MS/ETD System Getting Started Manual

MS/ETD System Getting Started Manual 98000-97003 Rev A December 2006

2006 Thermo Electron Corporation. All rights reserved. Microsoft and Windows are registered trademarks of Microsoft Corporation. Teflon is a registered trademark of E.I. du Pont de Nemours & Company. Unimetrics is a registered trademark of Unimetrics Company. Tygon is a registered trade mark of Saint-Gobain Performance Plastics Company. Rheodyne and the Rheodyne logo ( ) are registered trademarks of Rheodyne, L.P. All other trademarks are the property of Thermo Electron Corporation and its subsidiaries. This document is provided to customers who have purchased Thermo Fisher Scientific Inc equipment to use in the operation of such Thermo Fisher Scientific equipment. This document is copyright protected and any reproduction of this document or any part of this document is strictly prohibited, except as Thermo Fisher Scientific may authorize in writing. 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 Fisher Scientific 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 Fisher Scientific and the purchaser of an LC/MS system. In the event of any conflict between the provisions of this document and those contained in Thermo Fisher Scientific 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 December 2006. Software Revision: Xcalibur 2.0, LTQ 2.3

Regulatory Compliance Thermo Electron San Jose performs complete testing and evaluation of its products to ensure full compliance with applicable domestic and international regulations. When the system is delivered to you, it meets all pertinent electromagnetic compatibility (EMC) and safety standards as described below. EMC Directive 89/336/EEC EMC compliance has been evaluated by TUV Rheinland of North America, Inc. EN 61000-3-2 1995, A1; 1998, A2; 1998, A14; 2000 EN 61000-4-4 IEC 61000-4-4 1995, A1; 2001, A2; 2001; A2-1995 EN 61000-3-3 1998 EN 61000-4-5 1995, A1; 2001 IEC 61000-4-5 2005 EN 61326-1 1998, A3 EN 61000-4-6 1996, A1; 2001 IEC 61000-4-6 2004 EN 61000-4-2 2000 EN 61000-4-11 1994, A1; 2001 IEC 61000-4-2 2001 IEC 61000-4-11 2001-03 FCC Class A, CFR 47 Part 15 2005 CISPR 11 1999, A1; 1999, A2; 2002 Low Voltage Safety Compliance This device complies with Low Voltage Directive 73/23/EEC and harmonized standard EN 61010-1:2001. Changes that you make to your system may void compliance with one or more of these EMC and safety standards. Changes to your system include replacing a part or adding components, options, or peripherals not specifically authorized and qualified by Thermo Electron. To ensure continued compliance with EMC and safety standards, replacement parts and additional components, options, and peripherals must be ordered from Thermo Electron or one of its authorized representatives.

FCC Compliance Statement THIS DEVICE COMPLIES WITH PART 15 OF THE FCC RULES. OPERATION IS SUBJECT TO THE FOLLOWING TWO CONDITIONS: (1) THIS DEVICE MAY NOT CAUSE HARMFUL INTERFERENCE, AND (2) THIS DEVICE MUST ACCEPT ANY INTERFERENCE RECEIVED, INCLUDING INTERFERENCE THAT MAY CAUSE UNDESIRED OPERATION. CAUTION: Read and understand the various precautionary notes, signs, and symbols contained inside this manual pertaining to the safe use and operation of this product before using the device. Notice on Lifting and Handling of Thermo Electron San Jose Instruments For your safety, and in compliance with international regulations, the physical handling of this Thermo Electron San Jose instrument requires a team effort for lifting and/or moving the instrument. This instrument is too heavy and/or bulky for one person alone to handle safely. Notice on the Proper Use of Thermo Electron San Jose Instruments In compliance with international regulations: If this instrument is used in a manner not specified by Thermo Electron San Jose, the protection provided by the instrument could be impaired. Notice on the Susceptibility to Electromagnetic Transmissions Your instrument is designed to work in a controlled electromagnetic environment. Do not use radio frequency transmitters, such as mobile phones, in close proximity to the instrument.

WEEE Compliance This 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. WEEE Konformität Dieses 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 mit Verwertungs-/Entsorgungsfirmen in allen EU- Mitgliedsstaaten getroffen, damit dieses Produkt durch diese Firmen wiederverwertet oder entsorgt werden kann. Mehr Information über die Einhaltung dieser Anweisungen durch Thermo Electron, über die Verwerter, und weitere Hinweise, die nützlich sind, um die Produkte zu identifizieren, die unter diese RoHS Anweisung fallen, finden sie unter www.thermo.com/ WEEERoHS.

Conformité DEEE Ce 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.

Contents Preface... ix About This Guide... ix Related Documentation... ix Safety and Special Notices... ix Contacting Us... x Assistance... x Changes to the Manual and Online Help... x Chapter 1 Introduction...1 Chapter 2 ETD System Reagent Vial Installation...3 Place the MS/ETD System in Off Condition and Service Mode... 4 Install/Exchange the Reagent Vials... 8 Chapter 3 Powering On the ETD Module, View Reagent Ion Spectra...11 Powering ON the ETD Module... 12 Turning ON the Reagent Ion Source and Viewing Reagent Ion Spectra... 13 View Reagent Ion Spectra... 15 Chapter 4 Tuning the ETD Optics...17 Tuning The Reagent Ion Source... 18 Viewing the Reagent Ion Optics Settings... 23 Saving Your ETD Tune Method... 25 Chapter 5 Performing an ETD Infusion Experiment...27 Injection Control: Reagent Ion AGC Target and Ion Time Settings.. 28 Obtaining an ETD Spectrum for Angiotensin I... 30 Optimizing the Reagent Ion Reaction Time... 34 Chapter 6 Creating an Xcalibur Instrument Method That Uses ETD Activation...37 Chapter 7 Turning Off the Reagent Ion Source: What to Expect...43 Thermo Electron Corporation MS/ETD System Getting Started vii

