Orbitrap Mass Analyser - Overview and Applications in Proteomics

1. Orbitrap Mass Analyser - Overview and Applications in Proteomics Alexander Makarov, Michaela Scigelova Thermo Electron Corporation

2. Outline • Orbitrap mass analyser • Linking orbitrap to linear ion trap • Flexibility of use of LTQ Orbitrap • Focus on: • High resolution • Sensitivity • Speed • Dynamic range • Conclusion

3. Principle of Trapping in the Orbitrap • The Orbitrap is an ion trap – but there are no RF or magnet fields! • Moving ions are trapped around an electrode • Electrostatic attraction is compensated by centrifugal force arising from the initial tangential velocity • Potential barriers created by end-electrodes confine the ions axially • One can control the frequencies of oscillations (especially the axial ones) by shaping the electrodes appropriately • Thus we arrive at … Orbital traps Kingdon (1923)

4. Orbitrap – Electrostatic Field Based Mass Analyser r z φ Korsunskii M.I., Basakutsa V.A. Sov. Physics-Tech. Phys. 1958; 3: 1396. Knight R.D. Appl.Phys.Lett. 1981, 38: 221. Gall L.N.,Golikov Y.K.,Aleksandrov M.L.,Pechalina Y.E.,Holin N.A. SU Pat. 1247973, 1986.

5. Ion Motion in Orbitrap • Only an axial frequency does not depend on initial energy, angle, and position of ions, so it can be used for mass analysis • The axial oscillation frequency follows the formula w = oscillation frequency k = instrumental const. m/z = …. what we want! A.A. Makarov, Anal. Chem. 2000, 72: 1156-1162. A.A. Makarov et al., Anal. Chem. 2006, 78: 2113-2120.

6. Ions of Different m/z in Orbitrap • Large ion capacity - stacking the rings • Fourier transform needed to obtain individual frequencies of ions of different m/z

7. How Big Is Orbitrap?

8. Getting Ions into the Orbitrap • The “ideal Kingdon” field has been known since 1950’s, but not used in MS. Why? There is a catch • how to get ions into it ? • Ions coming from the outside into a static electric field will zoom past, like a comet from the outer space flies through a solar system • The catch: The field must not be static when ions come in! • A potential barrier stopping the ions before they reach an electrode can be created by lowering the central electrode voltage while ions are still entering • Thus we arrive at the principle of Electrodynamic Squeezing • A.A. Makarov,Anal. Chem. 2000, 72: 1156-1162. • A.A. Makarov, US Pat. 5,886,346, 1999. • A.A. Makarov et al., US Pat. 6,872,938, 2005.

9. Curved Linear Trap (C-trap) for ‘Fast’ Injection Push Trap Pull Lenses Orbitrap Gate Deflector • Ions are stored and cooled in the RF-only C-trap • After trapping the RF is ramped down and DC voltages are applied to the rods, creating a field across the trap that ejects along lines converging to the pole of curvature (which coincides with the orbitrap entrance). As ions enter the orbitrap, they are picked up and squeezed by its electric field • As the result, ions stay concentrated (within 1 mm3) only for a very short time, so space charge effects do not have time to develop • Now we can interface the orbitrap to whatever we want! • A.A. Makarov et al., US Pat. 6,872,938, 2005. • A. Kholomeev et al., WO05/124821, 2005.

10. Outline • Orbitrap mass analyser • Linking orbitrap to linear ion trap • Flexibility of use of LTQ Orbitrap • Focus on: • High resolution • Sensitivity • Speed • Dynamic range • Conclusion

11. Linking Linear Trap with Orbitrap • Combining the features of the Finnigan LTQ… • ESI, nanospray, APCI, APPI ionsation methods • outstanding sensitivity • MSn operation • Ruggedness and ease of use It adds capabilities for the most demanding analyses • …with excellent performance of orbitrap • High resolution • Accurate mass determination It is fast - even with high resolution/accurate mass detection

12. LTQ Orbitrap Operation Principle 1. Ions are stored in the Linear Trap 2. …. are axially ejected 3. …. and trapped in the C-trap 4. …. they are squeezed into a small cloud and injected into the Orbitrap 5. …. where they are electrostatically trapped, while rotating around the central electrode and performing axial oscillation The oscillating ions induce an image current into the two outer halves of the orbitrap, which can be detected using a differential amplifier Ions of only one mass generate a sine wave signal

