph View.

Since the gate current depends on whether the ECD unit is placed inside or outside the magnet, the ECD cathode activation has to be performed when the ECD unit is in the magnet.

Figure 7-5. ECD monitor parameters displayed in the Graph View during an ECD cathode activation

ECD Monitor (Plotting STOPPED)

50 100 150 200 250 300 350 400 450

Time [min]

Heater Resistance [Ohm] Gate Current [10uA] Heater Current [A] Vacuum Quality

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

9.0

9.5

10.0

10.5

11.0

11.5

12.0

12.5

13.0

13.5

14.0


ECDECD Calibration and Tuning Solution

ECD Calibration andTuning Solution

For the ECD calibrations and tuning procedures, a solution of Substance P dissolved in methanol/water with 0.1% formic acid at a concentration of 2 pmol/µL is used.

Note A vial of Substance P is shipped with the instrument. Refer to the Material Safety Data Sheet (MSDS) for proper handling of Substance P. To order more of this compound, contact Sigma-Aldrich. The product number of Substance P is S6883-##, for example S6883-1MG. Note that Substance P is not included in the standard LTQ API accessory kit.

Caution CAUTION. AVOID EXPOSURE TO POTENTIALLY HARMFUL MATERIALS.

Always wear protective gloves and safety glasses when you use solvents or corrosives. Also contain waste streams, and use proper ventilation. Refer to your supplier's Material Safety Data Sheet (MSDS) for proper handling of a particular solvent.

To prepare the ECD calibration solution, use the following procedure:

1. Stock Solution: Substance P

a. Obtain the vial of Substance P.

b. Dissolve 1 mg in 715 µL of 50:50 methanol:water with 0.1% formic acid.


d. Label the vial Substance P stock solution (1 nmol/µL) and store it in a freezer until it is needed.

2. ECD calibration solution (Substance P)

Prepare 10 mL of the ECD calibration solution as follows:

a. Pipet 10 mL of 50:50 methanol:water with 0.1% formic acid into a vial.

b. Add 20 µL of the Substance P stock solution.

c. Label the vial ECD calibration solution (Substance P, 2 pmol/µL) and store it in a refrigerator until it is needed.


ECDFT ECD Calibration Page

FT ECD CalibrationPage

Figure 7-6 shows the FT ECD page of the Calibrate dialog box.

Calibrate Heater Current The purpose of the ECD heater current calibration is to determine the heater current, which is necessary to get a suitable emission of electrons. This calibration can be performed when the instrument is On or in Standby since no ions are needed for this calibration. The emission is controlled by the gate current, which is the current measured on a grid placed in front of the cathode. The target gate current is usually 0.030 mA and can be changed on the FT Settings page of the LTQ FT Configuration dialog box.

A heater current calibration is automatically performed at the conclusion of an ECD cathode activation procedure. (See topic “ECD Cathode Activation” on page 7-8 for further details.) During operation the heater current is automatically corrected if the gate current deviates from the target gate current. Thus, it is usually not necessary to repeat the heater current calibration.

Figure 7-6. FT ECD page of the Calibrate dialog box


ECDFT ECD Calibration Page

Since the gate current depends on whether the ECD unit is placed inside or outside the magnet, the ECD heater current calibration has to be performed when the ECD unit is in the magnet.

Calibrate Potentials andTiming

The purpose of this calibration is to optimize the ECD efficiency and to determine an energy offset, which ensures that the optimum ECD energy value is 5.0. For this calibration, the ECD/IRMPD calibration solution, i.e. a Substance P solution, has to be used. Note that during the ECD potentials and timing calibration, the ECD duration time is used. This is set in the Define Scan dialog box; the default is 70 ms.

Check Heater Current The ECD heater current check verifies that the heater current can produce a suitable emission of electrons. No ions are needed to perform the heater current check. The gate current depends on whether the ECD unit is placed inside or outside the magnet. Therefore, the ECD heater current check has to be performed when the ECD unit is in the magnet.

Check Potentials andTiming

The potentials and timing check tests the ECD efficiency at the current ECD calibration parameters. During this check, that ECD duration time is used which is set in the Define Scan dialog box. The default ECD duration time is 70 ms. For this check, the ECD/IRMPD calibration solution (Substance P solution) must be used.


ECDFT ECD Dialog Box

FT ECD Dialog Box The look of the toolbar button of the FT ECD dialog box depends on the current ECD status, as shown in Figure 7-7.

Figure 7-7. Toolbar button of the FT ECD dialog box for ECD heater, a: off, b: standby, c: heating or not ready, and d: ready

Off The heater current is at 0 A, i.e. the ECD cathode is at room temperature. Apart from a planned venting it is usually not recommended to switch the ECD heater off. However, if you plan to vent the system, switch off the ECD heater at least 30 minutes before venting.

Standby The heater current is at 0.4 A, i.e the ECD cathode is hot but not emitting electrons. If the ECD option will not be used for a longer period, i.e. for several weeks, it is recommended to set the ECD heater into standby mode.

Heating If the ECD heater is switched on and was off or in standby mode before, it requires several minutes to reach the operating temperature of the ECD cathode. If the ECD heater was in standby before the warming up usually takes about 15 minutes.

Not ready The instrument is not ready, if one of the following conditions is active:

• ECD cathode heating procedure during bakeout,

• ECD cathode activation, or

• ECD calibration.Ready The heater current is set to that value that provides

sufficient electron emission of the cathode for ECD experiments. Usually this current is between 0.9 and 1.2 A.

a b c d



The FT ECD dialog box displays set points and readbacks of the most important ECD relevant parameters, i.e. the set value of the heater current, the heater resistance, the target gate current, and the actual gate current. See Figure 7-8. For further details about the heater current, heater resistance, and gate current, please refer to topic “ECD Status Monitor” on page 7-3.

In addition, the FT ECD dialog box shows the current status of the ECD heater. Table 7-2 shows the heater current in dependence on the ECD status.

If you use the ECD option on a regular basis, the ECD status should be On although the instrument is placed into Standby, because in this case the ECD is always ready. Otherwise, if the ECD heater starts from the standby current (0.4 A) or even from 0 A, you have to wait 15 minutes or longer before you are able to acquire ECD spectra.

However, if the ECD option will not be used for a longer period (i.e. for several days), it is recommended to set the ECD heater into standby mode.

Figure 7-8. FT ECD dialog box

Table 7-2. ECD heater current in dependence on ECD status

ECD STATUS HEATER CURRENT

On 0.9–1.2 A (calibration parameter)

Standby 0.4 A

Off 0 A



If the instrument is switched from On to Standby or vice versa using the On/Standby button of the Tune plus window, the ECD status is not changed. If the instrument is switched Off by means of the Control menu, the ECD heater is switched Off. If the instrument was switched Off and is placed into Standby or is switched On, the ECD heater remains Off until it is placed into Standby or On manually.

The ECD heater should only be switched On if the ECD cathode was activated before (at least once after the last venting). If the vacuum is not good enough, the ECD heater is placed into Standby automatically.

Caution If a venting of the LTQ FT Ultra is planned, it is necessary to switch Off the ECD heater at least 30 minutes before venting to ensure the ECD cathode is cold during venting. Otherwise, the ECD cathode may be damaged!

To switch off the ECD heater, use the FT ECD dialog box or turn off the MS detector in the Control menu. (Or switch off the FT electronics. See the LTQ FT Ultra Hardware Manual for further details.)

Before venting, you should also check whether the nitrogen venting kit is mounted correctly and whether enough nitrogen gas is available. Please refer to the LTQ FT Ultra Hardware Manual for further details.


ECDDefine Scan Dialog Box with ECD Option

Define Scan DialogBox with ECD Option

If ECD is configured in the LTQ FT Ultra Configuration dialog box and FTMS is chosen as analyzer, an extra grid is displayed on the Define Scan dialog box. See Figure 7-9.