Contents Appendix A Fluoranthene...45 Appendix B Troubleshooting...47 Appendix C Angiotensin I Solutions...49 Angiotensin I Solutions...50 Angiotensin I Stock Solution...50 Angiotensin I Test Solution...50 Index...51 viii MS/ETD System Getting Started Thermo Electron Corporation

Preface About This Guide Welcome to the Thermo Electron, MS/ETD System. The MS/ETD System is a member of the Thermo Electron family of MS detectors. This MS/ETD System Getting Started manual provides information on how to set up, calibrate, and tune the MS/ETD System, and how to acquire ETD data. All of these procedures can be performed from the Xcalibur Tune Plus window of the MS/ETD System. Related Documentation In addition to this manual, Thermo Electron provides the following documents for the MS/ETD System: MS/ETD System Preinstallation Guide MS/ETD System Getting Connected ETD Module Hardware Manual LTQ XL Manual set Online Help is also available from within the software. Safety and Special Notices Make sure you follow the precautionary statements presented in this guide. The safety and other special notices appear in boxes. Safety and special notices include the following: CAUTION Highlights laser-related hazards to human beings. It includes information specific to the class of laser involved. Each DANGER notice is accompanied by the international laser radiation symbol. CAUTION Highlights hazards to humans, property, or the environment. Each CAUTION notice is accompanied by an appropriate CAUTION symbol. IMPORTANT Highlights information necessary to avoid damage to software, loss of data, invalid test results, or information critical for optimal performance of the system. Thermo Electron Corporation MS/ETD System Getting Started ix

Preface Note Highlights information of general interest. Note Helpful information that can make a task easier. Contacting Us There are several ways to contact Thermo Electron. Assistance For new product updates, technical support, and ordering information, contact us in one of the following ways: Visit Us on the Web www.thermo.com/finnigan Contact Technical Support Phone: 1-800-685-9535 Fax: 1-561-688-8736 techsupport.finnigan@thermo.com Contact Customer Service In the US and Canada for ordering information: Phone: 1-800-532-4752 Fax: 1-561-688-8731 International contacts for ordering information: Visit www.thermo.com/finnigan for the current listing, Changes to the Manual and Online Help To suggest changes to this guide or to the Help, use either of the following methods: Fill out a reader survey online at www.thermo.com/lcms-techpubs Send an e-mail message to the Technical Publications Editor at techpubs.finnigan-lcms@thermo.com x MS/ETD System Getting Started Thermo Electron Corporation

Chapter 1 Introduction The MS/ETD System is a member of the Thermo family of MS detectors. The MS/ETD System consists of the LTQ XL MS Detector and the ETD Module. With the MS/ETD System you can perform electron transfer dissociation (ETD) mass spectroscopy on analytes of interest. The LTQ XL MS detector is an advanced analytical instrument that includes a linear ion trap, a syringe pump, a divert/inject valve, an atmospheric pressure ionization (API) source, and the Xcalibur data system. The ETD Module is a source of reagent ions that react with analyte molecules in the linear ion trap of the LTQ XL MS detector. The ETD Module consists of the Reagent Ion Source, ETD Module electronics, ETD Module power supply, ETD Module forepump, and the hardware that connects the ETD Module to the LTQ XL MS detector. The Reagent Ion Source contains two reagent vials, CI/carrier gas (nitrogen) handling hardware and flow restrictors, the ion volume and filament, ion optics, and heaters for these components. The flow restrictors keep the internal pressure of the reagent vials below atmospheric pressure. This prevents the contents of the reagent vials from being expelled to the laboratory atmosphere. Note The nitrogen gas serves two functions in the ETD process. It is both a carrier gas and a chemical ionization (CI) vehicle. As a carrier gas, the nitrogen sweeps the reagent from the vial to the ion source where reagent radical anions are formed. As a chemical ionization (CI) vehicle, the nitrogen undergoes collisions with 70eV electrons from the filament in the ion volume. These 70eV electrons from the filament knock electrons off of the nitrogen molecules (nitrogen ions are created). The secondary electrons resulting from these collisions have near thermal kinetic energies. These thermal electrons are captured by the fluoranthene to form reagent radical anions. The reagent radical anions are transported by the ETD optics to the Linear Ion Trap where they react with the analyte. Thermo Electron Corporation MS/ETD System Getting Started 1

Chapter 2 ETD System Reagent Vial Installation To change the reagent vials they are first cooled by placing the MS/ETD System in Off Condition. After the vials have cooled, the system is placed in Service mode. This chapter contains the following sections: Place the MS/ETD System in Off Condition and Service Mode Install/Exchange the Reagent Vials The ETD reagent vials are designed to keep the ETD reagent out of the laboratory environment. Removing and reinserting ETD reagent vials when they are not empty causes excessive puncturing of the septums and can reduce their integrity. This could result in ETD reagent (fluoranthene) entering the laboratory environment. Prevent this from occurring by removing and replacing the ETD reagent vials only when they are empty. CAUTION To preserve the integrity of the ETD reagent vial septums, remove and replace the ETD reagent vials only when they are empty. Do not reinstall used vials. CAUTION Store and handle all chemicals in accordance with standard safety procedures. The Material Safety Data Sheet (MSDS) describing the chemicals being used should be freely available to lab personnel for them to examine at any time. Material Safety Data Sheets (MSDSs) provide summarized information on the hazard and toxicity of specific chemical compounds. MSDSs also provide information on the proper handling of compounds, first aid for accidental exposure, and procedures for cleaning spills or dealing with leaks. Producers and suppliers of chemical compounds are required by law to provide their customers with the most current health and safety information in the form of an MSDS. Read the MSDS for each chemical you use. Dispose of all laboratory reagents in the appropriate way (see the MSDS). Safety information about fluoranthene is given in Appendix A, Fluoranthene on page 45. Thermo Electron Corporation MS/ETD System Getting Started 3

