1. LTQ Orbitrap Series: Calibration, Tuning, Operations
This talk will cover the calibration, tuning, and operation of the LTQ-Orbitrap series of instruments.
Calibration sets the correct values on the mass spectrometer components to produce a proper qualitative spectrum.
Tuning adjusts the voltages on the source and ion optics to maximize the instrument’s sensitivity.
LTQ Orbitrap Series: Calibration, Tuning, Operations
I Orbi

2. Agenda Preparation for Calibration Mass Spectrometer Calibration
Solutions
Tuning for Calibration
Mass Spectrometer Calibration
Lock Mass
Sample Tuning
ETD – Instrument Preparation
Testing LC-MS System Performance
This presentation will cover the following topics: see the slide.

3. Tuning vs. Calibration Tuning …Compound optimization
Getting ions efficiently from source to ion trap
Tune file must be manually saved after the tuning process
Compound / Application / Method dependent
Can have multiple tune files per method using segments
Calibration …Mass spectrometer optimization
The setting of parameters to insure the instrument runs within specifications
One active calibration at any one time
Good calibration routines are saved automatically
“Mass calibration” – adjustment of m/z scale only
Tuning and Calibration are distinctly different. Calibration is qualitative and tuning is quanitative.

4. Preparation for Calibration

5. Prepare for Calibration (Ready, Set, Go!)
Instrument must be warmed up for 80 minutes in the respective polarity
Scan in FTMS mode for 80 minutes prior to calibration
Does not require a spray voltage or liquid flow
Instrument reset and changing polarity DO reset the timer
Use correct calibration solution for your instrument
The warming is required since the Orbitrap is heated, but the electronics are cold.

6. Calibration Solution – Orbitrap
Directions are in “Getting Started Manual”
Positive ion mode:
Caffeine Methanol Solution (Sigma C6035-1ML)
MRFA (Met-Arg-Phe-Ala Acetate Salt) (Sigma M1170-5MG)
Ultramark® (ABCR AB172435)
N-Butylamine (needed for LTQ Velos / Velos Pro) (Sigma ML)
Negative ion mode:
Sodium Dodecyl Sulfate (Sigma L G)
Sodium Taurocholate Hydrate (Sigma T MG)
Ultramark® 1621
If using other supplier, insure mass spec grade
Please be aware that manuals are shipped online on the instrument computer.

7. Tips for Preparing Calibration Solutions
Make final calibration solution just prior to calibration
Use a light protected flask or vial for calibration solution storage
Use only solvents that have been dedicated & controlled for the MS
Do not use community solvents or chemicals
Only use glass or metal syringes when pipetting glacial acetic acid
Possible contamination from plastic pipette tips
Dedicated syringes
Emphasize that out of date or poorly made solutions are on the biggest problems blocking a successful calibration.

8. LTQ Orbitrap XL, Discovery, Classic Calibration Solutions
88322
Pierce LTQ ESI Positive Ion Calibration Solution
Solution of 20µg/mL caffeine, 1µg/mL MRFA and 0.001% Ultramark
Pierce now offers these calibration solutions.
The LTQ ESI Positive Ion Calibration Solution can be used to calibrate the LTQ series, the LTQ Orbitrap series, the LXQ, LCQ FLEET and the Exactive Mass Spectrometer instruments. The ESI Negative Ion Calibration Solution can be used to calibrate the LTQ series, LTQ Velos series, the LTQ Orbitrap series and Exactive Mass Spectrometer instruments.
88324
Pierce Negative Ion Calibration Solution
Solution of 2.9µg/mL sodium dodecyl sulfate, 5.4µg/mL sodium taurocholate and 0.001% Ultramark 1621

9. Orbitrap Velos and Elite Calibration Solutions
88323
Pierce LTQ Velos ESI Positive Ion Calibration Solution
Solution of 2µg/mL caffeine, 1µg/mL MRFA, 0.001% Ultramark 1621 and % n-butylamine
The LTQ Velos ESI Positive Ion Calibration Solution can be used to calibrate the LTQ Velos/Velos Pro, the LTQ Orbitrap Velos/Orbitrap Velos Pro, the Orbitrap Elite, and the QExactive mass spectrometer instruments. While there are two positive solutions, there is only one negative solution.
88324
Pierce Negative Ion Calibration Solution
Solution of 2.9µg/mL sodium dodecyl sulfate, 5.4µg/mL sodium taurocholate and 0.001% Ultramark 1621

