LTQ Orbitrap Operations, Maintenance, and Method Building

1. LTQ Orbitrap Operations, Maintenance, and Method Building Craig Dufresne, Ph.D. Thermo Scientific Training Institute

2. Chapter 1 Operations

3. Prepping the Instrument for Data Collection • Calibration • Tuning • Instrument Method Building • Xcalibur Sequence Building

4. Steps to Success Calibration Calibration • Tune • Glu Fibrinopeptide B • Human Endorphin • Ubiquitin

5. Chapter 2 Calibration

6. 5 Types of Calibration • LTQ Electron Multipliers • 1 / Month • Requires MRFA, but not Caffeine and Ultramark • LTQ Full Calibration • 1 / Quarter • Use new recipe for Calibration Mix (Separate Positive and Negative Solutions) • Orbitrap Mass Calibration • 1 / Week for most machines • Orbitrap Storage Calibration (LTQ to C-Trap)Orbitrap Transmission Calibration (C-Trap to Orbitrap) • Check 1 / Year • Calibrate if score fails All Calibrations REQUIRE A STABILE ELECTROSPRAY

7. Sigma Sells Cal Mix !!!

8. Preparing for Calibration Target Values Ion Trap Orbitrap MS 3e4 3e5 – 1e6 MSn 1e4 5e4 – 2e5 SIM 1e4 5e4 – 1e5 ZOOM 3e3 MAX IT Time MS 100 ms 500 ms MSn 100 ms 300 ms SIM 50 ms 300 ms ZOOM 50 ms Cap Temp = 275 °C ESI Source IT Time Variance – 10% or Less Fresh Cal Mix Wrap in Aluminum Foil (Light Sensitive) and Store in Cold (Heat Sensitive)

9. Using Lock Mass to Enhance Calibration • Why Use Lock Mass? • Extend time between calibrations • Lower mass errors due to space charge • Total mass error = inherent mass error + error from space charging • What Do I Lock On? • Choose one lock • Software does absolute shift not shaped • Usually a lab air peak (371 or 445 is most common) • Needs to present across gradient (often problematic)

10. Using Proteome Discoverer to Reveal Mass Error Information Positive Shift Due to Space Charge

11. How Does it Work • Lock Masses are isolated in the LTQ with a reduced inject time • The C-Trap is filled with them. The reduced inject time adjusts the lock mass intensity to approximately 5% • The LTQ and then the C-Trap are filled a second time using the full inject time • Ion populations are mixed and shot into the Orbitrap • After the transient acquisition and data processing the already existing calibration function is adapted based on the measured frequencies of the lock masses • Lock masses will be searched in a 10 ppm window initially • If the lock masses are not found, the instrument will start to expand the search window and injection times. This can lead to lower coverage's in complex mixtures. If no locks are found, then the external calibration is used.

12. How to Use an Internal Calibration 1. Check this box 2. Enter accurate masses of known compounds in here

13. Chapter 3 Maintenance

14. Daily Maintenance Daily: • Check the vacuum gauge pressures and make sure the vacuum system is operational. Typical values are: • Convectron pressure: 1 - 1.5 Torr • Ion gauge pressure: 0.75 – 1.5 x 10-5 Torr • Last ion gauge pressure (Penning gauge): < 6 x 10-10 • Pirani gauge pressure: ~1 Torr • If bottled nitrogen gas is being used, check nitrogen gas pressure. The recommended value for the LTQ is 80 – 120 psi. • Check the ESI fused silica sample tube. Make sure the fused silica is not elongated. Trim the tube if necessary. • Check HPLC solvent levels. • Make sure the solvent waste bottle for drain tube is empty. • After completion of analysis place system in stand-by mode.

15. Weekly Maintenance Weekly: • Check the mechanical pump oil level. • Fill the mechanical pump as needed. • Ballast the mechanical pump to drain oil back into pump. • Replace LC solvents to maintain low background levels. • Clean the ion transfer tube.

16. Monthly Maintenance Monthly: • Check the helium gas tank pressure. The recommended value is 30 – 50 psi. Replace the tank as needed. • Check the nitrogen gas supply. Replace or refill as needed. • Exchange ESI fused silica sample tube if necessary. • Calibrate LTQ electron multipliers. After calibration: - Clean or replace ion transfer tube. - Clean the API stack. Wipe tube lens and skimmer clean with methanol or isopropanol. Do not sonicate the tube lens and skimmer in acidic solutions! • Tune instrument after cleaning the API stack (optional). • Check the fluid in the chiller.

17. Quarterly Maintenance Quarterly: • Calibrate the LTQ. After calibration: - Clean or replace the ion transfer tube. - Clean the API stack. • Replace mechanical pump oil. • West Marine Fluid Extraction Kit • Back up data and defragment the PC. • Check the Thermo website for available software updates/fixes: - Thermo Fisher Scientific Fast LC and LC/MS Support http://www.thermo.com/ => Search for “Fast LC and LC/MS” - Thermo Fisher Scientific Knowledge Base http://198.173.130.188/thermofinnigandb/thermo.nsf • Thermo Fisher Scientific Customer Download Site http://mssupport.thermo.com/ Yearly: • Exchange lubricant reservoir in turbopumps.