If at least one parent mass is defined in the MSn Settings, you can set the following ECD relevant parameters:

Figure 7-9. Define Scan dialog box with ECD option

Active This check box allows you to specify whether to perform an ECD experiment of the last MSn experiment instead of a CID experiment. If ECD is activated here, the fields Normalized Collision Energy, Activation Q, and Activation Time of the last MSn setting are grayed out and are not effective any longer. The selected parent mass and isolation width of the last stage are now used for the ECD experiment.

Energy The Energy column allows you to select the electron energy (in arbitrary units). The valid range is 0.0 to 100.0. The default energy value is 5.0. The ECD potential and timing calibration ensures that this default energy value is the optimum value in terms of ECD efficiency for doubly protonated Substance P.



It is possible to perform MSn experiments combining CID and ECD in succession. Note that ECD is always the last MSn stage in such experiments. As example, Figure 7-10 on page 7-17 shows an experimental setup corresponding to an MS3 experiment. In this experiment, the parent mass 674 is fragmentated by CID at a normalized collision energy of 35%, followed by an ECD experiment of the product ion 455.

Energy(cont.)

The actual nominal potential of the ECD cathode can be calculated by means of the ECD energy offset which is determined in the ECD potentials and timing calibration. The current value of the ECD offset is one of the calibration parameters which can be displayed by choosing Diagnostics > Tools > Display Settings > Display Calibration Settings. The ECD offset corresponds to the nominal cathode potential at an ECD energy of 0.

For example, if the ECD offset is 3.5, an ECD energy of 5.0 corresponds to a cathode voltage of

(3.5–5.0) V = -1.5 V,

which corresponds roughly to an ECD electron energy of about 1.5 eV.

If the ECD offset is 2.6, an ECD energy of 9.5 corresponds to a cathode voltage of

(2.6–9.5) V = -6.9 V,

which corresponds roughly to an ECD electron energy of about 6.9 eV.

Delay The Delay column allows you to select the delay (in ms) between trapping the ions and electron exposure. The default delay value is 0 ms. The ECD potential and timing calibration ensures that a delay value of 0 ms is the optimum value in terms of ECD efficiency. If both ECD and IRMPD are available and configured at your instrument, you can use this delay setting to arrange the timing in an experiment in which both ECD and IRMPD are activated.



Scan Range of ECD Scans If ECD is set as last event in the Define Scan dialog box, the scan range can be chosen within 50–4000. Nevertheless, if the first mass is below m/z 98, ions beyond m/z 2500 are not observable.

Figure 7-10. Define Scan dialog box, showing a setup for a CID experiment followed by an ECD experiment


ECDTuning of ECD Energy and Exposure Time

Tuning of ECD Energyand Exposure Time

A tuning of the ECD energy and duration can be performed by means of the Scan Activation Parameters dialog box. See Figure 7-11. You get access to this page by clicking on the Activation button on the Define Scan dialog box.

The Scan Activation Parameters dialog box allows you to perform experiments in which you investigate the effects of variations in the ECD exposure time or electron energy on the formation of product ions. Note that the optimum exposure time or electron energy is not saved anywhere.

Scan Time Settingswith ECD Option

If ECD is configured at your instrument, the Scan Time Settings dialog box displays the current settings of the number of microscans and the maximum inject time for FT ECD experiments independently from the FT MSn (CID) target value. See Figure 7-12. It also allows changing these settings.

Figure 7-11. Scan Activation Parameters dialog box with ECD option


ECDInjection Control Box with ECD Option

Injection Control Boxwith ECD Option

If ECD is configured at your instrument, the FT tab of the Injection Control dialog box allows you to set the AGC target value for ECD experiments independently from the FT MSn (CID/PQD) target value. See Figure 7-13. It is recommended to use an ECD target value of 2.0E+05.

Figure 7-12. Scan Time Settings dialog box: FT tab with ECD option

Figure 7-13. Injection Control dialog box: FT tab with ECD option


ECDSet Device Page with ECD Option

Set Device Page withECD Option

If ECD is configured in the LTQ FT Configuration dialog box, the Set Device Page of the Diagnostics dialog box allows you to select several ECD devices and parameters from the list and to set the value for that device or parameter. See Figure 7-14.

Note If you have changed an ECD parameter on the Set Device page and run a successful ion trap or FT calibration procedure afterwards, the new value will be saved automatically in the calibration file.

Caution Changing the ECD settings, especially if followed by saving the calibration parameters (manually or at conclusion of a calibration procedure) can harm the ECD performance. Thus, this option should only be used by very advanced users.

Figure 7-14. Set device page of the Diagnostics dialog box with ECD option


ECDData Dependent Settings with ECD Option

Data DependentSettings with ECD

Option

This section contains the following topics:

• “Activate ECD for Data Dependent Scans” on page 7-21

• “ECD Settings” on page 7-21

• “Charge State Dependent ECD Time” on page 7-22

Activate ECD for DataDependent Scans

If FT is chosen as analyzer for the dependent scan, an ECD check box is displayed on the Activation page of the Data Dependent Settings dialog box. See Figure 7-15. If ECD is activated here, an ECD scan event is performed instead of CID or PQD. If ECD is activated, the CID/PQD relevant settings for activation type, normalized collision energy, activation Q, and activation time are disabled, whereas the default charge state and the isolation width can be chosen on this page.

ECD Settings The FT ECD/IRMPD page of the Data Dependent Settings dialog box allows you to specify the ECD energy, ECD delay and ECD duration for the current scan event. See Figure 7-16. On this page, you can also select whether the first mass of the ECD dependent scan is fixed to a specific

Figure 7-15. Activation page of the Data Dependent Settings dialog box with ECD option



m/z value or chosen relative to the parent mass. The last mass of the ECD dependent scan is calculated automatically, taking into account the charge state of the precursor ion. Regarding the first mass selection, one should note that ions beyond m/z 2500 are not observable, if the first mass is below m/z 98.

Charge State DependentECD Time

If the check box Enable charge state dependent ECD time is activated on the Charge State page of the Data Dependent Settings dialog box (See Figure 7-17 on page 7-23), the ECD duration is set in dependence on the charge state of the precursor, i.e. the duration is decreased with increasing charge according to the equation:

where the specified ECD duration is the ECD duration that is selected on the FT ECD/ IRMPD page of the Data Dependent Settings dialog box. The actual ECD duration used in an ECD scan can be seen in the FT Analyzer Settings of the corresponding raw file scan header, refer to “FT Analyzer Settings” on page A-2.

Figure 7-16. FT ECD/IRMPD page of the Data Dependent Settings dialog box with ECD option

(ECD duration) 4 (specified ECD duration) (charge state)2⁄×=



Note The check box Enable charge state dependent ECD time is only available on the Charge State page if the Advanced Features are turned on in the LTQ FT Ultra menu of the Instrument Setup.

Figure 7-17. Charge State page of the Data Dependent Settings dialog box with ECD option and Advanced Features On


Chapter 8 IRMPD

This chapter describes the changes in the software when the IRMPD option is installed on your instrument. IRMPD is not a standard option of the LTQ FT Ultra. This chapter contains only the additional IRMPD relevant topics. Reading this chapter is only necessary if your instrument is equipped with the IRMPD option.

It is assumed that the previous chapters of this manual are read and understood. If your instrument is equipped with the IRMPD option, read Appendix B: “Safety Information for Optional Hardware” before you start working.