10. Preparation for Calibration: LTQ Tuning

11. General Tips LTQ Tuning
Use an IonMax source for LTQ calibration (not a Nanospray)
Properly tune ESI source prior to mass spec. calibration
The ion source settings are optimized for the flow rate to be used (refer to next slide)
You must have a stable spray: the API stability evaluation should be used (below 10% RSD)
Let instrument scan scans before making a stability determination
The preparation for the calibration procedure is a crucial step for its successful completion.
Make sure you use a fresh calibration solution. Please follow the procedure from the manual for the solution preparation. It is essential, that the calibration solution contains all calibrant compounds!
If you plan to calibrate in positive or negative ion modes, check that you use the correct calibration mixture and that you have the masses of calibrants calculated correctly; pay special attention to the difference in mass caused by a difference of 1 electron – your Orbitrap might see that small difference!
Start infusing the calibration solution into the and wait for the signal to stabilise. Adjust the probe position if necessary to get a stable and intense signal. You can monitor the stability of the electrospray by watching the values of ‘IT’ (inject time) and intensity of the ‘NL’ (normalised largest peak).

12. Example of ESI Source Settings for MS Calibration
Calmix flow: ~ 3-5 µL/min
Orbitrap XL
(+) mode
(-) mode
Lower Calmix syringe flow  lower gas flows and higher Spray voltage
For negative ion mode: usually Calmix flow >10uL/min. 12

13. LTQ Positive Ion Spectrum (mass range 130-2000 m/z)
If you use the HESI probe, make sure the temp is not too high, or it degrades the calibrant. Set it to lowest temp.

14. LTQ Velos Positive Ion Spectrum (Low mass range)
Caffeine fragment
If the N-Butylamine is not visible, it may be necessary to lower the Multipole RF from 800 to 600 or 400 Vp-p.
N-Butylamine

15. Ion Optics - Orbitrap Velos and Elite
Make sure the difference between
Mulitpole 00 and 0 are at least 7 volts
For calibration, the user can change this value to 60% to make more 138 which may make calibration easier to pass.
S-Lens value affects the ion ratio’s. A lower RF% will lower the higher mass response.
S-Lens: ~45% is recommended for tuning/calibration.
When running samples, a lot of proteomics users raise the S-Lens RF to increase upper m/z transmission, with the addition of a small amount of in source fragmentation. Most protein characterization groups will drop the value down almost to 30% and completely eliminate all in source fragmentation.
The recommended setting is 800 volts for samples. In some cases, this setting may be to too high to produce a strong 74 peak for calibration and may need to be dropped to 600 volts temporarily. Non-Velos instruments should be run at 400 volts. 15

16. Mass Spectrometer Calibration

17. Calibration Backup BACK UP CURRENT CALIBRATION
As the calibration procedure automatically replaces original calibration file, it is sometimes useful to keep the original file backed up. To do this please go to File/Backup Current Calibration.

18. Good Calibration Practice
Issue: persistent contamination with Ultramark
Note: You contaminate just the source, not the instrument!
Prevention of contamination:
Use a dedicated syringe and infusion line for calibration
Use a dedicated ion transfer tube for calibration routine
If calibration done with the IonMax source, your Nanospray source is safe
If Orbitrap mass (only) calibration done with the static nanospray – no contamination issue exists
The most frequently quoted issue related to the calibration procedure is an occasional persistent contamination of the source with the polymer (ultramark) species. It is important to realise that the contamination happens in the source region, and that the other parts of the LTQ are not affected. There are a few simple steps to reduce/eliminate such post-calibration contamination from the source.
As the calibration of LTQ and possibly also the mass calibration of the Orbitrap are done with the standard ESI source, then if the subsequent analysis is to be done with a nanospray source, there should be minimal contamination.
Nevertheless, the ion transfer tube can be a possible source of Ultramark contamination. If possible you should use a dedicated ion transfer tube as well as a dedicated syringe and infusion line for the calibration routine.
If the mass calibration of orbitrap was done using static nanospray then there is usually no contamination problem.

