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LTQ OrbitrapOperations, Maintenance, and

Method Building

Craig Dufresne, Ph.D.Thermo Scientific Training Institute

The world leader in serving scienceChapter 1

Operations

3

Prepping the Instrument for Data Collection

Calibration Tuning Instrument Method Building Xcalibur Sequence Building

4

Steps to Success

Tune- Glu Fibrinopeptide B- Human Endorphin- Ubiquitin

Calibration

Calibration

The world leader in serving scienceChapter 2

Calibration

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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

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Sigma Sells Cal Mix !!!

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Preparing for Calibration

Target Values Ion Trap Orbitrap

MS 3e4 3e5 – 1e6MSn 1e4 5e4 – 2e5SIM 1e4 5e4 – 1e5ZOOM 3e3

MAX IT Time

MS 100 ms 500 msMSn 100 ms 300 msSIM 50 ms 300 msZOOM 50 ms

Cap Temp = 275 °CESI Source

IT Time Variance – 10% or LessFresh Cal Mix

Wrap in Aluminum Foil (Light Sensitive) and Store in Cold (Heat Sensitive)

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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)

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Using Proteome Discoverer to Reveal Mass Error Information

Positive Shift Due to Space Charge

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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

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Maintenance

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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.

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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.

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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.

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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.

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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

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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.

Recommended Maintenance Schedule

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Maintenance: API Stack Cleaning

1. First, put the instrument in standby.

2. Turn off the electronics (orange switch) and then the main power (green switch).

3. Pull out the API stack.

4. Remove the skimmer and then the tube lens

API Stack, Back View(with the skimmer and tube lens removed)

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Skimmer Lens and Tube Lens (Front View)

skimmer lens

tube lens

Pin for orientation

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Tube Lens (In API Stack)

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Skimmer (Put In API Stack After Tube Lens)

1. Put API stack back into the instrument.

2. Unplug one of the LTQ forepumps from the LTQ.

3. Turn on the main power (green), wait and then plug the forepump back into the LTQ

4. Turn on the electronics (orange).

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Maintenance ChartDaily Weekly Monthly Quarterly Biannually Yearly As needed Check

Check Valves and Seals on LC Pump           x    

New Rotorseal on Autosampler           x    

Autosampler needle             x  

Insure Center Piercing of Vial               x

UV Lamp (High Use)             x  

LTQ Rotorseal (injection port)             x  

Clean API Stack     x         

Replace Ion Transfer Tube         x      

Change Roughing Pump Oil *       x        

Clean Fan Inlets         x      

Change Mobile Phases   x            

Change Mobile Phase Filters           x    

Remove Precipitants from Source x              

Calibrate Electron Multipliers*     x          

Calibrate Orbi Mass x

Check Transmission x

Full Calibration of the LTQ*       x        

Change Chiller Fluid x

Oil mist filters           x    

Ballast the pumps             x  

The world leader in serving scienceChapter 4

Instrument Method Building

26

LTQ-Orbitrap Parallel Detection Scheme

Pre-scan InjectIT

FT

MS/MS#3

400 500 600 700 800 900 1000 1100 1200

584.81877.42

748.44

“Free”(in parallel)

MS/MS Data

MS/MS#2

MS/MS#1

Snap Shot@ 15k RP

m/z

Time 0.2 0.4 0.6 0.8 1.0s

Continue FT Acquisition @ 60k RP

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MiPS

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Monoisotopic Precursor Selection

1. Changes Preview Spectra from Set of Peaks to Set of Isotopic Clusters

2. Calculates Monoisotopic Peak for each Cluster3. Decides whether to do MS/MS

1. If MS/MS is Done, then the Monoisotopic Peak is Put in the Scan Header

4. Places Monoisotopic Peak on Dynamic Exclusion List if Fragmented. Prevents Redundant Data Collection in Future Scan Cycles

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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.

113

22

3

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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

Control_5b #2588 RT: 34.27 AV: 1 NL: 1.91E5F: FTMS + p ESI Full ms [400.00-2000.00]

933.4 933.6 933.8 934.0 934.2 934.4 934.6 934.8 935.0 935.2 935.4 935.6 935.8 936.0 936.2 936.4 936.6 936.8 937.0m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

934.15

934.49

933.82

934.82

935.16

935.49

935.82

934.33 936.16934.07 934.63

933.91

936.49 936.83

Averagine = C4.94H7.76N1.36O1.48S0.04

M.W. Senko, et al. JASMS 1995, 6, 229-233.

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Dynamic ExclusionRT: 22.03 - 22.96

22.1 22.2 22.3 22.4 22.5 22.6 22.7 22.8 22.9Time (min)

0

5

10

15

20

25

30

35

40

45

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70

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Rel

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bund

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22.57

22.7822.21 22.91

22.59

22.2422.17

22.55

22.80

22.62

Prevents Redundant Data

Collection Between Scans

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Dynamic Exclusion Parameters

How many MS/MS per peptide

How long to look for the peptides(1.5 * LC Peak Width)

How long to ignore the peptide once found(1.5 * LC Peak Width)

Set to 500

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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.

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Scan Header Information – MS/MS

Control_3 #2551 RT: 0.0029Total Ion Current: 15429.54Scan Low Mass: 275.00Scan High Mass: 2000.00Scan Start Time (min): 32.73Scan Number: 2551Base Peak Intensity: 3332.23Base Peak Mass: 1230.84Scan Mode: ITMS + c ESI d Full ms2 1038.58@cid35.00 [275.00-2000.00]LTQ Orbitrap XL Data:=====================AGC: OnMicro Scan Count: 1Ion Injection Time (ms): 100.000Scan Segment: 1Scan Event: 2Master Index: 1Elapsed Scan Time (sec): 0.34API Source CID Energy: 0.00Average Scan by Inst: NoCharge State: 3Monoisotopic M/Z: 1038.2439MS2 Isolation Width: 3.0FT Analyzer Settings:FT Analyzer Message:FT Resolution: 0Conversion Parameter I: 0.000Conversion Parameter A: 0.000

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Acknowledgements

Detlef Schuman Jonathan Ferguson

www.thermo.com/education