2 ETD System Reagent Vial Installation Place the MS/ETD System in Off Condition and Service Mode Place the MS/ETD System in Off Condition and Service Mode The LTQ XL MS power switches control power to the MS/ETD System (LTQ XL MS and the ETD Module). The ETD Module power switches control the power to the ETD Module only. When the MS/ETD system is fully operational (all systems On), the LTQ XL Main Power switch is in the On position and the MS Service switch is in the Electronics Normal (On) position. Normally the ETD Module Power and Service switches remain ON. Use the Service switch on the LTQ XL MS unit to place the MS/ETD system in Service mode. Turn the MS/ETD system (both the ETD and LTQ XL) ON and OFF with the LTQ XL power switch. CAUTION When the LTQ XL and ETD Module system is turned On, the flow restrictor and transfer line heaters can be heated to 200 C and the ion source heater can be heated to 160 C. The vial heaters can be at 130 C (or set point). Do not attempt to replace reagent vials or service heated components until you have determined that they have cooled to a safe temperature for handling. To place the MS/ETD System in Off Condition and Service mode and verify that the vials are safe to handle 1. Make sure no analyte is flowing into the API source. 2. If Tune Plus is not open, choose Start > Programs > Xcalibur > LTQ XL Tune from the taskbar to open Tune Plus. The Tune plus window appears (Figure 1). On Off Standby You can determine the state of the MS detector by observing the state of the On/Standby button on the Control/Scan Mode toolbar (Figure 1). The three different states of the On/Standby button are shown at the left. 4 MS/ETD System Getting Started Thermo Electron Corporation

2 ETD System Reagent Vial Installation Place the MS/ETD System in Off Condition and Service Mode On/Standby button Reagent Ion Source instrument control icon Figure 1. Tune Plus window 3. Choose Control>Off from the Tune Plus pull-down menu to place the system in OFF Condition. When the MS detector is in OFF Condition, the LTQ XL MS system turns off the ion source sheath gas, auxiliary gas, high voltage and all of the ETD Module heaters. CAUTION It is important to choose Control>Off from the Tune Plus pull-down menu in order to shut down all of the ETD Module heaters. 4. Click the Reagent Ion Source portion of the MS/ETD System instrument control icon at the top of the Tune Plus window (Figure 1). The Reagent Ion Source window appears (Figure 2). Thermo Electron Corporation MS/ETD System Getting Started 5

2 ETD System Reagent Vial Installation Place the MS/ETD System in Off Condition and Service Mode actual vial temperature in C Figure 2. Tune Plus Reagent Ion Source window Observe the temperature of Vial 1 in the Actual column of the Tune Plus Reagent Ion Source window (Figure 2). Nitrogen cooling gas will flow until the vial reaches 70 C (see Chapter 7, Turning Off the Reagent Ion Source: What to Expect ). Allow up to 90 minutes for the vial temperature to reach ambient temperature (about 30 C). CAUTION Do not attempt to handle the vials or vial holders when the cooling nitrogen stops. They are still too hot to handle when the cooling nitrogen stops at a vial temperature of 70 C. CAUTION Allow the vials to cool to about 30 C (allow up to 90 minutes after the cooling gas stops) before proceeding with the next step and handling the vials. 5. Toggle the LTQ XL MS Service switch to Service mode (Off ) when the vial has reached a temperature that is safe for handling (about 30 C). Toggling the LTQ XL MS Service switch to Service mode turns off all components except 6 MS/ETD System Getting Started Thermo Electron Corporation

2 ETD System Reagent Vial Installation Place the MS/ETD System in Off Condition and Service Mode the turbo pump and the fore pump in both the LTQ XL MS and the ETD Module. Note Do not place the ETD Module Service switch into its Service Mode (Off) position while the LTQ XL MS switches are left in their On positions. This action could cause communication problems between the LTQ XL MS and the ETD Module. The ability to control the Service mode for both the LTQ XL MS and the ETD Module at one point (at the LTQ XL MS Service switch) is a safety feature. CAUTION Do not place the system in Service mode until the vials reach a safe temperature (about 30 C). System temperature monitoring will stop when the system is placed in Service mode. Do not attempt to handle the vials, vial holders or heater assembly until a safe temperature is reached (about 30 C). The MS/ETD System is now in Service mode and the vials are at a safe temperature for handling. Thermo Electron Corporation MS/ETD System Getting Started 7

2 ETD System Reagent Vial Installation Install/Exchange the Reagent Vials Install/Exchange the Reagent Vials After the reagent vial heaters have cooled to room temperature, the reagent vials are ready to be installed or exchanged. To install or exchange the Reagent vials 1. Remove the back panel from the ETD Module (see the section Removing the ETD Main Access Panel and Side Access Panel in Chapter 4 of the ETD Module Hardware Manual). This exposes the Reagent Inlet Source heating unit which has its own cover (Figure 3). CAUTION Follow the procedures in Place the MS/ETD System in Off Condition and Service Mode on page 4 before removing the back panel of the ETD Module. Removing the back panel before the system is placed in service mode opens the panel electrical interlocks and stops all system activity including temperature monitoring. In the absence of temperature monitoring you might attempt to handle the vials before it is safe to do so. 2. Make sure that the vial heater cover is cool to the touch. CAUTION Verify that the vial heater cover is cool to the touch before removing it. The the vial heaters can be at 130 C (or set point). Allow sufficient time for the vials to cool (up to 90 minutes) and then place the system in Service mode (see Place the MS/ETD System in Off Condition and Service Mode on page 4). Verify that the vial heater cover is safe to handle before attempting to remove the vial holders and reagent vials. Reagent Inlet Source Unit Vial Heater Cover Figure 3. ETD Module with back panel removed 8 MS/ETD System Getting Started Thermo Electron Corporation