19. Calibration Frequencies – Check 1 / Year
Once a year, the performance of the Orbitrap should be evaluated for transmission to the Orbi from the LTQ and the HCD cell. This should be a done as a ‘Check’ and not a calibration.
Use the ‘Check’ tab

20. Calibration Frequencies – Calibrate 1 / Month
Effects full MS sensitivity
‘Transfer Lenses’ can be run as a ‘Check’ to evaluate trap cleanliness
The Electron Multiplier calibration effects how many ions are trapped and hence it is necessary to also calibrate the pAGC at the same time. New instruments with heavy use may require an EM calibration every week or two.
The Transfer Lens calibration can also be used to evaluate where contamination has occurred in instruments that show reduced performance. Please discus the results of the test with a member of Thermo Fisher’s tech support group or your local engineer if they are available.
Effects MS/MS sensitivity
Use the ‘Semi-Automatic’ tab
Calibrate EM Gain and pAGC as a group
On new instruments, the EMs and pAGC may need to be calibrated more often [every 2-5 days]

21. How Long Does it Take to Reach Plateau on EMs?
Until your electron multipliers have reached a plateau, they will require calibrations more often.
Plateau Voltage: ~1000 v (ITT style), ~1800 v (SGE style, on Velos Pro Only)

22. Calibration Frequencies – Calibrate 1 / Week
If you require a higher mass accuracy, you may need to calibrate more often. Most users calibrate mass once per week.
Use the ‘Semi-automatic’ tab !!!
Run as a group
Some instruments may require more frequent calibration

23. Use of Lock Mass for Internal Calibration

24. Internal Calibration – How It Works
…. Lock mass …. “mass recalibration on the fly” … using background ions.
Lock mass ions are isolated in the LTQ with a short inject time
The C-Trap is filled with these.
The short inject time adjusts the lock mass intensity to appr. 5%
The LTQ is filled for a second time using the inject time
Ions from the second filling are moved to the C-trap and mixed with the previous ion lock mass population
Ions are injected into the orbitrap
After transient acquisition and data processing are done, the calibration function is corrected based on the measurement of the lock mass
There are different types of lock massing available. This is describing the traditional Thermo approach.

25. Internal Calibration – How It Works
Mixing of ion populations and ejection
Injection of the calibrant
Injection of analyte
Detection
This simple animation shows the steps performed by the instrument resulting in internally calibrated spectrum. They were described on the prior slide.
Olsen, Mann et al. Mol. Cell. Proteomics 2005, 4:
“Parts per million mass accuracy on an orbitrap mass
spectrometer via lock-mass injection into a C-trap.”

26. Internal Calibration: “Tune Page“
In the Tune Page, the lock mass is engaged by ticking the box. Insert the mass of the lock mass into the relevant window. Our tech support group maintains a list of possible lock masses normally observed by users.
3. Enter lock mass
1. Locking ON
2. Hit the button

27. Internal Calibration: “Instrument Setup“
Enable lock mass in the current method ‘L’
Can edit lock masses
The lock mass chosen needs to be available across the elution profile if a gradient is being used.

28. Getting the Most From Your Lock Mass
Using only 1 lock mass is recommended
If multiple lock masses are used, multiple fills are necessary. This increases the time penalty in the experiment. This does not always improve mass accuracies.
Lock masses are searched within a +/- 10 ppm window.
If the LM is not found it widens the search window with 0.1 ppm/scan until the maximum window size is reached
If no lock mass on the LTQ Orbitraps, then the last external calibration is applied.
Another option available for Orbitrap Velos (see next slide)
Helps correct for space charge induced mass shifts and differences in the instrument compared to when calibration was done
To see the lock mass in your spectrum in Qual Browser, you need to turn on Exception Peaks under ‘Ranges’.

