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

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

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)

Orbitrap – Electrostatic Field Based Mass Analyser z φ r Electrostatic field mass analyser 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.

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.

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

How Big Is Orbitrap?

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.

Curved Linear Trap (C-trap) for ‘Fast’ Injection 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! Push Trap Pull Lenses Orbitrap Gate Deflector A.A. Makarov et al., US Pat. 6,872,938, 2005. A. Kholomeev et al., WO05/124821, 2005.

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

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

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

How Big Is LTQ Orbitrap? Electrostatic field mass analyser

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)

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

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’

“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

“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

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

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

MS/MS of m/z 1031 FQS*EEQQQTEDELQDK Orbitrap detector 982.43205 m/z 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance 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

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

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

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

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

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

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

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

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%).

Internal Calibration in LTQ Orbitrap Detection Mixing of ion populations and ejection Injection of analyte Injection of the calibrant 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.

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

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’

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

Parallel Detection in Orbitrap and 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 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 RT: 41.56 High resolution Full scan # 4869 High resolution full scan in Orbitrap and 5 MS/MS in linear ion trap 0.0 0.5 1.0 1.5 2.0 2..5 Time [sec] Total cycle is 2.4 seconds 1 High resolution scan with accuracies < 5 ppm External calibration 5 ion trap MS/MS in parallel

Resolving Power vs Cycle Time 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 20 40 60 80 100 Relative Abundance 785.8419 R=5901 786.3435 R=5900 786.8447 787.3463 R=6000 787.8453 R=5800 785.5934 R=6200 785.8421 R=23801 786.3434 R=23900 786.8446 R=24000 787.3457 R=24100 787.8471 R=15600 785.5992 R=24300 R=48101 R=47700 R=48200 787.3458 R=47500 787.8477 R=42000 785.5994 R=47100 785.8413 R=94801 786.3428 R=95200 786.8442 R=93600 R=98000 785.5989 R=95800 R=89200 RP 7500 0.2 s RP 30000 0.5 s RP 60000 0.9 s RP 100000 1.6 s

Sensitivity

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% Myoglobin 71% BSA 45%

Protein digest mix: 1 fmol each on column Peptide m/z 653 (2+) at RT: 24.93 min Base Peak Chromatogram NanoLC run with 75 um PicoFrit column, flow rate: 200 nl / min Fast gradient (20 min) 1 LTQ Orbitrap Full Scan resolution 60,000 6 DD LTQ MS/MS Scan Cycle time 2.6 seconds

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

Assigned Fragment Ions by SEQUEST

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

Angiotensin 10 pmol/ul + Glu-fibrinogen 10 fmol/ul Concentration Difference 1000x 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 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 784.5 785.0 785.5 786.0 786.5 787.0 787.5 788.0 m/z Measured 785.8419 Calculated 785.8421 Dm = -0.2 ppm 80 75 70 65 60 55 Relative Abundance 50 45 40 641.8381 35 30 25 20 785 ? 15 10 633.3358 385.7010 513.2818 5 269.1610 800 1000 652.8230 770.3946 915.6690 1014.5159 1282.6699 1221.9934 1305.6428 1552.9739 1711.2153 1804.3352 400 600 1200 1400 1600 1800 2000 m/z

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

Dynamic Range in a Single Spectrum (0.75 sec Acquisition)

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

About the Authors Dr. Alexander Makarov Dr. Michaela Scigelova 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