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Methods for LTQ Orbitrap A Guided Tour with Examples

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

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Methods for LTQ Orbitrap A Guided Tour with Examples

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  1. Methods for LTQ OrbitrapA 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 • Survey MS in Orbitrap • MS/MS in Orbitrap For de novo sequencing, PTM discovery

  5. 0.9 s 0.1 s 0.35 s 0.2 s Mass Accuracy and Resolution Settings Parallel Mode Serial Mode

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

  7. AGC settings orbitrap • Full MS 5.00e+05 - 1.00e+06 IT 500ms • SIM 1.00e+05 - 2.00e+05 IT 500ms • MSn 1.00e+05 - 2.0e+05 IT 500ms

  8. Settings for HCD, Lock mass • Act.Q 0.13-0.14 • 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. Can be used for directing MS/MS to a preferred region – e.g. 800-1300 in glycopeptide analysis Parallel Method – DDA Scan event 2

  16. Parallel Method – DDA Scan event 2

  17. Ignore Parallel Method – DDA Scan event 2

  18. Parallel Method – DDA Scan event 2 This is the key to PARALLEL mode

  19. Parallel Method – DDA Scan event 2

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

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

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

  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, 493-450

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

  34. Can resolve 4+ ions 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 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, 2010-2021

  42. Want ppb Mass Accuracy? • Use Lock mass in the method Olsen et al., MPC 2005, 4, 2010-2021

  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, 13417-22

  46. NL MS3 Method – starting the method

  47. NL MS3 Method – Survey MS

  48. NL MS3 Method – MS2

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

  50. NL MS3 Method – MS2 NOTE: phosphopeptide analysis

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