2 ETD System Reagent Vial Installation Install/Exchange the Reagent Vials 3. Remove the vial heater cover (the screws that need to be removed require a 3/32 inch or 2.38mm hex driver). The vial heater cover is located on the right side of the ETD Module as you view it from the back of the MS/ETD System (Figure 3). 4. Remove the vial holder by gently pulling it out of the vial heater. Remove the empty vial if it is present. The vial holder is a cylindrical tube with a handling knob at one end and ribs along its length. These ribs prevent the vial holder from rotating once it is placed into the vial heater. Figure 4 shows the tab and ribs of a vial holder in the vial heater. Dispose of an empty fluoranthene vial in accordance with its MSDS. CAUTION Store and handle all chemicals in accordance with standard safety procedures. The Material Safety Data Sheet (MSDS) describing the chemicals being used are to be freely available to lab personnel for them to examine at any time. Material Safety Data Sheets (MSDSs) provide summarized information on the hazard and toxicity of specific chemical compounds. MSDSs also provide information on the proper handling of compounds, first aid for accidental exposure, and procedures for cleaning spills or dealing with leaks. Producers and suppliers of chemical compounds are required by law to provide their customers with the most current health and safety information in the form of an MSDS. Read the MSDS for each chemical you use. Dispose of all laboratory reagents in the appropriate manner (see the MSDS). Vial Heater Ribs Vial 1 Heater Vial Holder Tab Vial 2 Heater Figure 4. ETD Module with Vial Heater Cover removed Thermo Electron Corporation MS/ETD System Getting Started 9

5. Take a vial containing the ETD reagent (fluoranthene) from its box and place it into a vial holder. 6. Place this ETD reagent vial and its vial holder into the Vial 1 heater (top vial heater). Gently slide the vial holder into the vial heater. 7. Place the empty vial from the box into the other vial holder if an empty vial is not already installed. 8. Place this empty vial and its holder into the Vial 2 heater (bottom vial heater) if an empty vial is not already installed. CAUTION The empty vial in the Vial 2 heater is an integral part of the carrier/ci gas system. It is necessary to keep the carrier/ci gas system closed to the laboratory. If no vial is placed in the Vial 2 heater: a. The carrier/ci gas containing the reagent may escape to the laboratory causing a safety problem. b. The ETD Module will not operate correctly and the filament will burn out. 9. Replace the vial heater cover over the vial heaters. 10. Reinstall the back panel of the ETD Module (see the section Removing the ETD Main Access Panel and Side Access Panel in Chapter 4 of the ETD Module Hardware Manual). The ETD Module will not turn on unless the back panel is installed. 11. Start the system. a. Toggle the LTQ XL MS Service switch to the ON position. The system will boot in to Standby mode. The the Ion Source heater, flow restrictor and transfer line heaters will start heating. Monitor these temperatures in the Status View on the right side of the Tune Plus window (Figure 1). They will have green check marks when they have reached their operating temperatures. b. Check the Reagent Ion Source On check box in the Tune Plus Reagent Ion Source window (Figure 2) when the Ion Source heater, flow restrictor and transfer line heaters have green check marks in the Status View on the right side of the Tune Plus window (Figure 1). The MS/ETD System is now ready for use.

Chapter 3 Powering On the ETD Module, View Reagent Ion Spectra After the reagent vials have been installed, powered on the ETD Module by placing the LTQ XL MS in On mode. Turn On the Reagent Ion Source to view the Reagent Ion Spectra. This chapter contains the following sections: Powering ON the ETD Module Turning ON the Reagent Ion Source and Viewing Reagent Ion Spectra View Reagent Ion Spectra Thermo Electron Corporation MS/ETD System Getting Started 11

3 Powering On the ETD Module, View Reagent Ion Spectra Powering ON the ETD Module Powering ON the ETD Module Power On the ETD Module 1. Toggle the ETD Module Service switch to the Operating Mode (ON) position if it is not already in this position. 2. Toggle the LTQ XL MS Service switch to the Electronics Normal (ON) position. This turns the ETD Module ON if the ETD Module Service switch is already in the Operating Mode (ON) position. The ETD Module status LEDs indicate the ETD Module condition as it powers up: The ETD Module Power LED is solid green when the ETD Module power is ON. The Vacuum LED is solid green when the ETD Module vacuum pressure is in the proper range as measured by both the ion and convection gauges. The Filament LED is OFF when the filament is OFF. The ETD Module System LED will flash yellow when all of the following occur: a. The ETD Module electronics are ON. b. The restrictor, transfer line and ion source heaters are ON but not at their target temperatures. c. The reagent vial heaters are OFF. The ETD Module System LED switches to solid yellow when the restrictor, transfer line and ion source heaters reach their target temperatures and the reagent vial heaters are OFF. The carrier/ci gas turns ON. See Chapter 2 (Display PCB section) in the ETD Module Hardware Manual for more about the ETD Module status LEDs. 12 MS/ETD System Getting Started Thermo Electron Corporation

3 Powering On the ETD Module, View Reagent Ion Spectra Turning ON the Reagent Ion Source and Viewing Reagent Ion Spectra Turning ON the Reagent Ion Source and Viewing Reagent Ion Spectra To Turn On the Reagent Ion Source 1. Click the Reagent Ion Source portion of the MS/ETD System instrument control icon at the top of the Tune Plus window (Figure 5). The Reagent Ion Source window appears (Figure 6). Reagent Ion Source instrument control icon Figure 5. Tune Plus window showing the Reagent Ion Source instrument control graphic 2. Click the Reagent Ion Source On check box (Figure 6). If the reagent vials are not at their target temperature, a Please Wait window appears (Figure 7). The System LED flashes green to indicate that the reagent vial heaters are turned on but not at their target temperatures (the other heaters are at their target temperatures). The System LED is solid green when the reagent vial heaters reach their target temperatures. When the reagent vials reach their target temperature, voltage is applied to the ETD Module ion optics. The filament automatically turns on (the Filament On check box automatcally shows a check mark and its actual condition switches from Off to On) and the Filament LED illuminates solid green. Thermo Electron Corporation MS/ETD System Getting Started 13

