Mass Analyzers. Want to do MS or MS/MS? Need a Mass Spectrometer. Árpád Somogyi. Summer Workshop. Mass analyzer. Ionization source.

Transcription

1 Mass Analyzers Árpád Somogyi Summer Workshop Want to do MS or MS/MS? Need a Mass Spectrometer Ionization source Mass analyzer Detector inlet all ions sorted ions Data system 1

2 Ionization source Mass analyzer Detector inlet all ions sorted ions Data system Ion movement Charged metal plates (electrodes, lenses) used to move ions between ion source and analyzers Electrodes and physical slits used to shape and restrict ion beam Good sensitivity is dependent on good ion transmittance efficiency in this area Ionization source Mass analyzer Detector inlet all ions sorted ions Data system 2

3 Ion detection Ions can be detected efficiently w/ high amplification by accelerating them into surfaces that eject electrons Ionization source Mass analyzer Detector inlet all ions sorted ions Data system Ion energy Kinetic energy of ions defined by E = zev = qv = ½ mv 2 E = kinetic energy m = mass v = velocity e = electronic charge ( e -19 C) z = nominal charge V = accelerating voltage 3

4 Learning Check Consider two electrodes, one at 1000 V and one at ground (0 V) ❸ 1000 V 0 V + ion will travel with kinetic energy of Mass Resolution Mass spectrometers separate ions with a defined resolution/resolving power Resolving power- the ability of a mass spectrometer to separate ions with different mass to charge (m/z) ratios. 4

5 Example of ultrahigh resolution in an FTICR J. Throck Watson Introduction to Mass Spectrometry p. 103 Resolution defined at 10% valley M R=M/ M M 5

6 General about mass spectrometers Common features Accelerated charge species (ions) interact with Electrostatic field (ESA, OT) Magnetic field (B, ICR) Electromagnetic (rf) fields (Q, IT, LT) Or ions just fly (TOF) Desirable mass spectrometer characteristics 1) They should sort ions by m/z 2) They should have good transmission (improves sensitivity) 3) They should have appropriate resolution (helps selectivity) 4) They should have appropriate upper m/z limit 5) They should be compatible with source output (pulsed or continuous) 6

7 For each of the following applications, choose the most appropriate mass analyzer from the following list. Use each analyzer only once. orbitrap (OT) time-of-flight (TOF) quadrupole (Q) FTICR Analyzer Purpose synthetic organic chemist wants exact mass of compound biochemist wants protein molecular weight of relatively large protein (MW 300,000) EPA (Environmental Protection Agency) wants confirmation of benzene in extracts from 3000 soil samples Petroleum chemist wants to confirm the presence of 55 unique compounds at one nominal mass/charge value in a mass spectrum Mass spectrometers record m/z values m/z values are determined by actually measuring different physical parameters Type of analyzer electric sector magnetic sector quadrupole, ion trap time-of-flight FT-ion cyclotron resonance Physical parameter used as basis for separation kinetic energy/z momentum/z m/z flight time m/z (resonance frequencies) 7

8 Types of Mass Analyzers Magnetic (B) and/or Electrostatic (E) (HISTORIC/OLDEST) Time-of-flight (TOF) Quadrupole (Q) Quadrupole Ion Trap (IT) Linear Ion Trap (LT) Orbitrap Fourier Transform-Ion Cyclotron Resonance (ICR) Performance Advantages / Disadvantages / $$$ TOF Quadrupole Quadrupole Ion trap FTICR Orbitrap 8

9 Notice: accelerating voltages vary with analyzer (has consequences for MS/MS) High voltage (kev energy range) magnet (B) electrostatic (E) time-of-flight (TOF) Low voltage (ev energy range) quadrupole (Q) ion trap (IT, LT) ion cyclotron resonance (ICR) MS and MS/MS revisited Ionization Analysis MS: Collide with target to produce fragments MS/MS: Ionization Selection Activation Analysis 9