• “Instrument Configuration for IRMPD Option” on page 8-2

• “IRMPD Tuning Solution” on page 8-3

• “FT IRMPD Dialog Box” on page 8-4

• “Define Scan Dialog Box with IRMPD Option” on page 8-4

• “Tuning of IRMPD Energy and Duration” on page 8-7

• “Scan Time Settings with IRMPD Option” on page 8-7

• “Injection Control Dialog Box with IRMPD Option” on page 8-8

• “IRMPD Efficiency Test” on page 8-9

• “Data Dependent Settings with IRMPD Option” on page 8-10

• “ECD and IRMPD” on page 8-12


IRMPDInstrument Configuration for IRMPD Option

InstrumentConfiguration for

IRMPD Option

If your instrument is equipped with IRMPD, you have to configure IRMPD in the LTQ FT Ultra Configuration dialog box to get access to all IRMPD relevant topics of Tune plus. See further details in Chapter 6: “Instrument Configuration”.



IRMPDIRMPD Tuning Solution

IRMPD TuningSolution

For the IRMPD tuning procedures and the IRMPD efficiency test, a solution of Substance P dissolved in methanol/water with 0.1% formic acid at a concentration of 5 pmol/µL is used. It is the same solution that is used as ECD calibration and tuning solution. Thus it is not necessary to prepare both an ECD and an IRMPD tuning solution.

Note A vial of Substance P is added to the LTQ accessory kit shipped with the instrument. Refer to the Material Safety Data Sheet (MSDS) for proper handling of Substance P. To order more of this compound, contact Sigma-Aldrich. The product number of Substance P is S6883-##, for example S6883-1MG. Note that Substance P is not included in the standard LTQ API accessory kit.

To prepare the IRMPD tuning solution, use the following procedure:

1. Stock Solution: Substance P

a. Obtain the vial of Substance P in your accessory kit.

b. Prepare the stock solution of Substance P by dissolving 1 mg in 715 µL of 50:50 methanol:water with 0.1% formic acid.


d. Label the vial Substance P stock solution (1 nmol/µL) and store it in a freezer until it is needed.

2. IRMPD tuning solution (Substance P)

Prepare 10 mL of the IRMPD tuning solution, as follows:

a. Pipet 10 mL of 50:50 methanol:water with 0.1% formic acid into a vial.

b. Add 20 µL of the Substance P stock solution.

c. Label the vial IRMPD tuning solution (Substance P, 2 pmol/µL) and store it in a refrigerator until it is needed.

Warning AVOID EXPOSURE TO POTENTIALLY HARMFUL MATERIALS.

Warning Always wear protective gloves and safety glasses when you use solvents or corrosives. Also contain waste streams, and use proper ventilation. Refer to your supplier's Material Safety Data Sheet (MSDS) for proper handling of a particular solvent.


IRMPDFT IRMPD Dialog Box

FT IRMPD Dialog Box The FT IRMPD dialog box shows the status of the IRMPD option. See Figure 8-2.

Define Scan DialogBox with IRMPD

Option

If IRMPD is configured in the LTQ FT Ultra Configuration dialog box, an extra grid is displayed on the Define Scan dialog box. See Figure 8-3.

Figure 8-2. FT IRMPD dialog box

Figure 8-3. Define Scan dialog box with IRMPD option


IRMPDDefine Scan Dialog Box with IRMPD Option

If at least one parent mass is defined in the MSn Settings, the following IRMPD relevant parameters can be set:

It is possible to perform MSn experiments combining CID and IRMPD. Note that IRMPD is always the last MSn stage in such experiments. As example, Figure 8-4 on page 8-6 shows an experimental setup corresponding to an MS3 experiment. In this experiment, the parent mass 674 is fragmentized by CID at a normalized collision energy of 35%, followed by an IRMPD experiment of the product ion 624.

Active This check box allows you to specify whether to perform an IRMPD experiment of the last MSn experiment instead of a CID experiment. If IRMPD is activated here, the fields Normalized Collision Energy, Activation Q, and Activation Time of the last MSn setting are grayed out and are not effective any longer. The selected parent mass and isolation width of the last stage are now used for the IRMPD experiment.

Energy The Energy column allows you to select the relative laser intensity. The valid range is 0.0 to 100.0. The default energy value is 60.

Delay The Delay column allows you to select the delay (in ms) between trapping the ions and firing the IR laser. The default delay value is 0 ms. If both ECD and IRMPD are available and configured at your instrument, you can use this delay setting to arrange the timing in an experiment in which both ECD and IRMPD are activated.

Duration The Duration column allows you to select the IRMPD duration (in ms). The default value is 50 ms.


IRMPDDefine Scan Dialog Box with IRMPD Option

Scan Range of IRMPDScans

If IRMPD is set as last event in the Define Scan dialog box, the scan range can be chosen within 50–4000. Nevertheless, if the first mass is below m/z 98, ions beyond m/z 2500 are not observable.

Figure 8-4. Define Scan dialog box, showing a setup for a CID experiment followed by an IRMPD experiment


IRMPDTuning of IRMPD Energy and Duration

Tuning of IRMPDEnergy and Duration

A tuning of the IRMPD energy and duration can be performed by means of the Scan Activation Parameters dialog box. See Figure 8-5. You get access to this page by clicking on the Activation button on the Define Scan dialog box.

The Scan Activation Parameters dialog box allows you to perform experiments in which you investigate the effects of variations in the IRMPD exposure time or laser intensity on the formation of product ions.

Scan Time Settingswith IRMPD Option

If IRMPD is configured at your instrument, the Scan Time Settings dialog box allows changing the current settings of the number of microscans and the maximum inject time for FT IRMPD experiments independently from the FT MSn (CID) target value. See Figure 8-6.

Figure 8-5. Scan Activation Parameters dialog box with IRMPD option

Figure 8-6. Scan Time Settings dialog box: FT tab with IRMPD option


IRMPDInjection Control Dialog Box with IRMPD Option

Injection ControlDialog Box with

IRMPD Option

If IRMPD is configured at your instrument, the FT tab of the Injection Control dialog box allows you to set the AGC target value for IRMPD experiments independently from the FT MSn (CID/PQD) target value. See Figure 8-7. It is recommended to use an IRMPD target value of 2.0E+05.

Figure 8-7. Injection Control dialog box: FT tab with IRMPD option


IRMPDIRMPD Efficiency Test

IRMPD Efficiency Test By means of the system evaluation procedure FT IRMPD efficiency test (under Diagnostics > Tools), it is possible to determine the fragmentation efficiency of IRMPD at the current settings of the instrument. This efficiency test is only applicable for an infusion experiment with the IRMPD tuning solution.

The IRMPD efficiency test determines two properties of the IRMPD option. As standard for comparing intensities, the software uses the doubly protonated species of Substance P ([M+2H]2+, m/z 674.4).

Fragmentation Efficiencyat the current Settings

An IRMPD experiment is performed with the settings as entered in the Define Scan dialog box. In the resulting spectrum, the software searches for a number of predefined product ions. It compares their intensities with the intensity of the precursor ion. The resulting efficiency should be larger than 8%.

Pulse Duration requiredfor Halving the Intensity of

the Precursor Ion

With the laser energy set to 100%, the instrument performs a number of IRMPD experiments. After each experiment, the software measures the remaining intensity of the precursor ion. In the following experiment, the duration of the laser pulse is increased. The test is finished when the remaining intensity falls below 50%. The pulse duration of the last experiment should be less than 150 ms.


IRMPDData Dependent Settings with IRMPD Option

Data DependentSettings with IRMPD

Option

This section contains the following topics:

• “Activate IRMPD for Data Dependent Scans” on page 8-10

• “IRMPD Settings” on page 8-10

Activate IRMPD for DataDependent Scans

If FT is chosen as analyzer for the dependent scan, an IRMPD check box is displayed on the Activation page of the Data Dependent Settings dialog box. See Figure 8-8. If IRMPD is activated here, an IRMPD scan event is performed instead of CID or PQD. If IRMPD is activated, the CID/PQD relevant settings for activation type, normalized collision energy, activation Q, and activation time are disabled, whereas the default charge state and the isolation width can be chosen on this page.