3 Powering On the ETD Module, View Reagent Ion Spectra Turning ON the Reagent Ion Source and Viewing Reagent Ion Spectra Reagent Ion Source On View reagent ion spectra Figure 6. Reagent Ion Source window Figure 7. Reagent Vial NOT At Temperature! Please Wait window 14 MS/ETD System Getting Started Thermo Electron Corporation

3 Powering On the ETD Module, View Reagent Ion Spectra Turning ON the Reagent Ion Source and Viewing Reagent Ion Spectra View Reagent Ion Spectra To view the Reagent Ion Spectra, click the View Reagent Ion Spectra check box (Figure 6). Reagent ion peaks appear in the Tune Plus spectrum view (Figure 8). Spectrum view Figure 8. Tune Plus window showing the fluoranthene radical anion mass spectrum Thermo Electron Corporation MS/ETD System Getting Started 15

Chapter 4 Tuning the ETD Optics This chapter describes how to tune the ETD Reagent Ion Optics settings to obtain optimized Reagent Ion Source performance. This chapter contains the following sections: Tuning The Reagent Ion Source Saving Your ETD Tune Method Thermo Electron Corporation MS/ETD System Getting Started 17

4 Tuning the ETD Optics Tuning The Reagent Ion Source Tuning The Reagent Ion Source Automatically tuning the Reagent Ion Source assures the best ion optics settings for optimum transmission of reagent ions (fluoranthene). Automatically Tune the Reagent Ion Source 1. Open the Tune Plus window (Figure 9). Tune button Figure 9. Tune Plus window showing the fluoranthene radical anion mass spectrum 2. If the Reagent Ion Source is not on, turn it on as described in the section Turning ON the Reagent Ion Source and Viewing Reagent Ion Spectra on page 13. 3. Click View Reagent Ion Spectra (see Figure 6 on page 14). 4. Click the Tune button (Figure 9). The Tune window appears (Figure 10). The left side of the underlying view shows the Reagent Ion Spectra (fluoranthane). The center view shows one of the parameters that are automatically tuned (back lens potential tuned to maximize the m/z=202 signal intensity). 18 MS/ETD System Getting Started Thermo Electron Corporation

4 Tuning the ETD Optics Tuning The Reagent Ion Source Automatic tab Figure 10. Tune Plus with Tune window Automatic page displayed. 5. Click the Automatic tab in the Tune window if it is not already the active tab (Figure 10). The Automatic page of this window appears. 6. Click Start in the Automatic page of the Tune window. The system will begin automatically tuning the ion optics of the Reagent Ion Source. The Status portion of the window indicates that automatic tuning is completed by displaying the message, Optimization Complete. This message will also indicate the percentage change in the reagent ion signal (m/z=202) intensity relative to the prior value. A typical reagent signal intensity is about 1-2E7 in profile mode when the system has been cleaned and the ion volume is new. 7. Rerun Automatic Tune if the percentage change is greater than 20%. This is an iterative process. At some point there will be no more improvements in signal intensity. Thermo Electron Corporation MS/ETD System Getting Started 19

4 Tuning the ETD Optics Tuning The Reagent Ion Source Automatically tuning the Reagent Ion Source is the best method for most situations. In some cases it may be appropriate to perform manual tuning. Choose manual tuning to manually optimize Reagent Ion Optics Parameters and Reagent Ion Source parameters not automatically tuned such as Emission Current, Electron Energy, and CI gas pressure. Manual tuning is done by observing the effects of adjusting these parameters on the reagent ion signal intensity. Manually Tune the Reagent Ion Source 1. Click the Display Graph View icon in Tune Plus. 2. If the Reagent Ion Source is not on, turn it on, and open the Reagent Ion Source window as described in the section Turning ON the Reagent Ion Source and Viewing Reagent Ion Spectra on page 13. 3. Click View Reagent Ion Spectra (see Figure 6 on page 14). 4. Click the Reagent Ion Optics icon at the top of the Tune Plus window (Figure 11). The Reagent Ion Optics window appears (Figure 12). Reagent Ion Optics icon Figure 11. Tune Plus window showing the Reagent Ion Optics graphic 20 MS/ETD System Getting Started Thermo Electron Corporation

4 Tuning the ETD Optics Tuning The Reagent Ion Source Figure 12. Reagent Ion Optics window 5. Click the Tune button in Tune Plus (Figure 9). The Tune window appears. You can also tune the Reagent Ion Source. When the Reagent Ion Source window is open (Figure 6) click on the Tune button in Tune Plus. Thermo Electron Corporation MS/ETD System Getting Started 21

4 Tuning the ETD Optics Tuning The Reagent Ion Source Figure 13. Reagent Ion Source Tune window, manual view 6. Click the Manual tab in the Tune window (Figure 13) if the Manual Tune view is not already visible. 7. Check Reagent Ion from Vial 1. 8. Click Start. The display graph view (Figure 14) displays a plot of the reagent ion intensity. You can observe the response of the reagent ion intensity to changes in the lens parameters (Reagent Ion Optics window) and Emission Current, CI gas flow, and Electron Energy (Reagent Ion Source window). Adjust these parmeters to achieve the maximum reagent ion signal intensity. 22 MS/ETD System Getting Started Thermo Electron Corporation

4 Tuning the ETD Optics Tuning The Reagent Ion Source Display Graph View Figure 14. Tune Plus window showing the Display Graph view for manual tuning of the Reagent Ion Source Viewing the Reagent Ion Optics Settings To view the current Reagent Ion Optics settings 1. Click the Reagent Ion Optics instrument control icon in the Tune Plus window (Figure 15) to open the Reagent Ion Optics window. The parameters in the Reagent Ion Optics window have been optimized by the Auto Tune process. Thermo Electron Corporation MS/ETD System Getting Started 23