10 Current popular MS/MS arrangements (2009) Tandem in Space QqQ Q Trap Q TOF TOF TOF Tandem in Time Ion Trap (2 or 3 D) FT-ICR (FT) 10

11 Decision factors when choosing a mass spectrometer S p e e e e e e e e e e e e e d R e s o l u t i o n Sensitivity Dynamic range Co$t 11

12 TOF Quadrupole Quadrupole Ion trap FTICR Orbitrap Time of Flight D m/z Detector V KE = zev = ½mv 2 v = D/t ½m(D/t) 2 = zev t = m 1 / 2 2zeV D m = mass V = velocity D = distance of flight t = time of flight KE = kinetic energy e = charge 12

13 How does the ion generation step in TOF influence m/z analysis? Consider MALDI hυ Target + Matrix Analyte Analyte ion may have (1) Kinetic Energy distribution or (2) Spatial distribution Ions of same m/z How will KE spread influence the spectrum? Has slightly greater KE Effect is broad peaks c/o Cotter 13

14 How will KE spread influence the spectrum? Solution to peak broadening caused by kinetic energy spread: Reflectron (ion mirror) Series of ring electrodes, typically with linear voltage gradient Time-of-Flight Reflectron Increased resolution by compensating for KE spread from the source 14

15 Reflectron TOF Mass Analyzer (TOF) Detector Reflectron Ion Source (MALDI) x x x x x High resolution KE = ½ mv 2 Reflectron TOF Mass Analyzer (TOF) Detector Reflectron Ion Source (MALDI) x High resolution KE = ½ mv 2 15

16 We talked about how to deal with kinetic energy spread. How do we deal with ions formed at different locations in the source (spatial distribution)? 16

17 Delayed Extraction 10 KV 10 KV KV Low mass (below 40 k Da) High resolution Continuous vs. Delayed Extraction Continuous TOF Resolution = 700 (FWHM) Delayed Extraction Resolution = 6000 (FWHM) 17

18 Recent TOF designs: improved resolution, better sensitivity and mass accuracy (Ultraflex III MALDI TOF-TOF) In te n s. [a.u.] Resolution: 25,000 S/N: 46 * Pepmix\0_N6\1\1SRef m/z Typical TOF Specs m/z Range: unlimited u Resolution: ~20,000 (Reflectron) Mass Accuracy: ~ 3-10 ppm (300-4,000 u) Scan Speed: 10 6 u/s Vacuum: 10-7 Torr Quantification: low - medium Positive and Negative Ions Variations: Linear, Reflectron, Tandem w/ quads, TOFs and/or sectors 18

19 Can an MS/MS instrument be constructed if the only analyzer type available is TOF? TOF-TOF 19

20 TOF TOF (high energy, kev, collisions) More fragmentation than QqQ or trap (low energy, ev, collisions) Advantages and Disadvantages Mass Spectrometer Advantages Disadvantages TOF-TOF high resolution, high m/z fragment ions kev CID easier de novo peptide sequencing d n and w n to distinguish Ile/Leu Large size Not ideal for continuous ionization source $$$ 20

21 Analyzers TOF Quadrupole Quadrupole Ion trap FTICR Orbitrap 21

22 Quadrupole (Q) Quadrupole (Q) four parallel rods or poles fixed DC and alternating RF voltages only particular m/z will be focused on the detector, all the other ions will be deflected into the rods scan by varying the amplitude of the voltages (AC/DC constant). rf voltage +dc voltage rf voltage 180 out of phase -dc voltage 22

23 Quadrupole Field Animation Negative rod Positive rod Positive rod Negative rod negative DC offset -DC positive DC offset + +DC 23

24 Ion Motion in Quadrupoles - a qualitative understanding + DC, ions focused to center - DC, ions defocused +DC w/ rf, light ions respond to rf, eliminated (high mass filter) -DC w/ rf, light ions respond to rf, focused to center (low mass filter) TSQ to 2000 TSQ Quantum 2001 to c/o ThermoFinnigan Corp. 24