IRMPD Settings The FT ECD/IRMPD page of the Data Dependent Settings dialog box allows you to specify the IRMPD energy, IRMPD delay and IRMPD duration for the current scan event. See Figure 8-9. On this page, you can also select whether the first mass of the IRMPD dependent scan is fixed to a specific m/z value or chosen relative to the parent mass. The last mass of the IRMPD dependent scan is calculated automatically, taking into

Figure 8-8. Activation page of the Data Dependent Settings dialog box with IRMPD option


IRMPDData Dependent Settings with IRMPD Option

account the charge state of the precursor ion. Regarding the first mass selection, one should note that ions beyond m/z 2500 are not observable, if the first mass is below m/z 98.

Figure 8-9. FT ECD/IRMPD page of the Data Dependent Settings dialog box with IRMPD option


IRMPDECD and IRMPD

ECD and IRMPD All topics mentioned in Chapter 7: “ECD” and this chapter are also valid if both ECD and IRMPD are configured. In addition, the items described in the following topics have to be taken into account.

Instrument Configurationfor ECD and IRMPD

If your instrument is equipped with both ECD and IRMPD, you have to configure ECD and IRMPD in the LTQ FT Ultra Configuration dialog box to get access to all ECD and IRMPD relevant topics of Tune plus. See Figure 8-10. See further details in Chapter 6: “Instrument Configuration”. For ECD it is recommended to use the default value of the target gate current, which is 0.030 mA.

Define Scan Dialog Boxwith ECD and IRMPD

Option

If ECD and IRMPD are configured in the LTQ FT Ultra Configuration dialog box, an extra grid is displayed on the Define Scan dialog box. If at least one parent mass is defined in the MSn Settings, both the ECD and the IRMPD relevant parameters can be set independently, i.e. it is possible to perform an experiment in which both ECD and IRMPD are active. By means of the Delay column, it is possible to choose whether the ions are exposured with electrons (ECD) or IR radiation (IRMPD) simultaneously, delayed, or consecutively.

Figure 8-11 on page 8-13 shows an example in which an IRMPD experiment with a duration of 70 ms and an ECD experiment with a duration of 120 ms are performed consecutively with a delay of 20 ms between these two experiments.



IRMPDECD and IRMPD

Scan Time Settings withECD and IRMPD Option

If both ECD and IRMPD are configured at your instrument, the Scan Time Settings dialog box displays and allows changing the current settings of the number of microscans and the maximum inject time for experiments in which both ECD and IRMPD are activated. See Figure 8-12.

Figure 8-11. Define Scan dialog box with ECD and IRMPD option

Figure 8-12. Scan Time Settings dialog box: FT tab with ECD and IRMPD option


IRMPDECD and IRMPD

Injection Control DialogBox with ECD and IRMPD

Option

If ECD and IRMPD are configured at your instrument, the FT tab of the Injection Control dialog box allows you to set the AGC target value for experiments in which both ECD and IRMPD experiments are activated. See Figure 8-13. It is recommended to use an ECD & IRMPD target value of 2.0E+05.

Data Dependent Settingswith ECD and IRMPD

Option

If FT is chosen as analyzer for the dependent scan, an ECD check box as well as an IRMPD check box are displayed on the Activation page of the Data Dependent Settings dialog box. See Figure 8-14 on page 8-15. If ECD and/or IRMPD are activated here, an ECD scan event and/or an IRMPD scan event are performed instead of CID or PQD. If ECD and/or IRMPD are activated, the CID/PQD relevant settings for activation type, normalized collision energy, activation Q, and activation time are disabled, whereas the default charge state and the isolation width can be chosen on this page.

Figure 8-13. Injection Control dialog box: FT tab with ECD and IRMPD option


IRMPDECD and IRMPD

The FT ECD/IRMPD page of the Data Dependent Settings dialog box allows you to specify the energy, delay and duration for both ECD and IRMPD for the current scan event. See Figure 8-15 on page 8-16. On this page you can also select whether the first mass of the ECD and/or IRMPD dependent scan is fixed to a specific m/z value or chosen relative to the parent mass. The last mass of the ECD and/or IRMPD dependent scan is calculated automatically, taking into account the charge state of the precursor ion. Regarding the first mass selection, one should note that ions beyond m/z 2500 are not observable, if the first mass is below m/z 98.

Figure 8-14. Activation page of the Data Dependent Settings dialog box with ECD and IRMPD option


IRMPDECD and IRMPD

Figure 8-15. FT ECD/IRMPD page of the Data Dependent Settings dialog box with ECD and IRMPD option


Appendix A Miscellaneous Information

This appendix contains supplemental information for the previous chapters. It contains the following topics:

• “FT Analyzer Information in Scan Header” on page A-2

• “Data Size of FT Raw Files” on page A-4

Thermo Electron Corporation LTQ FT Ultra Getting Started A-1

Miscellaneous InformationFT Analyzer Information in Scan Header

FT AnalyzerInformation in Scan

Header

The Qual Browser window allows you to open a raw file and to display scan header information for a selected scan in any of the cells. Choose View > Scan Header to display the Scan Header of the current scan in the active cell.

FT Analyzer Settings The scan header information of an FT scan includes information about the FT Analyzer Settings, which is not available in the usual reports (Tune method, Instrument method, Status log, or Error log):

T=1e5 AGC Target for this scan (here: 1e+05)PsIT=0.65 Prescan inject time (here: 0.65 ms)Tog=(…) Manual diagnostic toggles are set different from

their default values. See Table A-1 below for detailed information.

iWf Inject waveform on for this scanWsr=3 Wide scan range mode activated with two or more

(here: 3) sections for this analytical scan.Mtof=0.80 TOF time set manually via Diagnostics (here: 0.80)PvR=3e4 Preview analysis active for this scan ECDnotREADY ECD scan activated while ECD heater not readyECD=15ms Actual ECD exposure time (here: 15 ms)IRMPD=30ms Actual IRMPD exposure time (here: 30 ms)NSR Active noise reduction is activatedDiagManualSettings Calibration parameters were manually changed

under Diagnostics.

Table A-1. Actual settings of manual toggles

Tog = (…) Relevant Toggle Current setting Default setting

ApoOff FT apodization Off On

DetEx FT detect during excite On Off

ExOff FT excite amplifier Off On

TrExp FT include transient On Off

Rf1off FT multipole 1 and 2 RF Off On

Rf2off FT multipole 3 RF Off On

FullP FT profile mode Full Reduced

IWFoff FT SIM and MSn injection waveforms

Off On

TICcorr FT TIC correction On Off

A-2 LTQ FT Ultra Getting Started Thermo Electron Corporation

Miscellaneous InformationFT Analyzer Information in Scan Header

FT Analyzer Messages The scan header of an FT scan includes also so-called FT Analyzer Messages:

If the Analyzer information check box of the FT page of the Spectrum Display Options dialog box is selected (refer to “Spectrum View” on page 2-3 and Figure 2-3 on page 2-4), the FT Analyzer Messages are also displayed above the spectrum graph in the Tune Plus window.

Freq FT view frequency On Off

Offset FT zero offset On Off

Table A-1. Actual settings of manual toggles, continued

Tog = (…) Relevant Toggle Current setting Default setting

Ufill=0.45 Maximum ion time reached. Here: the real number of ions is only ~45% of the target value.