13. How Big Is LTQ Orbitrap?

14. What LTQ Orbitrap Delivers • Mass resolution > 60,000 at m/z 400 at 1 sec cycle • Max. resolution over 100,000 (FWHM) • Mass accuracy < 5 ppm external calibration • Mass accuracy < 2 ppm internal calibration • Mass range 50 – 2,000; 200 – 4,000 • Sensitivity sub-femtomole on column • Throughput 4 scans per second (1 high-resolution scan in the orbitrap + 3 MS/MS scans in the LTQ)

15. Outline • Orbitrap mass analyser • Linking orbitrap to linear ion trap • Flexible method design for LTQ Orbitrap • Focus on: • High resolution • Sensitivity • Speed • Dynamic range • Conclusion

16. MS/MS with precursor accurate mass only Setup for highest MS/MS productivity Cycle time 1 second SE1 Full Scan MS SE2 MS/MS SE3 MS/MS SE4 MS/MS 1 LTQ Orbitrap high resolution full scan and in parallel 3 low resolution ion trap MS/MS scans SE denotes a ‘scan event’

17. “All-round accurate mass” MS/MS methods Setup for high mass accuracy Cycle time 2 seconds SE1 Full Scan MS SE2 MS/MS SE3 MS2 (or MS3) SE4 MS2 (or MS3) 1 LTQ Orbitrap high resolution full scan and sequentially 3 high resolution LTQ Orbitrap MS/MS scans External mass calibration

18. “All-round accurate mass” MS/MS methods Setup for highest mass accuracy Cycle time 2.2 seconds SE1 Full Scan MS SE2 MS/MS SE3 MS2 (or MS3) SE4 MS2 (or MS3) 1 LTQ Orbitrap high resolution full scan and sequentially 3 high resolution LTQ Orbitrap MS/MS scans Internal mass calibration

19. Various combinations of MS/MS methods SE1 Full Scan MS Example: phosphopeptides analysis SE2 MS/MS SE4 MS/MS SE3 MS3 SE5 MS3 1 Orbitrap high resolution full scan and { high resolution Orbitrap MS/MS scan and neutral loss triggered Low-resolution ion trap MS3 scan } x2 External mass calibration

20. Precursor phosphopeptides m/z 831: -S1 Casein 121-134; m/z 1031: -Casein 33-48 1031.92296 PP_28092005_10-POS # 22-49 RT: 0.31-0.70 AV: 14 NL: 5.93E3 1031.42128 F: FTMS + p NSI Full ms [ 800.00-1800.00] 830.90315 831.40519 1032.42430 1032.92600 831.90689 1031.0 1032.0 1033.0 1034.0 m/z 832.40787 830.0 831.0 832.0 833.0 834.0 835.0 m/z 800 850 900 950 Orbitrap detector z=2 100 95 90 85 1042.91402 80 z=2 1031.42128, + 3.3 ppm 75 70 65 60 z=2 55 50 Relative Abundance 45 40 35 841.89392 830.90313, + 2.5 ppm 30 z=2 25 1050.89741 20 z=2 15 10 1062.38000 z=2 5 0 1000 1050 1100 1150 m/z Samples: Dr. Martin Larsen, Prof. Ole N Jensen University of Southern Denmark

21. MS/MS of m/z 1031 FQS*EEQQQTEDELQDK 100 95 90 85 80 75 70 65 60 55 Relative Abundance 50 45 40 35 30 25 20 15 10 5 0 m/z Orbitrap detector 982.43205 Neutral loss exactly detected 982.4320 +2.7 ppm 977.43825 976 977 978 979 980 981 982 983 984 985 986 m/z 400 600 800 1000 1200 1400 1600 1800 2000 S* denotes dehydroalanine

