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

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Presentation on theme: "Orbitrap Mass Analyser - Overview and Applications in Proteomics Alexander Makarov, Michaela Scigelova Thermo Electron Corporation."— Presentation transcript:

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

2 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 3 Principle of Trapping in the Orbitrap Orbital traps Kingdon (1923) 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 …

4 4 Orbitrap – Electrostatic Field Based Mass Analyser z φ r 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 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 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 7 How Big Is Orbitrap?

8 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 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 mm 3 ) 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 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 11 Linking Linear Trap with Orbitrap Combining the features of the Finnigan LTQ… –ESI, nanospray, APCI, APPI ionsation methods – outstanding sensitivity –MS n 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 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 13 How Big Is LTQ Orbitrap?

14 14 What LTQ Orbitrap Delivers Mass resolution> 60,000 at m/z 400 at 1 sec cycle Max. resolutionover 100,000 (FWHM) Mass accuracy< 5 ppm external calibration Mass accuracy < 2 ppm internal calibration Mass range50 – 2,000; 200 – 4,000 Sensitivitysub-femtomole on column Throughput4 scans per second (1 high-resolution scan in the orbitrap + 3 MS/MS scans in the LTQ)

15 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 16 MS/MS with precursor accurate mass only Setup for highest MS/MS productivity Cycle time 1 second 1 LTQ Orbitrap high resolution full scan and in parallel 3 low resolution ion trap MS/MS scans SE1 Full Scan MS SE2 MS/MS SE3 MS/MS SE4 MS/MS SE denotes a ‘scan event’

17 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 MS 2 (or MS 3 ) SE4 MS 2 (or MS 3 ) 1 LTQ Orbitrap high resolution full scan and sequentially 3 high resolution LTQ Orbitrap MS/MS scans External mass calibration

18 18 “All-round accurate mass” MS/MS methods Setup for highest mass accuracy Cycle time 2.2 seconds 1 LTQ Orbitrap high resolution full scan and sequentially 3 high resolution LTQ Orbitrap MS/MS scans Internal mass calibration SE1 Full Scan MS SE2 MS/MS SE3 MS 2 (or MS 3 ) SE4 MS 2 (or MS 3 )

19 19 Various combinations of MS/MS methods Example: phosphopeptides analysis SE1 Full Scan MS SE2 MS/MS 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 SE3 MS 3 SE4 MS/MS SE5 MS 3

20 20 Precursor phosphopeptides m/z 831:  -S1 Casein 121-134; m/z 1031:  -Casein 33-48 1000105011001150 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance 1031.42128, + 3.3 ppm z=2 1042.91402 z=2 830.90313, + 2.5 ppm 841.89392 z=2 1050.89741 z=2 1062.38000 z=2 830.0831.0832.0833.0834.0835.0 m/z 830.90315 831.40519 831.90689 832.4078 7 z=2 Samples: Dr. Martin Larsen, Prof. Ole N Jensen University of Southern Denmark Orbitrap detector

21 21 MS/MS of m/z 1031 FQS*EEQQQTEDELQDK Neutral loss exactly detected 976977978979980981982983984985986 m/z 982.4320 977.43825 400600800100012001400160018002000 982.43205 +2.7 ppm S* denotes dehydroalanine Orbitrap detector

22 22 MS 3 of m/z 982 triggered upon the accurate neutral loss detection 40060080010001200140016001800 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance 672.3 328.1 747.3 1620.7 632.5 965.8 1619.6 632.4 876.3 836.9 1332.2 965.0 1619.5 965.9 1818.6 1216.5 836.8 1817.4 966.3 1361.6 1087.2 1689.7 827.8 964.8 1234.3 1089.3 1490.7 503.3 900.4 1105.5 544.8 1106.5 584.3 345.2 1702.3 1574.8 390.2 1281.3 1461.5 1198.7 1070.9 456.3 1817.3 1820.7 1715.4 968.0 1836.3 1836.6 Linear ion trap detector

23 23 Interpretation of fragments from MS 3 experiment Complete y and b series are observed

24 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 25 High Resolution & Accurate Mass.. confident ID, PTMs, de novo sequencing, top-down

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

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

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

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

30 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 31 Speed..while delivering accurate mass in MS, MS/MS and MS n

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

33 33 Complex Mixture - Selecting Ions for Fragmentation MS/MS

34 34 Parallel Detection in Orbitrap and 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 RT: 41.56 High resolution Full scan # 4869 High resolution full scan in Orbitrap and 5 MS/MS in linear ion trap RT: 41.57 MS/MS of m/z 598.6 Scan # 4870 RT: 41.58 MS/MS of m/z 547.3 Scan # 4871 RT: 41.58 MS/MS of m/z 777.4 Scan # 4872 RT: 41.59 MS/MS of m/z 974.9 Scan # 4873 RT: 41.60 MS/MS of m/z 1116.5 Scan # 4874 0.0 0.5 1.0 1.5 2.0 2..5 Time [sec]

35 35 Resolving Power vs Cycle Time 0.9 s 1.6 s RP 7500 0.2 s RP 30000 0.5 s RP 60000 RP 100000

36 36 Sensitivity

37 37 Horse Cytochrome C, Horse Myoglobin Bovine Serum Albumin, 1 fmol on column m/z 653 (2+) theory: 653.361701 measured: 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% Myoglobin71% BSA45%

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

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

40 40 Assigned Fragment Ions by SEQUEST

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

42 42 Angiotensin 10 pmol/ul + Glu-fibrinogen 10 fmol/ul Concentration Difference 1000x Angio10pmol_Glufib10fmol_Res30000 #6RT:0.09AV:1 NL:1.18E8 T:FTMS + p ESI Full ms [ 215.00-2000.00] 40060012001400160018002000 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance 428.2281 641.8381 513.2818 633.3358 1282.6699 269.1610 385.7010 1305.6428 8001000 652.8230 770.3946 915.6690 1014.5159 1221.99341552.97391711.21531804.3352 785 ? 0 10 20 30 40 50 60 70 80 90 100 Relative Abundance 785.5992 785.8419 786.3431 786.6021 786.8450 787.6064 787.3463 NL: 9.35E4 784.5785.0785.5786.0786.5787.0787.5788.0 m/z Measured 785.8419 Calculated 785.8421  m = -0.2 ppm

43 43 627.3 bb 8 +1 887.3 bb 5 +1 515.2 480.2558 684.3457 813.3882 333.1879 942.4313 yy 12 +2 692.8 246.1558 y2 y4 y3 y5 y6 y7 MS/MS of Glu-Fibrinogen @10 fmol/ul Measured 246.1558 Calculated 246.1561  m = -1.2 ppm

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

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


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