4 Tuning the ETD Optics Tuning The Reagent Ion Source Reagent Ion Optics instrument control icon Figure 15. Reagent Ion Optics window in Tune Plus 2. Click OK to close the Reagent Ion Optics window. 24 MS/ETD System Getting Started Thermo Electron Corporation

4 Tuning the ETD Optics Saving Your ETD Tune Method Saving Your ETD Tune Method After either automatic or manual tuning ( Tuning The Reagent Ion Source on page 18), save the ETD Tune parameters in a tune method. To save in the currently used tune file, click File>Save in Tune Plus. To save the parameters under a specific name, click File>Save As, enter the desired file name, and save this file to the desired location. Thermo Electron Corporation MS/ETD System Getting Started 25

5 Performing an ETD Infusion Experiment Chapter 5 Performing an ETD Infusion Experiment Procedures for performing an ETD infusion experiment are described in the following sections: Injection Control: Reagent Ion AGC Target and Ion Time Settings Obtaining an ETD Spectrum for Angiotensin I Optimizing the Reagent Ion Reaction Time Thermo Electron Corporation MS/ETD System Getting Started 27

5 Performing an ETD Infusion Experiment Injection Control: Reagent Ion AGC Target and Ion Time Settings Injection Control: Reagent Ion AGC Target and Ion Time Settings The number of reagent ions admitted into the ion trap of the LTQ XL mass spectrometer is regulated by the parameters in the Reagent tab in the Injection Control window (Figure 16). Open the Reagent tab in the Injection Control window by clicking the Injection Control instrument control graphic (Figure 17) (or click Setup>Injection Control>Reagent). Figure 16. Injection Control window The ETD Reagent Injection control consists of two parameters, the Reagent Ion Automatic Gain Control (AGC) Target value and the Maximum Injection time. The Reagent Ion Automatic Gain Control (AGC) Target value sets the target number of reagent anions to be injected into the trap in order to perform ETD. The default value for this parameter is 1E5. The Maximum Injection time specifies the maximum amount of time that the system allows for anions to be injected into the trap. The default value for this time is 100ms. The Reagent Ion Source injects reagent anions into the trap until the ETD AGC target is reached. The time allowed to reach the ETD AGC target can not exceed the Maximum Injection time (the Maximum Injection time takes precedence over the AGC target). If the AGC target is not reached due to the Maximum Injection time limit, the system displays an error message advising you that the AGC target has not been reached within the specified time limit (Maximum Injection time limit exceeded). This implies that the sensitivity of the Reagent Ion Source is too low. There are several ways of dealing with this error. 1. To increase the sensitivity of the source, run automatic tuning of the Reagent Ion Source (see Tuning The Reagent Ion Source on page 18). 28 MS/ETD System Getting Started Thermo Electron Corporation

5 Performing an ETD Infusion Experiment Injection Control: Reagent Ion AGC Target and Ion Time Settings 2. The sensitivity decrease might be due to a dirty ion volume. A sufficiently contaminated Ion Volume causes the Maximum Injection time limit to be exceeded. Clean or change the Ion Volume. See the ETD Module Hardware Manual for the procedure to do this. 3. Clean the Reagent Ion Source and its optics (see Chapter 4 in the ETD Module Hardware Manual for the procedure to do this). The sensitivity decrease might be due to a dirty Reagent Ion Source and its optics. A sufficiently contaminated Reagent Ion Source and its optics causes the Maximum Injection time limit to be exceeded. Clean or change the Reagent Ion Source and its optics. 4. The Emission Current can be increased. However, doing this may decrease the filament life. 5. Increase the Maximum Injection time limit. This is a temporary way to eliminate the error message. The Maximum Injection time limit can be increased up to the limits imposed by the overall scan cycle time. The Maximum Injection Time limit and AGC Target influence the ETD reaction results. Thermo Electron Corporation MS/ETD System Getting Started 29

5 Performing an ETD Infusion Experiment Obtaining an ETD Spectrum for Angiotensin I Obtaining an ETD Spectrum for Angiotensin I This section assumes that you are infusing Angiotensin I into the MS/ETD System according to the procedures in the LTQ XL Getting Started manual. The recipe for this solution is given in Appendix C, Angiotensin I Solutions on page 49. To Obtain an ETD Spectrum of Angiotensin I 1. Open the Tune Plus application (Figure 17). The Tune Plus window appears. On/Off/Standby Define Scan Injection Control instrument control graphic Figure 17. Tune plus window showing a mass scan of infused Angiotensin I On Off Standby 2. Click On/Off/Standby to On. The mass spectrometer scans the infused analyte and produces a mass spectrum (Figure 17). 3. Turn on the Reagent Ion Source as explained in Turning ON the Reagent Ion Source and Viewing Reagent Ion Spectra on page 13. 4. Click Define Scan. The Define Scan window appears (Figure 18). 30 MS/ETD System Getting Started Thermo Electron Corporation

5 Performing an ETD Infusion Experiment Obtaining an ETD Spectrum for Angiotensin I Figure 18. Define Scan window in Tune Plus 5. Enter the Parent ion m/z of the 3+ charge state of Angiotensin I in the n=2 line of the Define Scan window. The molecular weight of Angiotensin I (acetate hydrate) is 1296u and the (M + 3H) 3+ parent m/z=433.0. Thermo Electron Corporation MS/ETD System Getting Started 31

5 Performing an ETD Infusion Experiment Obtaining an ETD Spectrum for Angiotensin I Activation Type=ETD Figure 19. Define Scan window with the Activation Type pull-down active 6. Select ETD from the Activation Type pull down in the Define Scan window (Figure 19). 7. Click Apply in the Define Scan window (Figure 19). 8. Click OK in the Define Scan window (Figure 19). The Define Scan window closes and the ETD MS/MS spectrum of Angiotensin I appears (Figure 20). 32 MS/ETD System Getting Started Thermo Electron Corporation

