1. Methods for LTQ Orbitrap A Guided Tour with Examples
Dr. Michaela Scigelova

2. Outline Data acquisition strategies Method setup and examples
OT_LTQ_Big6
MS in Orbi + 6 MS/MS in ion trap
OT_3OT
MS in Orbi + 3 MS/MS in Orbi
NL_MS3_AccMass
Phosphopeptides with accurate NL
MS in Orbi, MS2 in Orbi, and MS3 in ion trap
OT_MSA
Phosphopeptides
‘composite’ MS2 and MS3 spectrum = MSA
MS in Orbi, MSA in LTQ

3. What is the objective of the analysis?
One or more of these:
Maximise the protein ID
Boost the sequence coverage
Phosphorylation
De novo sequencing
Quantitation

4. Data Acquisition Strategies
Parallel mode
Survey MS in Orbitrap
MS/MS in LTQ
For highest protein ID and coverage
assuming very complex mixtures
Serial mode
MS/MS in Orbitrap
For de novo sequencing, PTM discovery

5. Mass Accuracy and Resolution Settings
Parallel Mode
Resolution
Mass Accuracy
Survey MS (OT)
60,000
3 ppm
MS/MS (LTQ)
Unit
~200 ppm
0.9 s
0.1 s
0.35 s
0.2 s
Serial Mode
Resolution
Mass Accuracy
Survey MS (OT)
15,000
3 ppm
MS/MS (OT)
7,500

6. AGC settings LTQ Full MS 3.00e+04 IT 50ms SIM 1.00e+04 IT 100ms
MSn e+04 IT 100ms

7. AGC settings orbitrap Full MS 5.00e+05 - 1.00e+06 IT 500ms
SIM e e+05 IT 500ms
MSn e e+05 IT 500ms

8. Settings for HCD, Lock mass
Act.Q
Normalized Collision Energy 60-70
specify the FT lock mass abundance (%)
Diagnostics/Set Device
Default value 10%

9. OT_LTQ_Big6
MS in Orbi + 6 MS/MS in ion trap

10. Parallel Mode – Method Setup
Step-by-step guide
Method BIG 6
Tune settings
Max fill time 100 ms
Number of microscans 1

11. Parallel Method – starting the method

12. Parallel Method – Survey MS

13. Parallel Method – Add another 6 scan events

14. Parallel Method – BIG 6
Save yourself the typing: use copy and paste!

15. Parallel Method – DDA Scan event 2
Can be used for directing MS/MS to a preferred region –
e.g in glycopeptide analysis

16. Parallel Method – DDA Scan event 2

17. Parallel Method – DDA Scan event 2
Ignore
Exclusion list size – use max number available. The list will contain also all the isotopes of excluded ions so it fills up quite quickly.
Exclusion duration - should be about ½ peak width – this ensures another MS/MS spectrum is taken hopefully around the peak apex.
Exclusion mass width - is set very narrow. Isotopes will be also excluded.

18. Parallel Method – DDA Scan event 2
‘most intense if no parent masses found’ will be used for NL MS3 method for phosphopeptides (peak corresponding to the detected neutral loss of phosphate is considered as a parent mass for this purpose)
‘exclude parent mass from MSn selection’ – used mainly for drugs. When parent fragmentation is not efficient, the leftover parent ion is still the biggest in the spectrum – when ticking this button the system will ignore such parent ion leftover and go for the next biggest ion in the MSn spectrum.
This is the key to PARALLEL mode

19. Parallel Method – DDA Scan event 2
The NCE settings can vary for different MSn levels: good for phosphopeptides where MS2 energy should be set lower (i.e. 20) to just knock off the labile modification, and MS3 energy can go back to normal 30 to fragment the dephosphorylated peptide with maximum efficiency.

20. Parallel Method – DDA Scan event 2
Import lists of target parent ions (parent list) or contaminants (reject list)

21. Parallel Method – DDA Scan event 2
Reject ‘1+’ and ‘unassigned’ for max number of PROTEIN ID

22. Parallel Method – DDA Scan event 2
Leave unticked if going for max sequence COVERAGE

23. Parallel Method – DDA Scan event 2
Even better COVERAGE: if you inject sample 3 times and set the following:

24. Parallel Method – DDA Scan event 2

25. Parallel Method – DDA Scan event 3
Default settings:
WRONG!!!!!

26. Parallel Method – DDA Scan event 3

27. Parallel Method – DDA Scan event 4

28. Parallel Method – DDA Scan event 5

29. Parallel Method – DDA Scan event 6

30. Parallel Method – DDA Scan event 7

31. When you have built your method.. Check it!

32. Who Is Working this Way? John Yates (Scribbs Inst.)
Ruedi Aebersold (ETH Basel)
David Goodlet (ISB Washington)
Yates et al., AC 2006, 78,

33. OT_3OT
MS in Orbi + 3 MS/MS fragmented
in LTQ and measured in OT

34. Serial Method Setup Full scan in Orbi + 3 MS/MS in Orbi Tune settings
Max fill time 100 ms
Number of microscans 2 to improve S/N in MSn
Can resolve 4+ ions
Resolution
Mass Accuracy
Survey MS (OT)
15,000
3 ppm
MS/MS (OT)
7,500
0.35 s
0.2 s