22. MS3 of m/z 982 triggered upon the accurate neutral loss detection Linear ion trap detector 672.3 100 95 90 328.1 747.3 85 1620.7 632.5 965.8 80 75 1619.6 632.4 70 876.3 836.9 1332.2 965.0 65 1619.5 965.9 60 1818.6 55 1216.5 836.8 50 1817.4 Relative Abundance 966.3 1361.6 1087.2 45 1689.7 827.8 40 964.8 1234.3 1089.3 35 1490.7 503.3 900.4 1105.5 544.8 30 1106.5 584.3 345.2 1702.3 25 1574.8 390.2 1281.3 1461.5 1198.7 1070.9 456.3 1817.3 1820.7 20 1715.4 15 968.0 1836.3 10 5 1836.6 0 400 600 800 1000 1200 1400 1600 1800 m/z

23. Interpretation of fragments from MS3 experiment Complete y and b series are observed

24. Outline • Orbitrap mass analyser • Linking orbitrap to linear ion trap • Flexibility of use of LTQ Orbitrap • Focus on: • High resolution and mass accuracy • Sensitivity • Speed • Dynamic range • Conclusion

25. High Resolution & Accurate Mass .. confident ID, PTMs, de novo sequencing, top-down

26. High Mass Resolution and Accurate Mass (in 1 second) 312.12181 312.13272 NOTE: All mass accuracies in this presentation are with external calibration theoretical R= 82,000 + 0.7 ppm measured

27. High Masses and Mass Accuracy: Apomyoglobin, charge state 10+ measured All mass accuracies < 2 ppm theoretical

28. High Masses and Mass Accuracy: Carbonic Anhydrase, charge state 21+ measured All mass accuracies < 3 ppm theoretical

29. Long-term stability of external calibration Deviation, ppm 3 ppm 4 hours Time, hours (m/z 1422 at 100%; m/z 524 at <0.02%).

30. Internal Calibration in LTQ Orbitrap Injection of the calibrant Injection of analyte Mixing of ion populations and ejection Detection Olsen, J.V.; de Godoy, L.M.; Li, G.; Macek, B.; Mortensen, P.; Pesch, R.; Makarov, A.A.; Lange, O.; Horning, S.; Mann, M. “Parts per million mass accuracy on an orbitrap mass spectrometer via lock-mass injection into a C-trap.” Mol. Cell. Proteomics 2005, 4: 2010-2021.

31. Speed ..while delivering accurate mass in MS, MS/MS and MSn

32. Complex Protein Digests: ‘Big 5’ Experiment Digging deep into the baseline for low abundant co-eluting peptides Total time 2.4 seconds SE1 Full Scan MS SE2 MS/MS SE3 MS/MS SE4 MS/MS SE5 MS/MS SE6 MS/MS 1 LTQ Orbitrap high resolution full scan and 5 fast ion trap MS/MS scans SE denotes a ‘scan event’

33. Complex Mixture - Selecting Ions for Fragmentation 600.9776 100 Relative Abundance 50 598.6563 0 804.3450 599.0 600.0 601.0 602.0 603.0 558.7548 100 m/z 100 Relative Abundance Relative Abundance 50 50 777.3942 547.6516 0 0 775 780 785 790 795 800 805 810 548 550 552 554 556 558 560 562 m/z m/z MS/MS MS/MS MS/MS MS/MS MS/MS

34. Parallel Detection in Orbitrap and Linear Ion Trap RT: 41.57 MS/MS of m/z 598.6 Scan # 4870 0.0 0.5 1.0 1.5 2.0 2..5 RT: 41.58 MS/MS of m/z 547.3 Scan # 4871 RT: 41.59 MS/MS of m/z 974.9 Scan # 4873 Time [sec] RT: 41.60 MS/MS of m/z 1116.5 Scan # 4874 RT: 41.58 MS/MS of m/z 777.4 Scan # 4872 RT: 41.56 High resolution Full scan # 4869 High resolution full scan in Orbitrap and 5 MS/MS in linear ion trap • Total cycle is 2.4 seconds • 1 High resolution scan with accuracies < 5 ppm • External calibration • 5 ion trap MS/MS in parallel