MCal=4d Mass calibration for this scan is several days old (here: 4). “MCal=99d” indicates that no valid FT mass calibration is available (if, for example, a manual transmission calibration is not followed by a mass calibration)

Est=0x24 Machine-readable result message for post-processing toolsDAC=0.98 FT transient measurement near saturation, this might

result in spectral harmonics (typically target value too high)

PkOvf Internal Peak detection overflow in the FT spectrum analysis

Thermo Electron Corporation LTQ FT Ultra Getting Started A-3

Miscellaneous InformationData Size of FT Raw Files

Data Size of FT RawFiles

The data size of a raw file with FT data depends on many parameters, for example on the number of scans, the resolution setting, the data format and on the status of the Active Noise Reduction. Table A-2 below displays typical data sizes (per scan) of an FT spectrum (standard LTQ FT calibration solution, scan range 190–1900, AGC target 5E5, 1 microscan, resolution setting 100 000) at different FT data formats and with Active Noise Reduction on or off.

Table A-2. Typical data sizes (per scan) of an FT spectrum

FT Data Format Active Noise Reduction Typical data size / scan

Centroid On ca. 10 kB

Reduced Profile On ca. 20 kB

Centroid Off ca. 35 kB

Reduced Profile Off ca. 80 kB

Full Profile On/Off ca. 2800 kB

A-4 LTQ FT Ultra Getting Started Thermo Electron Corporation

Appendix B Safety Information for Optional Hardware

This appendix gives safety information for the optional hardware that is available for the LTQ FT Ultra MS detector. When your instrument is equipped with the IRMPD option, read this appendix before you start working.

The following topics are described in this appendix:

• “Safety Components for IRMPD” on page B-2

• “Safety Instructions for IRMPD” on page B-4

Complies with FDA performance standards for laser products except for deviations pursuant to Laser Notice No. 50, dated july 26, 2001.

Thermo Electron Corporation LTQ FT Ultra Getting Started B-1

Safety Information for Optional HardwareSafety Components for IRMPD

Safety Componentsfor IRMPD

If your LTQ FT Ultra is equipped with the IRMPD option, the instrument is supplemented by various safety components that are described in this section.

Safety Interlocks Covered by the left side panel, a safety interlock at the rear side of the support frame disconnects the power supply for the electronics when you extract the vacuum system from the magnet (for instrument bakeout, for example).

Another safety interlock disconnects the power supply for the laser, when the beam enclosure is opened. See Figure B-2.

Figure B-1. Safety interlock at support frame (side panel removed)

B-2 LTQ FT Ultra Getting Started Thermo Electron Corporation

Safety Information for Optional HardwareSafety Components for IRMPD

Safety Lock forElectronics Cabinet

To prevent unauthorized access to the hazardous IRMPD components in the electronics cabinet, the cabinet panel is equipped with a safety lock that can be opened with a special tool only. See Figure B-3 on page B-3.

Figure B-2. Mirror unit for IRMPD, showing the safety interlock and the adjusting knobs

Safety interlock

Protective caps for adjusting knobs

Figure B-3. Safety lock for electronics cabinet


Safety Information for Optional HardwareSafety Instructions for IRMPD

Safety Instructions forIRMPD

This topic describes the hazards posed by the IRMPD components and gives instructions for the safe operation of the LTQ FT Ultra. See also the manual provided by the laser’s manufacturer.

Laser Safety The instrument is designed for the safe operation in a laboratory. Because the laser radiation is completely shielded, the instrument is assigned to class 1. For adjusting operations, an additional class 2 (class 3R) diode laser is used; it is disconnected from the power line during normal operations. A class 4 laser is integrated into the instrument. When the instrument is operated in compliance with the safety regulations, this laser does not pose a danger for users and environment.

General Advice for SafeOperation

For the safe operation of the instrument always keep the housing closed. We recommend acquiring the habit of not opening the housing when the instrument is operated. This is especially the case for covers that cannot be opened without a tool.

For performing maintenance, we recommend switching off the instrument with the main switch. Prevent unintentional rebooting of the instrument by padlocking the main switch.

Do not modify the instrument. Never change the mechanical design particularly that of the beam guide area. Never change the wiring; it might change security relevant components.

Under no circumstances defeat the safety interlocks. Deactivating the safety interlocks is never required for performing maintenance or adjustment operations.

Direct and scattered radiation of the built-in laser is dangerous for eyes and skin. Combustible materials can be ignited by the laser. Always comply with the safety regulations and never override safety mechanisms. Only by doing so, you prevent risks of injury and fire hazards.

Glass Cover The transparent cover of the mirror unit consists of a special material. The safety of the system is only assured by using undamaged original parts. It is essential to replace damaged covers (scratches, cracks, laser abrasives). Do not use the laser with damaged cover. In no case replace the cover by simple glass, it will burst upon laser bombardment. Using a Thermo Electron original replacement part is mandatory.



Diode Pointer (AdjustingLaser)

The diode pointer is only required for the first adjustment of the system by the Thermo Electron field service engineer. Afterward the diode pointer must be switched off and separated from the power supply. Class 2 laser radiation is emitted through the magnet bore when it is not covered by the position device as specified by the adjusting instruction. Do not gaze into the beam or its focussed reflections!

Optical Components The optical components are made of anti-reflex (AR) coated ZnSe and require special care. In addition to the information below, please consider the separate safety information by the manufacturer.

ZnSe - Body Material Health hazard: Toxic by inhalation. Danger of cumulative effects. Contact with dust can cause irritation of eyes and respiratory system.

Hazard by thermal decomposition or synthesis: At high temperatures (>400 °C); irritant and toxic fume (ZnOx, Se, SeOx, ZnH and H2Se)

Anti-Reflex Coating Anti-reflex coatings of the AR 10.6 type contain minimal amounts of the radioactive substance thorium. The thorium coating is embedded within other thin layers; it thereby presents a “sealed radioactive source“. That means that no radioactive material can leak from the source as long as it is not damaged.

Thorium is a potential health hazard if it is inhaled or swallowed. Eating, drinking or smoking should be prohibited in the application area. After each skin contact with the coated components, wash the affected body part immediately with plenty of water.

The coated components have an activity of about 4.2 Bq per square centimeter. Therefore, normal usage or cleaning of the optics is possible WITHOUT DANGER BY RADIOACTIVE RADIATION. Just take the same precautions as when touching or cleaning other sensitive optics; wearing vinyl gloves is sufficient.

Warning Take special precautions when grinding or polishing lenses, also when handling damaged or broken lenses. Refer to the safety instructions.



Hazard Labels It is essential to know the following hazard labels:

In most places on the instrument, the hazard labels are accompanied by the following label:

Warning Laser radiation: Do not stare into the beam.Follow the additional instructions near this label.

Hazard area Do not open covers signed with this label. The parts inside are to be serviced by trained personnel only, in compliance with the safety regulations.

Consult Manual Read the manual(s) that came with the instrument for usage and maintenance instructions. The instructions must be precisely followed!



Position of Safety Labels Table B-1 lists all safety labels for the optional hardware and their respective positions.

Table B-1. Safety labels for the optional hardware

Label description Label position

A: Back access door labelThis label is located on the back of the instrument, next to the handle of the door to the upper electronics compartment:

The door may be opened for the following maintenance operations:

• Baking the system

• Fine adjustment of the laser direction

B: LTQ front access labelThis label is attached to the front of the LTQ, next to the front access door and the ion source.

Cleaning the Q00 ion guide requires a shut down of the system (the assembly cannot be removed when the system is under vacuum). The system has to be shut down using the main switch on the back of the LTQ FT Ultra. The main switch should be secured in the off position during maintenance.

Note. Opening the front access door is considered a service operation.



C: Back access door inside labelThe label is attached to the laser housing. It is exposed when the upper back door is opened.The beam is still completely encapsulated. Only the maintenance operations mentioned in connection with the back access door label (A) are allowed. See above.

Note. Never attempt any changes to the system. Major adjustments or exchange of parts require a service call.

D: Diode pointerThis label is attached to the adjustment laser ("diode pointer"). The adjustment laser is disconnected from power during operation and maintenance.