29. Optional Lockmass Setting for Orbitrap Velos
When using this second option, the extra injection of the lock mass into the C-trap is not done.
If the lock mass is found in the actual scan, then it is used for the correction. If not, then the last correction used will be applied.
The lock mass must be within your scan range.
This is currently only in the Orbi Velos and later instruments.
Saved in the tune file.
Be sure to ‘Save’ the tune file before exiting. 1. 2.
4. Hit it
3. Type “0”

30. Sample Tuning

31. Steps to Tune
Tee into the LC stream the compound of interest, or if you will immediately try to follow with high sensitivity work, a compound that is similar in structure (avoids contamination of the source)
For nanospray, load a static tip, or use a syringe pump with a small volume syringe (such as 20 uL)
Optimize source conditions for best signal stability
Tune voltages for best peak height
Set microscans / max IT / Target Values
Determine best isolation widths, scan range cutoffs, and collision energies / activation times
While the tune file records the information in the top of the slide, the user should note the items on the bottom of the slide.

32. Using API Stability Test to Optimize the Source
Its often more important to gain stability (lower RSD) than gain a small increase in sensitivity.
To stabilize the signal:
Use more sheath and aux gas
Heat up the ion transfer tube if the compound is not temperature sensitive
Adjust the source position
In addition to the API stability evaluation, the user can monitor the Injection Time and the Normalized Level to determine stability.

33. Tune Ion Optics
The sweep gas usually needs to be done while watching the full ms to understand the difference in the S/N in the low mass region versus the region around the tune compound.
For chromatography where the spectrometer needs to scan below m/z 200, the sweep cone should be tested with a small amount of gas (3, 7, 10, etc) to remove cluster ions and improve S/N.
The recommended setting is 800 volts for samples. In some cases, this setting may be to too high to produce a strong 74 peak for calibration and may need to be dropped to 600 volts temporarily. Non-Velos instruments should be run at 400 volts.

34. Setting Microscans, Max IT, and Target Values
Ion Trap FT
Reagent
Full MS
3e4
1e6
5e5
SIM
1e4
2e5
MSn
5e4 – 2e5
Zoom
8000
Usually the most influential items are the MSn targets and Max IT. The dynamic range in qualitative identification experiments and small molecule work can be increased by reducing the number of ions trapped and the maximum time allowed to trap them. In experiments such as TMT / iTRAQ, the variance in the quantification can be lowered by increasing the target value.
Microscans
Max IT
IT Full MS 1
100
FT Full MS
200
IT MSn
FT MSn

35. Isolation Width – 10, 5, 3, 2, 1.5 Rule
Its often useful to maintain a wider width if you are going to analyze the MS/MS for charge state information or if your small molecule work has halogenated species (the retention of the isotopes can provide structural characterization aides).
The isolation width of an ion should be determined by monitoring the NL for loss of signal while decreasing the width from 10 to 5 to 3 to 2 to 1.5.
Sometimes the user may want to keep the width wider to maintain isotopes useful in the tandem ms intrepretation.
Labile molecules often require larger isolation widths
Increasing the Target Value will often require an opening of the width

36. CID, PQD, HCD – Fragmenting Through Collisions
Type
Pros
Cons
Mass Range Restriction
CID
Resonance Activation (Fixed Q)
High Efficiency
No energy tuning
- Can’t retain low mass product ions
~ 28% LMCO
PQD
Resonance Activation (Changing Q)
- Can retain lower mass product ions (TMT, iTRAQ)
Lower efficiency
Must tune energy
~ 4% LMCO
HCD
Similar to Beam
Can see lower mass ions
True energy control to fine tune MS/MS
Generational Fragmentation
Below 10% to avoid high mass drop-off
Collisional Induced Dissociation (CID) and Pulsed Q Dissociation (PQD) are both resonance activation types of fragmentation, while Higher Energy Collisional Dissociation is an example of a beam type instrument. All three care collisional activated dissociation techniques.