35. Serial Method – Full scan MS

36. Serial Method – MS + 3 MS/MS
Save yourself the typing: use copy and paste!

37. Serial Method – Major change in ‘Current Segment’
There is NO box ticked!

38. Serial Method – DDA Scan event 2

39. Serial Method – DDA Scan event 3

40. Serial Method – DDA Scan event 4

41. Who Is Doing It This Way? Matthias Mann (MPI Martinsreid)
Lock mass ‘trick’
Divide the mass range is sections
Roman Zubarev (Uppsala)
Olsen et al., MPC 2005, 4,

42. Want ppb Mass Accuracy? Use Lock mass in the method
Olsen et al., MPC 2005, 4,

43. Lock Mass - Caveats
Lock mass should be present in your analysis all the time
Can be a background ion
Or spike in a lock mass solution via a Y-connector (P-773,17 nL swept volume) with fused silica tubing attached to a syringe pump
Use multiple lock masses
For calibrating MS/MS spectra the lock mass closest to your parent mass of interest is taken (good for peptides)
Once you fill in the lock mass dialog box, you can export it, save it, and import it to other methods

44. NL_MS3_AccMass
MS in Orbi +
MS/MS in Orbi +
MS3 in LTQ

45. Strategy for Phosphopeptides
Enrichment!!!
Use prominent neutral loss of phosphate in MS2 to trigger MS3
Accurately defined neutral loss
Use Orbi for MS and MS2
Cuts down on false positive MS3 triggers
MS3 is highly informative for peptide sequence and PTM position
Data review is fast and easy – search MS3 scans only
Thorough search (MS2 + MS3) provides high confidence ID
Olsen and Mann, PNAS 2004, 101,

46. NL MS3 Method – starting the method

47. NL MS3 Method – Survey MS

48. NL MS3 Method – MS2

49. NL MS3 Method – MS2 NOTE: phosphopeptide analysis
Accurate NL determination avoids false triggers of MS3  more efficient analysis

50. NL MS3 Method – MS2 NOTE: phosphopeptide analysis
‘most intense if no parent masses found’ will be used for NL MS3 method for phosphopeptides (peak corresponding to the detected neutral loss of phosphate is considered a parent mass for this purpose)

51. NL MS3 Method – MS2 NOTE: phosphopeptide analysis
The NCE settings can vary for different MSn levels: good for phosphopeptides where MS2 energy should be set lower (i.e. 20) to just knock off the labile modification, and MS3 energy can go back to normal 30 to fragment the dephosphorylated peptide with maximum efficiency.

52. NL MS3 Method – MS2 NOTE: phosphopeptide analysis
The system recalculates NL for all relevant charge states

53. NL MS3 Method – MS2
NOTE: phosphopeptide analysis

54. NL MS3 Method – MS3

55. NL MS3 with accurate mass support – MS3

56. NL_MSA
MS in Orbi + MSA in LTQ

57. Multistage Activation - MSA
To improve the signal, it is advantageous to lump together the fragments from both MS2 and MS3 scans and search them as one ‘strong’ scan

58. Phosphopeptide analysis with MSA
APPDNLPSPGGpSR 5 fmol
CID MS²
CID MS³
[M+2H-98]2+
MSA
Weitere Gegenüberstellung: wie vorherhegehende Folie, nur auch mit pulsed-q fragmentierung. Bei phosphopeptid mit starken NL kein Vorteil sichtbar (keine zB. Immoniumionen oder iTRAQ)
1.) OT_14PP_CAD-ITnl-ms3x5_par14PP_MPSon dta
2.) OT_14PP_CAD-ITnl-ms3x5_par14PP_MPSon dta
3.) FTc2_CCBSAPP_mstgMS2_ dta
The problem for SEQUEST – does not label NL-fragments in MSA spectrum. Therefore our customers who want to use SEQUEST do not like MSA method and still use NL MS3 method. This is not a problem for Mascot – does NL-ion labeling really well.
Data courtesy of Karl Mechtler

59. MSA Method – starting the method

60. MSA Method – Survey MS

61. MSA – data dependent MS2

62. Dynamic exclusion

63. Neutral Loss – measured in LTQ

64. Current Segment

65. MSn Settings

66. Monoisotopic precursor selection

67. Neutral Loss – no need to spell it our for different charge states

68. Multistage Activation - ON

69. MSA - Conclusions
MSA enables to activate ions from MS2 that show neutral loss from parent, while still keeping the rest of the MS2 fragments in the trap. The MS3 fragments from activated NL candidates are ‘added’ to the rest of the MS2 fragments
Data can be processed by SEQUEST/BioWorks or Mascot

70. Who is Doing it this Way Donald Hunt (Uni Virginia)
Karl Mechtler (IMP, Vienna)
Andy West, Peter Francis (GSK, Stevenage)
Schoeder et al, AC2004, 76,