D1: Class 4 laser label in German or other national language, see photo in position C for details.

D1

Table B-1. Safety labels for the optional hardware, continued




E: Magnet openingThis label is attached next to the LTQ side of the magnet.

Service may include readjustment of the laser with the diode pointer. The laser power is reduced by incomplete reflection on the IR grade mirror. When the magnet opening is not covered, class 2 laser radiation could be emitted from the opening. As mentioned above (D), the diode pointer is always disconnected during operation and maintenance.

F: Do not open CoversThis label is located on the mirror unit (1). The mirror unit has to be closed during operation and maintenance. Copies of this label are on the transfer block (2) and the LTQ top cover (3) as reminders for service engineers that the system has to be shut down and secured correctly before performing service.

1)

2)





3)

G: Laser PowerThe upper sticker is attached to the laser power supply.

The other sticker is wrapped around the power chord and secured with transparent adhesive tape.




Glossary

The following abbreviations and terms are used in this manual.

A ampere

ac alternating current

ADC analog-to-digital converter

AGC Automatic Gain Control

AP acquisition processor

APCI atmospheric pressure chemical ionization

API atmospheric pressure ionization

APPI atmospheric pressure photo ionization

ASCII American Standard Code for Information Interchange

b bit

B byte (8 b)

baud rate data transmission speed in events per second

°C degrees Celsius

CI chemical ionization

CID collision induced dissociation

cm centimeter

cm3 cubic centimeter

CPU central processing unit (of a computer)

CRM consecutive reaction monitoring

<Ctrl> control key on the terminal keyboard

d depth

Da dalton

DAC digital-to-analog converter

dc direct current

DS data system

DSP digital signal processor

ECD electron capture dissociation

EI electron ionization

EMBL European Molecular Biology Laboratory

<Enter> Enter key on the terminal keyboard

ESD electrostatic discharge

ESI electrospray ionization

eV electron volt

f femto (10-15)

°F degrees Fahrenheit

.fasta file extension of a SEQUEST search database file

ft foot

FFT Fast Fourier Transformation

Thermo Electron Corporation LTQ FT Ultra Getting Started G-1

Glossary: FT

FT Fourier Transformation

FT-ICR Fourier Transform-Ion Cyclotron Resonance

FTMS Fourier Transformation Mass Spectroscopy

FTP file transfer protocol

FWHM full width at half maximum

g gram

G Gauss; giga (109)

GC gas chromatograph; gas chromatography

GC/MS gas chromatograph / mass spectrometer

GUI graphical user interface

h hour; height

HPLC high-performance liquid chromatograph

HV high voltage

Hz hertz (cycles per second)

ICIS™ Interactive Chemical Information System

ICL™ Instrument Control Language™

ICR ion cyclotron resonance

ID inside diameter

IEC International Electrotechnical Commission

IEEE Institute of Electrical and Electronics Engineers

in inch

I/O input/output

IRMPD infrared multiphoton dissociation

k kilo (103, 1000)

K kilo (210, 1024)

KEGG Kyoto Encyclopedia of Genes and Genomes

kg kilogram

l length

L liter

lb pound

LC liquid chromatograph; liquid chromatography

LC/MS liquid chromatograph / mass spectrometer

LED light-emitting diode

LHe liquid helium

LN2 liquid nitrogen

µ micro (10-6)

µL microliter

m meter; milli (10-3)

M mega (106); mole, molar

M+ molecular ion

MALDI matrix-assisted laser desorption/ionization

MB Megabyte (1048576 bytes)

MH+ protonated molecular ion

min minute

mL milliliter

G-2 LTQ FT Ultra Getting Started Thermo Electron Corporation

Glossary: mm

mm millimeter

MRFA A peptide with the amino acid sequence methionine–arginine–phenylalanine–alanine.

MS mass spectrometer; mass spectrometry

MS MSn power: where n = 1

MSDS Material Safety Data Sheet

MS/MS MSn power: where n = 2

MSn MSn power: where n = 1 through 10

m/z mass-to-charge ratio

n nano (10-9)

NCBI National Center for Biotechnology Information (USA)

NIST National Institute of Standards and Technology (USA)

OD outside diameter

OVC outer vacuum case

Ω ohm

p pico (10-12)

Pa pascal

PCB printed circuit board

P/N part number

ppm parts per million

PQD pulsed-Q dissociation

RAM random access memory

RF radio frequency

RMS root mean square

ROM read-only memory

s second

SIM selected ion monitoring

SRM selected reaction monitoring

TCP/IP transmission control protocol / Internet protocol

TIC total ion current

Torr torr

TWA time weighted average

u atomic mass unit

V volt

V ac volts alternating current

V dc volts direct current

vol volume

w width

W watt

WSR wide scan range

Thermo Electron Corporation LTQ FT Ultra Getting Started G-3

Index
Aacetic acid 3-6Activation button 7-18, 8-7Activation page 7-21, 8-10, 8-14activation Q 7-15, 7-21, 8-5activation time 7-15, 8-5active noise reduction 6-3, A-4actual ECD duration 7-22actual settings, of manual toggles A-2advanced calibration features 2-9, 3-2advanced features 5-4
calibration 2-9, 3-2Scan 2-10

AGC 2-13stability 4-5target compensation factor 4-6target values 2-14, 2-23, 8-14target values for ECD 7-19target values for IRMPD 8-8

All tab 2-7analyzer 2-10API accessory kit 3-5, 3-7Apply button 3-26automatic calibration 3-2

page 3-14procedures 3-14trapping voltage for the positive ion mode 3-24

automatic FT advanced calibration 3-24automatic gain control 2-13automatic magnetic field drift correction 6-3Automatic page 3-14automatic tune 3-10averaging, spectrum 2-17

Bbakeout 7-6buttons

Apply 3-26Execute 3-28FT Cryo Monitor 2-2Refresh 3-26Save 3-26Vacuum 2-2

CCalibrate dialog box 3-3, 3-16, 3-20

Automatic page 3-14Check page 3-18FT Advanced page 2-9, 3-2, 3-14, 3-22FT ECD page 7-10FT Manual page 3-20Semi-Automatic page 3-16

calibratingECD heater current 7-10for FT Measurements 3-1ion trap 3-10potentials and timing 7-11

calibrationautomatic 3-2, 3-8, 3-14, 3-17, 3-23excitation amplitudes 3-23files 3-2, 4-6, 4-13ion trap 3-10manual coarse two-point 3-17, 3-27negative ion mode 3-17, 3-19positive ion mode 3-19semi-automatic 3-2, 3-8, 3-17trapping voltage 3-23

calibration filesbackups 3-4history 3-4

calibration masses 3-2, 3-9, 3-21, 3-25selecting 3-2

calibration parameters 2-15, 3-2, 4-15checking all 3-19

calibration procedures 3-2calibration ranges 2-15, 3-2calibration solutions 3-5

automatic calibration 3-8contaminants 3-6ECD 3-9FT manual calibration 3-9ion trap calibration 3-8IRMPD 3-9LTQ 3-5LTQ FT 3-7–3-8, 3-12semi-automatic calibration 3-8

calibration values 3-4calibration, of the excitation amplitudes and the trapping voltages 3-23Centroid format 2-16

Thermo Electron Corporation LTQ FT Ultra Getting Started I-1

Index: D

Centroid/Profile 2-16changing, the instrument settings 4-12charge state 7-22charge state dependent ECD time 7-22charge state of peaks, in the FT spectrum 2-4Charge State page 5-4, 7-22–7-23Check page 3-18checks 4-1

heater current 7-11potentials and timing 7-11

CID 7-16, 7-21, 8-5compensation factor 4-6compressed profile format 2-16configuration changes 6-3consecutive reaction monitoring 2-13Control menu 2-9CRM 2-13cryogen fill levels 2-2, 2-20–2-21Current Scan Event page 5-2current segment 5-3