5 Performing an ETD Infusion Experiment Obtaining an ETD Spectrum for Angiotensin I Figure 20. ETD MS/MS spectrum of Angiotensin I Thermo Electron Corporation MS/ETD System Getting Started 33

5 Performing an ETD Infusion Experiment Optimizing the Reagent Ion Reaction Time Optimizing the Reagent Ion Reaction Time Typically the system default Reagent Ion Reaction Time of 100ms is appropriate for most analyses. In some cases it is helpful to obtain an optimized Reagent Ion Reaction Time for your specific analyte. The procedures presented in this section assume that your system is generating the reagent ions as described in Turning ON the Reagent Ion Source and Viewing Reagent Ion Spectra on page 13. To Obtain an Optimized Reagent Ion Reaction Time 1. Turn On ETD activation on for the analyte of interest (Angiotensin I in this case). 2. Open the Define Scan window. 3. Click Tune (Figure 21). Reagent Ion Reaction Time tab Tune Total Ion Current (TIC) Figure 21. Tune and Define Scan windows open in Tune Plus 4. Click the Reagent Ion Reaction Time tab in the Tune window (Figure 21). 5. Optimize on either the Total Ion Current of the product ions (TIC) or a particular Product Ion Mass. 34 MS/ETD System Getting Started Thermo Electron Corporation

5 Performing an ETD Infusion Experiment Optimizing the Reagent Ion Reaction Time a. Optimize on the Total Ion Current of the product ions (TIC). i. Click TIC in the What To Optimize On section of the Tune window (Figure 21). ii. Click Start. The software generates a graph of the product ion TIC versus reaction time. The Status portion of the Tune window shows the optimized Reagent Ion Reaction Time after the Tune process is completed. iii. A pop up dialog asks if you want to accept the optimized value. If you accept the optimized value, the Reagent Ion Reaction time is set to this optimized value in the Define Scan window. Otherwise, it is restored to its previous value. The Reagent Ion Reaction Time is now optimized based on the Total Ion Current. b. Optimize on the Produuct Ion Mass. i. Click Produuct Ion Mass in the What To Optimize On section of the Tune window (Figure 22). The window adjacent to Product Ion Mass becomes active. ii. Enter the m/z of the fragment of interest. iii. Click Start. The software generates a graph of intensity of the m/z of interest versus reaction time. The Status portion of the window shows a Reagent Ion Reaction Time after the Tune process is completed. iv. A pop up dialog asks if you want to accept the optimized value. If you accept the optimized value, the Reagent Ion Reaction time is set to this optimized value in the Define Scan window. Otherwise, it is restored to its previous value. The Reagent Ion Reaction Time is now optimized based on a particular Product Ion Mass. Thermo Electron Corporation MS/ETD System Getting Started 35

5 Performing an ETD Infusion Experiment Optimizing the Reagent Ion Reaction Time Product Ion Mass Figure 22. Tune window showing Product Ion Mass selected for Reagent Ion Reaction Time Optimization 36 MS/ETD System Getting Started Thermo Electron Corporation

Chapter 6 Creating an Xcalibur Instrument Method That Uses ETD Activation To create an Xcalibur instrument method that uses ETD activation 1. Open the Xcalibur application from the Windows desktop. The Roadmap Home Page appears (Figure 23). Figure 23. Xcalibur Roadmap Home Page 2. Click the Instrument Setup icon in the Xcalibur Roadmap-Homepage (Figure 23). Thermo Electron Corporation MS/ETD System Getting Started 37

6 Creating an Xcalibur Instrument Method That Uses ETD Activation 3. Click General MS or MSn in the Select Experiment Type section of the New Method view (Figure 24). Figure 24. New Method view in Xcalibur Instrument Setup 4. Click the MS Detector Setup tab if this is not already the selected tab in the Untitled-Instrument Setup window (Figure 25). 5. Load the appropriate Tune Method (such as a method saved per Saving Your ETD Tune Method on page 25). 38 MS/ETD System Getting Started Thermo Electron Corporation

6 Creating an Xcalibur Instrument Method That Uses ETD Activation Figure 25. Xcalibur MS Detector Setup view Thermo Electron Corporation MS/ETD System Getting Started 39

6 Creating an Xcalibur Instrument Method That Uses ETD Activation Scan Events Scan Event bar Dependent Scan Settings (active when Dependent Scan is checked) Figure 26. Xcalibur Instrument Setup 6. Choose Scan Events to be 2 or more in the Segment 1 Settings portion of the MS Detector Setup view. (Figure 26). 7. Select the Scan Event 2 bar (or the bar for a Scan Event >2), see Figure 26. 8. Click Dependent Scan at the lower left corner of the MS Detector Setup view (Figure 26). The adjacent Settings button becomes active. 9. Click Settings. A Data Dependent Settings window appears (Figure 27). 10. In the Data Dependent Settings window (Figure 27): a. Choose Scan Event>Activation in the menu on the left side of the window. 40 MS/ETD System Getting Started Thermo Electron Corporation

6 Creating an Xcalibur Instrument Method That Uses ETD Activation b. Select an Activation Type. Choose ETD, CID or PQD. The choice of Activation Type may be different for each Scan Event. c. For Default Charge State use values of 5 or more. d. For Isolation Width use values between 2-3. e. Leave the Normalized Collision Energy and Activation Q at their default values. They are not applicable to ETD. f. The Activation Time is either left at its default value or chosen as discussed in Optimizing the Reagent Ion Reaction Time on page 34. Figure 27. Data Dependent Settings window in MS Detector Setup 11. Click File>Save As to save your Xcalibur method under the file name of your choice (Figure 28). This method can be chosen and run when Sequence Setup is chosen in the Xcalibur Roadmap Home Page (Figure 23). Refer to your Thermo Electron Corporation MS/ETD System Getting Started 41