18. Biannual Chiller Maintenance • Change the Chiller Fluid Twice a Year • North America • Thermo Neslab 610000000005 Water Treatment Kit • NALCO 7330 for Biological Control (300 uL / Gallon) • Thermo100 for Corrosion Control (10 mL / Gallon) • Needs about 2 Gallons for the Fluid Change • Europe • Thermo Scientific 1245920 ‘Scania Engine Corrosion Inhibitor • Add 100 mL to the Chiller

19. Recommended Maintenance Schedule Notes: • Heavy use and dirty samples may require more frequent cleaning of the ion transfer tube and the API stack. • If the sensitivity starts to drop off and can not be restored by replacing the ion transfer tube, clean the API stack as needed. • As needed, perform system bakeout after shutting down instrument for cleaning (10-14 hour procedure): 1. With the system in standby, set the bakeout time by entering the desired time with the up/down arrow keys on the bakeout time. 2. Start the bakeout procedure by pressing the green start button on the right of the timer. The LTQ Orbitrap indicates a running bakeout procedure by flashing Vacuum and System LEDs on the front panel of the instrument. 3. The bakeout procedure is complete when the status LEDs (System and Vacuum) on the front panel has stopped flashing.

20. Maintenance: API Stack Cleaning API Stack, Back View(with the skimmer and tube lens removed) • First, put the instrument in standby. • Turn off the electronics (orange switch) and then the main power (green switch). • Pull out the API stack. • Remove the skimmer and then the tube lens

21. Pin for orientation Skimmer Lens and Tube Lens (Front View) skimmer lens tube lens

22. Tube Lens (In API Stack)

23. Skimmer (Put In API Stack After Tube Lens) • Put API stack back into the instrument. • Unplug one of the LTQ forepumps from the LTQ. • Turn on the main power (green), wait and then plug the forepump back into the LTQ • Turn on the electronics (orange).

24. Maintenance Chart

25. Chapter 4 Instrument Method Building

26. 0.2 0.4 0.6 0.8 1.0s Time Snap Shot @ 15k RP FT Continue FT Acquisition @ 60k RP Pre- scan MS/MS #1 MS/MS #2 MS/MS #3 IT Inject 584.81 877.42 “Free” (in parallel) MS/MS Data 748.44 400 500 600 700 800 900 1000 1100 1200 m/z LTQ-Orbitrap Parallel Detection Scheme

27. MiPS

28. Monoisotopic Precursor Selection • Changes Preview Spectra from Set of Peaks to Set of Isotopic Clusters • Calculates Monoisotopic Peak for each Cluster • Decides whether to do MS/MS • If MS/MS is Done, then the Monoisotopic Peak is Put in the Scan Header • Places Monoisotopic Peak on Dynamic Exclusion List if Fragmented. Prevents Redundant Data Collection in Future Scan Cycles

29. Viewing a Spectra as Clusters MiPS processes the preview scan and groups peaks into isotopic clusters rather than individual peaks. This prevents redundant data being collected in the same scan cycle. 1 1 3 2 2 3

30. Averagine = C4.94H7.76N1.36O1.48S0.04 M.W. Senko, et al. JASMS 1995, 6, 229-233. Calculates Monoisotopic Peak from Cluster • Runs ‘Averagine’ Analysis on Isotopic Cluster • Determines MW (Hence, Unassigned Charge States) • Calculates Theoretical Shape (See Red Circles) • Measures Fit Factor If the Fit Factor is too Low, Skip MS/MS of the Peak

31. RT: 22.03 - 22.96 100 22.57 95 90 85 80 75 70 65 60 55 Relative Abundance 50 45 40 35 30 25 20 15 22.62 22.59 22.55 10 22.80 5 22.78 22.21 22.91 22.17 22.24 0 22.1 22.2 22.3 22.4 22.5 22.6 22.7 22.8 22.9 Time (min) Dynamic Exclusion Prevents Redundant Data Collection Between Scans

32. Dynamic Exclusion Parameters How many MS/MS per peptide How long to look for the peptides (1.5 * LC Peak Width) Set to 500 How long to ignore the peptide once found (1.5 * LC Peak Width)

33. Setting the Mass Widths • MiPS on • Set to 10 ppm for each • MiPS places the monoisotopic m/z on the list. All ions compared to this will use their monoisotopic m/z also. • MiPS off • Set to 0.8 and 1.8 • This range generally covers the isotopic cluster size and prevents collecting redundant data of isotopes.

34. Scan Header Information – MS/MS Control_3 #2551 RT: 0.0029 Total Ion Current: 15429.54 Scan Low Mass: 275.00 Scan High Mass: 2000.00 Scan Start Time (min): 32.73 Scan Number: 2551 Base Peak Intensity: 3332.23 Base Peak Mass: 1230.84 Scan Mode: ITMS + c ESI d Full ms2 1038.58@cid35.00 [275.00-2000.00] LTQ Orbitrap XL Data: ===================== AGC: On Micro Scan Count: 1 Ion Injection Time (ms): 100.000 Scan Segment: 1 Scan Event: 2 Master Index: 1 Elapsed Scan Time (sec): 0.34 API Source CID Energy: 0.00 Average Scan by Inst: No Charge State: 3 Monoisotopic M/Z: 1038.2439 MS2 Isolation Width: 3.0 FT Analyzer Settings: FT Analyzer Message: FT Resolution: 0 Conversion Parameter I: 0.000 Conversion Parameter A: 0.000

35. Acknowledgements • Detlef Schuman • Jonathan Ferguson www.thermo.com/education