Ddata dependent FT narrow SIM scan 5-2data dependent settings 5-1Data Dependent Settings dialog box 7-22

Activation page 7-21, 8-10Current Segment page 5-3FT ECD/IRMPD page 7-21, 8-10–8-11, 8-15IRMPD 8-11

data formats 2-5, 4-8storage 2-25switching 2-16

data sizes, of an FT spectrum A-4DC offset voltages 3-23–3-24default charge state 8-10, 8-14default setting

ECD status 7-13default settings 4-8–4-9

resolution 2-11Define Scan dialog box 2-10, 2-14–2-16, 3-21, 3-27, 7-15, 8-4, 8-12–8-13

automatic tune of the ion trap 3-11ECD 7-15ECD and IRMPD 8-13IRMPD 8-4setup for a CID experiment followed by an ECD experiment

7-17setup for a CID experiment followed by an IRMPD

experiment 8-6delay 7-16, 8-5, 8-12

devices 4-11Diagnostics dialog box 3-27, 4-1–4-2, 4-11

FT ECD page 7-3Set device page 4-11System evaluation page 4-2Toggles page 2-5, 4-7

diagnostics/checks 4-1dialog boxes

Define Scan 2-10FT Cryo Monitor 2-21FT Transfer Ion Optics 2-18Injection Control 2-18–2-19Spectrum Averaging 2-17Spectrum Display Options 2-3–2-4User Status Display Configuration 2-8Vacuum 2-21

Display menu 2-17Display Settings page 4-14displaying

FT calibration settings 4-15FT Diagnostics 4-15FT instrument settings 4-15

Dynamic Exclusion 5-4

EECD 6-3–6-4, 7-1–7-2, 7-8, 7-11–7-13, 7-16

calibration and tuning solution 7-9calibration page 3-3calibration solution 7-9cathode activation 7-8cathode heating 7-6cathode heating, during bakeout 7-6cathode potential 7-16changes in Define Scan dialog box 7-15changing settings 7-20check box 7-21configuring 7-2data dependent settings 7-21delay 7-21duration 7-21duration equation 7-22duration time 7-11, 7-22efficiency 7-11, 7-15electron current 6-4energy 7-21exposure time 7-18gate current calibration 7-5heater 7-12, 7-14heater current 7-13heater current calibration 7-10–7-11heater current check 7-11

I-2 LTQ FT Ultra Getting Started Thermo Electron Corporation

Index: F

monitor parameters 7-6, 7-8monitor results 7-3parameters 7-13scan event 8-14scan time settings 7-18setting parameters 7-15status 7-12status monitor 7-3switching off the heater 7-14target value 7-19

ECD and IRMPDconfiguring 8-12data dependent settings 8-14scan ranges 2-15scan time setting 8-13scan time settings 2-16target value 8-14

electron current, for ECD 6-4electron energy 7-15, 7-18electron multiplier calibration 3-15electron multiplier gain calibration 3-10enable full scan injection waveforms 2-20energy offset 7-11error diagnostics 4-5ESI parameters 2-24excite amplifier 4-15

printed circuit board 4-9excite amplitudes 3-23

automatic calibration 3-23manual calibration 3-25

excite and detect electrodes, DC offset voltages 3-24Execute button 3-28

Ffactory-supplied mass lists 3-22, 6-6fill levels, of cryogens 2-2, 2-20flags, for inject waveform 2-24format

centroid 2-16compressed profile 2-16

frequency modes 2-4frequency, of the RF voltage 3-19FT Advanced calibration page 3-22FT Advanced page 2-9, 3-2, 3-14, 3-22FT analyzer

information in scan header A-2messages A-3settings A-2target values 2-19

FT apodization 4-8

FT calibration 3-7–3-8manual 3-9

FT CRM scansscan range 2-15

FT Cryo Monitor button 2-2FT Cryo Monitor dialog box 2-20–2-21FT detect during excite 2-5, 4-8FT dynamic range test 4-4FT ECD calibration page 7-10FT ECD command 2-22FT ECD dialog box 7-12–7-13FT ECD page 7-3, 7-6, 7-8, 7-10FT ECD/IRMPD page 7-21, 8-10–8-11, 8-15–8-16FT excite amplifier 4-9FT excite signal 2-5FT excite signal view 2-5FT Frequency Spectrum view 2-4FT include transients 4-9FT injection control 2-18FT injection waveforms 2-20FT instrument settings 4-15

displaying 4-15FT ion optics 2-18FT ion transmission calibration 2-18, 3-2, 3-15, 3-17, 3-19FT IRMPD command 2-22FT IRMPD dialog box 8-4FT isolation test 4-4FT manual calibration, in diagnostic mode 4-9FT Manual page 3-2, 3-20–3-21FT mass calibration 2-6, 3-2, 3-15, 3-17, 3-28, 4-6FT Mass Lists page 6-5FT mass spectrum 4-5

resolution 2-11FT Mass Spectrum view 2-4FT master scan 5-3FT measurements, targets 2-19FT MSn scans 4-10FT multipole 1 and 2 RF 4-9FT multipole 3 RF 4-9FT multipole and intermultipole lens DC offsets 4-5FT multipole RF calibration parameters 3-15FT Narrow SIM scans

scan range 2-15FT noise test 4-3–4-4

progress 4-4FT page 2-22, 8-13–8-14

ECD 7-19ECD and IRMPD 8-13–8-14

FT profile mode 2-16, 4-9FT sensitivity test 4-3


Index: G

FT Settings page 6-3, 7-2, 8-2, 8-12FT SIM and MSn injection waveforms 4-10FT SIM scans 4-10FT spectrum 3-28FT SRM scans, scan range 2-15FT stability test 4-4–4-5

result 4-5FT tab 2-4, 2-20FT Transfer Ion Optics dialog box 2-18, 4-12FT transfer ion optics parameter 4-13FT transfer ion optics parameters 4-12

changing 4-13FT transfer ion optics settings 4-13FT transfer optic electrodes 4-5FT transfer optics test 4-5FT transfer parameters 2-18FT transfer time 4-13FT transient view 2-5FT transmission calibrations 4-12FT tune procedures 3-2FT vacuum 2-21FT view frequency 2-4, 4-10FT Wide SIM scans, scan range 2-15FT zero offset 4-10FTMS, SIM modes 2-12full MS target 2-19Full Profile format 2-16

Ggate current 7-3, 7-5, 7-10–7-11Graph view 2-6, 4-4–4-5, 7-3, 7-5–7-6, 7-8

Hheater current 7-3–7-4, 7-13

calibration 7-10dependence on ECD status 7-13

heater resistance 7-3–7-4heater voltage 7-4high mass range 3-21

mode 4-6high resolution 5-3

Iinfusion experiment 4-4inject time 2-13, 3-12

maximum 2-23

inject waveform 2-24inject waveform flags 2-24Injection Control dialog box 2-18–2-19, 7-19, 8-8, 8-14

FT tab 2-20, 8-14FT tab with ECD option 7-19FT tab with IRMPD option 8-8Ion Trap tab 2-19, 7-19tabs 2-19

injection waveforms 2-20, 4-10enabling 2-20

instrument configurationECD 7-2ECD and IRMPD 8-12IRMPD 8-2

Instrument Configuration dialog box 6-2instrument control board 4-15instrument reset 4-12–4-13ion optic supply board 4-9, 4-15ion polarity 2-16

changing 2-16ion trap 2-19ion trap calibrations 3-10ion trap CRM scans, scan range 2-15ion trap SIM scans, scan range 2-15ion trap spectrum, of the LTQ FT calibration solution 3-12–3-13ion trap SRM scans, scan range 2-15Ion Trap tab 2-19, 2-21IRMPD 6-3–6-4, 8-1, 8-5