6 Creating an Xcalibur Instrument Method That Uses ETD Activation Xcalibur software online help and the LTQ XL MS Getting Started manual for information about the other Data Dependent settings. Figure 28. Save As window in Xcalibur Instrument Setup, MS Detector Setup view 42 MS/ETD System Getting Started Thermo Electron Corporation

Chapter 7 Turning Off the Reagent Ion Source: What to Expect The Reagent Ion Source controls are accessed as discussed in Place the MS/ETD System in Off Condition and Service Mode on page 4. When you deselect the Reagent Ion Source On check box in the Reagent Ion Source window (Figure 29) the ETD source and Reagent Heaters are placed in Standby. Reagent Ion Source On check box Figure 29. Reagent Ion Source On control in the Reagent Ion Source window Thermo Electron Corporation MS/ETD System Getting Started 43

7 Turning Off the Reagent Ion Source: What to Expect When the ETD Module is placed in Standby, the filament and vial heaters turn off. Simultaneously a valve opens to allow nitrogen gas to cool the reagent vials. This cooling nitrogen runs until the vials reach 70 C. You will hear the sound of escaping gas when this occurs. This sound is a normal part of the instrument operation. CAUTION The Reagent Vials are too hot to handle after the cooling nitrogen turns off at a vial temperature of 70 C. Verify that the reagent vials have cooled to a safe temperature before handling them. This can take up to 90 minutes after the cooling nitrogen has turned off. Other conditions that will cause the ETD Module to remain in standby: 1. Attempting to turn on the Reagent Ion Source when the restrictor heater, transfer line heater, and the source heater are not at their target temperatures. 2. Whenever either the LTQ XL MS or the ETD Module goes into standby mode. Reagent vial nitrogen cooling will turn on if the vials are at an elevated temperature. On Off Standby Exception: if the MS/ETD System is placed in Standby by clicking the Standby button in Tune Plus (see Standby icon in the margin), there is a 1 hour delay before the cooling nitrogen turns On. 3. Whenever the pressure in the LTQ XL MS or the ETD Module exceeds its protection limit. Reagent vial nitrogen cooling will turn on if the vials are at an elevated temperature. 4. Whenever the flow of reagent ions becomes insufficient as determined by the AGC setting. When this occurs the MS/ETD System completes the Xcalibur Sequence step in progress before going into standby mode. This prevents the loss of analysis results that may not be affected by the reduced reagent ion flow. 44 MS/ETD System Getting Started Thermo Electron Corporation

Appendix A Fluoranthene Fluoranthene is used as the Electron Transfer Dissociation (ETD) reagent in the ETD Module portion of the MS/ETD System. The fluoranthene radical anion is generated according to the reaction shown in Figure 30. + e - fluoranthene Figure 30. ETD Reagent (fluoranthene radical anion) generation from fluoranthene Fluoranthene is potentially hazardous. Use it in accord with its Material Safety Data Sheet (MSDS). CAUTION Store and handle all chemicals in accordance with standard safety procedures. The Material Safety Data Sheet (MSDS) describing the chemicals being used shoud be freely available to lab personnel for them to examine at any time. Material Safety Data Sheets (MSDSs) provide summarized information on the hazard and toxicity of specific chemical compounds. The MSDS also provides information on the proper handling of compounds, first aid for accidental exposure, and procedures for cleaning spills or dealing with leaks. Producers and suppliers of chemical compounds are required by law to provide their customers with the most current health and safety information in the form of an MSDS. Read the MSDS for each chemical you use. Dispose of all laboratory reagents in the appropriate way (see the MSDS). The fluoranthene in your ETD Reagent Kit (Thermo Reagent Kit P/N 98000-62008, Thermo fluoranthene P/N 00301-01-0013) is Sigma/Aldrich Supelco #48535. The fluoranthene MSDS is obtained by clicking the MSDS link at: http://www.sigmaaldrich.com/catalog/search/productdetail/supelco/48535 Thermo Electron Corporation MS/ETD System Getting Started 45

A Fluoranthene Thermo supplies fluoranthene as a two vial kit. One vial contains 150mg of fluoranthene and the other is the required empty vial. 46 MS/ETD System Getting Started Thermo Electron Corporation

Appendix B Troubleshooting The ETD Module uses consumables: the Ion Volume, filamant and the ETD reagent (fluoranthene). After a period of time, the ion volume might need to be cleaned or replaced, the filament might need to be replaced and the ETD reagent will need to be replenished. As the consumables are depleted, the ETD reagent m/z peak intensity diminishes (m/z=202 in negative mode). Periodically checking this peak intensity is a good way to monitor the ETD Module consumables. Follow the procedure in Turning ON the Reagent Ion Source and Viewing Reagent Ion Spectra on page 13 to view the ETD reagent m/z signal. The ion volume and filament should last for about 400-500 hours of operation. A vial of fluoranthene reagent should last for several months or more. Table 1 discusses some problem symptoms, their causes, and fixes. Table 1. ETD Module problem symptoms, causes and fixes Symptom Cause Fix No ions at 202 m/z with the emission current at the correct level. The 202 m/z signal intensity drops slowly over several days when the emission current is at the correct level. A system error message advising that the maximum injection time has been reached for the ETD AGC. Sudden and complete drop of 202 m/z level, low emission current. The 202 m/z is outside of the mass range. The Ion Volume needs to be cleaned or replaced. The AGC target has not been reached within the specified time limit. The Ion Volume needs to be cleaned or replaced. The filament may have just blown out. Set the starting mass lower. Clean or replace the Ion Volume when the ion injection time is over 100 ms. 1. Clean the Ion Volume. 2. Increase the Maximum Injection time limit. See the section Injection Control: Reagent Ion AGC Target and Ion Time Settings on page 28 for more explanation. Check the filamant. Replace it if necessary. Thermo Electron Corporation MS/ETD System Getting Started 47