35. Resolving Power vs Cycle Time 785.8419 R=5901 786.3435 100 R=5900 80 786.8447 60 R=5900 787.3463 40 787.8453 785.5934 R=6000 R=5800 20 R=6200 0 785.8421 786.3434 R=23801 100 R=23900 80 786.8446 60 R=24000 Relative Abundance 787.3457 40 785.5992 787.8471 R=24100 R=15600 20 R=24300 0 785.8419 786.3435 100 R=48101 R=47700 80 786.8446 60 R=48200 787.3458 40 787.8477 785.5994 R=47500 R=42000 20 R=47100 0 785.8413 786.3428 100 R=94801 R=95200 80 786.8442 60 R=93600 787.3458 40 785.5989 787.8477 R=98000 20 R=95800 R=89200 0 785.0 785.2 785.4 785.6 785.8 786.0 786.2 786.4 786.6 786.8 787.0 787.2 787.4 787.6 787.8 788.0 788.2 m/z RP 7500 0.2 s RP 30000 0.5 s RP 60000 0.9 s RP 100000 1.6 s

36. Sensitivity

37. Horse Cytochrome C, Horse Myoglobin Bovine Serum Albumin, 1 fmol on column m/z 653 (2+) theory: 653.361701measured: 653.36127 (+0.7 ppm) dd IT MSMS on this scan (scan 3588) m/z 653 nanoLC NewObjective 75 um PicoFrit column Flow rate: 200 nl / min From 98 % A (water, 0.1 % FA) to 60% B (Acetonitrile, 0.1 % FA) in 20 min Coverage Cytochrome C 67% Myoglobin 71% BSA 45%

38. Protein digest mix: 1 fmol each on column Peptide m/z 653 (2+) at RT: 24.93 min Base Peak Chromatogram

39. Data Dependent MS/MS of Peptide m/z 653 (2+)

40. Assigned Fragment Ions by SEQUEST

41. Dynamic Range ..detecting minor components in complex mixtures

42. Angiotensin 10 pmol/ul + Glu-fibrinogen 10 fmol/ulConcentration Difference 1000x NL: 9.35E4 785.5992 785.8419 100 786.3431 90 80 70 786.6021 60 50 Relative Abundance 40 786.8450 787.6064 30 787.3463 Measured 785.8419 Calculated 785.8421 Dm = -0.2 ppm 20 10 0 785 ? 652.8230 770.3946 915.6690 1014.5159 784.5 785.0 785.5 786.0 800 786.5 1000 787.0 787.5 788.0 m/z Angio10pmol_Glufib10fmol_Res30000 # 6 RT: 0.09 AV: 1 T: FTMS + p ESI Full ms [ 215.00-2000.00] 428.2281 100 NL: 1.18E8 95 90 85 80 75 70 65 60 55 50 Relative Abundance 45 40 641.8381 35 30 25 20 15 10 633.3358 385.7010 513.2818 5 1282.6699 269.1610 1305.6428 1221.9934 1552.9739 1711.2153 1804.3352 0 400 600 1200 1400 1600 1800 2000 m/z

43. MS/MS of Glu-Fibrinogen @10 fmol/ul y4 #199-199 RT:5.30-5.30 NL: 6.64E3 480.2558 100 95 90 y5 85 684.3457 80 75 y6 70 65 813.3882 60 55 y3 50 Relative Abundance 333.1879 45 40 +2 y y 12 35 y7 692.8 30 942.4313 25 20 627.3 15 y2 10 +1 b b 8 246.1558 887.3 +1 5 b b 5 515.2 0 200 300 400 500 600 700 800 900 1000 1100 1200 m/z Measured 246.1558 Calculated 246.1561 Dm = -1.2 ppm

44. Dynamic Range in a Single Spectrum (0.75 sec Acquisition)

45. Conclusion • The orbitrap mass analyzer is first fundamentally new mass analyzer introduced commercially in over 20 years • The last novel mass spectrometer introduction was the RF Ion Trap (Finnigan MAT) in the early1980’s • The main advantages of the orbitrap mass analyzer are: • Unsurpassed dynamic range of mass accuracy • High resolution • High sensitivity • High stability • Compact package • Maintenance-free • The LTQ Orbitrap is the first implementation of the orbitrap analyzer in a hybrid instrument • Isolation, fragmentation and MSn is provided mainly by the linear trap • The C-trap supports multiple ion fills, CID and future expansion • The orbitrap is and will be used as a detector

46. About the Authors Dr. Alexander Makarov The inventor of orbitrap mass analyser Research Manager at Thermo Electron in Bremen Dr. Michaela Scigelova LC/MS application expert at Thermo Electron in UK