AGC target value 8-8changes in Define Scan dialog box 8-4configuring 8-2data dependent settings 8-10delay 8-10duration 8-5, 8-10duration of lase pulse 8-9duration tuning 8-7efficiency test 8-9energy 8-10energy tuning 8-7exposure time 8-7fragmentation efficiency 8-9hazards B-4laser power supply B-2maximum inject time 8-7mirror unit B-3number of microscans 8-7relative laser intensity 8-5safety components B-2safety instructions B-4scan event 8-14scan range 8-6scan time settings 8-7


Index: L

setting parameters 8-5settings for current scan event 8-10settings for current segment 8-10target value 8-8tuning solution 8-3

isolation width 7-15, 8-10, 8-14

Llaser

intensity 8-7pulse duration 8-9

last successful check 3-20log file 2-21LTQ API accessory kit 3-7LTQ calibration solution 3-5, 4-5–4-6

preparing 3-5–3-6LTQ Configuration dialog box, FT Mass Lists page 6-5LTQ ESI calibration solution 3-7LTQ FT calibration solution 3-7–3-8, 3-12

preparing 3-7LTQ FT Ultra Configuration dialog box 6-2–6-3, 8-2

FT Mass Lists page 6-5FT Settings page 6-3, 7-2, 8-2

Mmanual calibration 3-25manual calibration, of excite amplitude 3-25Manual coarse calibration page 3-27manual coarse FT mass calibration 3-28manual coarse two-point mass calibration 3-27mass accuracy 2-19mass calibration 3-2

checking 3-19copying parameters 3-28

Mass calibration page 3-27mass list 3-21, 6-5mass range 2-11mass resolution 2-11mass-to-charge ratios 3-21Material Safety Data Sheet 8-3maximum AGC target value 4-4maximum inject time 2-13–2-14, 2-23menu bar 2-3menus

Display menu 2-17View menu 2-3

methods, tune 2-23microscans 2-13, 2-23

minimum resolution, for Tune Plus window 2-2monitor parameters 7-8monoisotopic peaks 5-4monoisotopic precursor selection 5-4MRFA alone solution 4-4MSDS 8-3MSn settings 2-14MSn target 2-19multiplier gain calibration 3-15multipole RF amplitudes 4-5

NNarrow SIM 2-12Narrow SIM scans, TIC correction 6-7Narrow SIM target 2-19negative ion mode 3-12

checks 3-18nitrogen venting kit 7-14Normalized Collision Energy 7-15, 8-5number, of decimals 2-4number, of microscans 2-13

Pparameters 2-23–2-24

calibration 2-15, 3-2FT transfer 2-18mass calibration 2-16not saved in a tunefile 2-24with differentiation between ion trap and FT scans 2-23without differentiation between ion trap and FT scans 2-24

parent mass 7-15performing diagnostics/checks 4-1positive/negative mode 2-16potential, of ECD cathode 7-16potentially harmful materials 7-9PQD 7-21precursor ions 5-4pressure 7-4preview mode 5-3procedure, system evaluation 4-2product ions 7-18Profile format, compressed 2-16protective gloves 8-3

QQual Browser A-2


Index: R

Rreadback values 4-15Reduced Profile format 2-16Refresh button 3-26removing unwanted high mass ions 2-20resistant noise peaks 4-3resolution 2-11

displaying 2-4FT mass spectrum 2-11

Ssafety glasses 8-3safety instructions, for IRMPD B-4safety interlocks B-2–B-3safety lock B-3Save button 3-26scan

ranges 2-15time 2-13type 2-12

Scan Activation Parameters dialog box 8-7displaying 7-18, 8-7ECD 7-18IRMPD 8-7

scan description 2-10scan features, advanced 2-10Scan Header A-2scan header A-3Scan Mode menu 2-10scan ranges 2-15

ECD scans 7-17ECD/IRMPD 2-15FT analyzer 2-11Full scans 2-15IRMPD scans 8-6SIM/SRM/CRM scans 2-15

scan rate 2-11scan speed 2-12scan time settings 2-13, 2-15Scan Time Settings dialog box 2-14–2-16, 7-18, 8-7, 8-13

displaying 2-14FT tab 8-13FT tab with ECD 7-19FT tab with IRMPD 8-7

scan type 2-12Scan widths page 5-2selected reaction monitoring 2-13selecting

calibration masses 3-2

experimental parameters 4-11semi-automatic calibration 3-2–3-3Semi-Automatic page 3-16Set device page 4-11, 7-20

ECD option 7-20setting

a new FT transfer time 4-13new FT transfer ion optics parameters 4-12

settings 4-14Define Scan dialog box 3-11manual toggles A-2

setupCID experiment followed by an ECD experiment 7-17CID experiment followed by an IRMPD experiment 8-6

Setup menu 2-18shortcut menu, of Spectrum view 2-5signal-to-noise ratio 4-4sodium dodecyl sulfate 3-7sodium taurocholate 3-7spectrum averaging 2-17Spectrum Averaging dialog box 2-17Spectrum Display Options dialog box 2-4

FT tab 2-4Spectrum view 2-3–2-4, 4-8

displaying views 2-4FT detect during excite toggle 4-8

SRM 2-13starting, instrument configuration 6-2statistic noise 6-3Status view 2-6

All page 2-6All tab 2-7User page 2-7

status, of a toggle 4-7Substance P 7-9, 8-3switching data formats 2-16system bakeout 7-6System evaluation page 4-2system evaluation procedures 4-2system performance 4-2

Ttarget

MSn 2-19SIM 2-19

target gate current 7-5, 7-10target values

additional 2-20AGC 2-23FT analyzer 2-19


Index: U

targets, for FT measurements 2-19testing

FT dynamic range 4-4FT isolation 4-4FT noise 4-3FT sensitivity 4-3

TIC correction 6-7toggles 4-7

status 4-7Toggles page 2-5, 4-7toolbar 2-3total ion current 4-4transfer time

displaying 4-13saving 4-13

transient view 4-8–4-9transients

acquiring 4-9averaging 2-17displaying 2-5

trapping voltages 3-23automatic calibration 3-24

tube lens 3-11–3-12manual adjustment 3-12

tune methods 2-23tune parameters 2-14Tune Plus version 1-1Tune Plus window 2-1–2-2, 3-4

functions 2-2minimum screen resolution 2-2

tune procedures, for FT 3-2tuning

ECD energy and exposure time 7-18ion trap 3-10ion trap for negative ion mode 3-12ion trap for positive ion mode 3-10IRMPD energy and duration 8-7

two-point calibration 3-28

UUser Status Display Configuration dialog box 2-7–2-8User tab 2-8using toggles 4-7

VVacuum button 2-2Vacuum dialog box 2-21

FT tab 2-22Ion Trap tab 2-21tabs 2-21

vacuum quality 7-3–7-4values, AGC target 2-23venting the instrument 7-14View menu 2-3views

FT Mass Spectrum 2-4

WWarnings

exposure to potentially harmful materials 8-3grinding or polishing lenses B-5laser radiation B-6

waveforms, injection 2-20wide scan range mode 2-14, 2-16, 2-24Wide SIM 2-12Wide SIM Target 2-19windows

Tune Plus 2-1–2-2, 3-4WSR 2-14, 2-16

XXcalibur raw file 4-9


Thermo Electron Corporation81 Wyman StreetP.O. Box 9046Waltham, Massachusetts 02454-9046United States

www.thermo.com

Analyze • Detect • Measure • Control™

LTQ FT Ultra - Thermo Fisher Scientific · Welcome to the Thermo Electron, LTQ FT Ultra™ system!...

Documents

Transcript of LTQ FT Ultra - Thermo Fisher Scientific · Welcome to the Thermo Electron, LTQ FT Ultra™ system!...