37. ETD – Instrument Preparation

38. Before ETD Tuning Calibration Mass calibration
Multiplier gain calibration, especially when the instrument has been vented
positive ion mode
negative ion mode
Reagent Ion Selection
The calibration of the negative ion mode can have a substantial effect on the signal height in ETD.
ETD uses the EM calibration in negative mode when observing the ETD anion signal.
If not calibrated, ETD anion may reflect a lower intensity response than expected.

39. Preparing the Instrument for Operation
Tuning
Fluoranthene signal intensity
Aim: Signal > 5e6
Measure in centroid mode (not profile)
1. Stabilize the ETD Signal
2. Use “Automatic tuning“ (must be done each time the source is cleaned)
3. Adjust CI gas pressure (must be done when ion volume is changed)
4. ETD related calibrations
Press the reset button on the LTQ if temperatures are not increasing. You may have lower temperatures here.

40. 1. Stabilizing the ETD Reagent Ion Signal
Before starting an Xcalibur method, it is easier to turn on the ETD source and let the temperature equilibrate before injection.
After turning on the Filament, start a manual tune and wait for the signal to plateau.

41. Step 2: Automatic Tuning Procedure
Automatic tuning should be done every few months or when the source is taken apart for cleaning or repair.
Select

42. Step 3: Reagent CI Gas Pressure Evaluation
The CI gas pressure evaluation should be done every time the ion volume or the source is removed.

43. Step 3: Adjust CI Gas Pressure
Set here optimal CI gas pressure
Make sure to set the value after optimization.
NOTE: Retune CI gas every time ion volume is changed!

44. Calibrate when ETD source is taken apart
Step 4: Calibrations
Calibrate when ETD source is taken apart
Check once / year

45. Newer Software Saves Values in master.LTQReagent
The movement of the ETD settings from the tune file to the master.LTQReagent file have made ETD easier to maintain amongst different users.
It’s a good practice to backup the LTQReagent file occasionally.

46. Determining Reaction Times (1, 50, 100 msec, SA)
The reaction time typed into instrument methods is for the doubly charged ions.
This can also be done using an autotune

47. Testing LC-MS System Performance
The last step in performance improvements involves chromatography.
Testing LC-MS System Performance

48. Thermo Fisher Peptide Retention Time Calibration Mixture
Part Number
Item
Amount
88320
Peptide Retention Time Calibration Mixture
50 uL of 0.5 uM
88321
200 uL of 5 uM
The solution can be bought from Thermo and is ready to use out of the box.

49. 15 Heavy Labeled Peptides#
Peptide Sequence
Mass
Hydrophobicity Factor (HF) 1
SSAAPPPPPR
7.56 2
GISNEGQNASIK
15.50 3
HVLTSIGEK
15.52 4
DIPVPKPK
17.65 5
IGDYAGIK
19.15 6
TASEFDSAIAQDK
25.88 7
SAAGAFGPELSR
25.24 8
ELGQSGVDTYLQTK
28.37 9
GLILVGGYGTR
32.18 10
GILFVGSGVSGGEEGAR
34.50 11
SFANQPLEVVYSK
34.96 12
LTILEELR
37.30 13
NGFILDGFPR
40.42 14
ELASGLSFPVGFK
41.18 15
LSSEAPALFQFDLK
46.66
The peptides are heavy labeled so as not to interfere with your real samples.

50. Running the Samples Orbi Velos Q Exactive
Works great for developing and measuring chromatography performance.
Most hydrophillic…first to be lost if there is an
issue with sample loading or resin choice
Most hydrophobic – first to demonstrate carry-over problems

51. What Can We Test Determine average peak capacity
Determine peak width for automated cycle time determination for QE experiments
Fill time analysis
Mass accuracy evaluation
Peak symmetry evaluation – i.e. overloading columns
CID/HCD performance against a spectral library
Troubleshooting sample reconstitution solutions
Troubleshooting LC gradient profiles (are we missing hydrophilic/phobic peptides?) and trap column performance
Works great as a Quantitative Standard
What can we use it for? Thermo is working on developing the solution as a standard. We will keep users up to date on our progress in the future.