25 Quadrupole animation QuickTime and a TIFF (LZW) decompressor are needed to see this picture. 25

26 RF only mode Initial kinetic energy affects ion motion in quad 26

27 Quadrupole Typical Specs m/z Range: u Resolution: Unit Mass Accuracy: ca +/- 0.1 u Scan Speed: 4000 u/s Vacuum: Torr Low Voltages: RF ~ V DC ~500V-840V Source near ground Quantification: good choice Positive and Negative Ions Variations: SingleQ, TripleQ, Hybrids What MS/MS instruments can be produced from Q? What MS/MS instruments can be produced from Q and TOF? 27

28 Triple Quadrupole - since 1970 s and still going strong! Q1 Q2 Q3 S D Attractive Features: Source near ground and operates at relatively high pressure Couples well to source and to chromatography Multiple scan modes easy to implement Triple Quadrupole (QQQ) Detection System Dynode Turbo Q3 Source Q0 Q1 Q3 Q2 Q2 Heated Capillary Q1 Q0 Q00 c/o Thermo Finnigan Corp. c/o Agilent Corp. 28

29 Quadrupole Time of Flight (Q-TOF) (outdated) Low-energy (ev) Collisions with Gas The third Q in QQQ is replaced by a TOF Advantages? 29

30 Q-TOF 80 fmol BSA digest QQQ Shevchenko, A., et al., Rapid. Commum. Mass Spectrom., 1997, 11: Another important use of a modified Q-TOF: Ion Mobility 30

31 Selection of [M+2H] +2 at m/z m/z Relative Intenisty GRGDS SDGRG Drift Time (ms) Transfer CID of [M+2H] +2 at m/z Drift Time (ms) y 3 y 4 Relative Abundance S i S i y 1 R R b 2 b 3 b 4 SDGRG dt = ms GRGDS dt = ms m/z

32 Analyzers TOF Quadrupole Quadrupole Ion trap FTICR Orbitrap 32

33 What is small, inexpensive and still gives great MSMS????? Quadrupole Ion Traps Miniature IT: Cooks, R. G. and coworkers Anal. Chem. 2002, 74, ; 2000, 72, Ion path in a trap 33

34 Quadrupole Ion Trap (QIT) Quadrupole Ion Trap (QIT) Quadrupole ion trap mass analyzer consists of three hyperbolic electrodes: the ring electrode, the entrance end cap electrode and the exit endcap electrode. Ions enter trap through inlet focusing system and entrance endcap electrode. An AC potential of constant frequency and variable amplitude is applied to the ring electrode to produce a 3D quadrupolar potential field within the trapping cavity which traps ions in a stable oscillating trajectory confined within the trapping cell. The oscillating trajectory is dependent on the trapping potential and the mass-to-charge ratio of the ions. During ion detection, the electrode system potentials are altered to produce instabilities in the ion trajectories and thus eject the ions. 34

35 Quadrupole Ion Trap (QIT) 3D - Quadrupole Ion Trap (Demo) 35

36 Activation: Low-energy (ev) Collisions with Gas IRMPD (Infrared Multiphoton Dissoc) Research: Micro CIT Array 1.8 µm Matt Blain, Sandia Cooks, Purdue 2.4 µm Top End Cap Array Ring Electrode Array Bottom End Cap Array Collector Array 36

37 QIT Typical Specs m/z Range: u Resolution: Unit Mass Accuracy: ca +/- 0.1 u Scan Speed: 4000 u/s Vacuum: 10-3 Torr Quantification: possible, not accurate Positive and Negative Ions Advantages and Disadvantages Mass Spectrometer Advantages Disadvantages QIT Compact Limited storage of ions limits the dynamic range of the ion trap (space charge effect) MS n Data dependent scanning Relatively inexpensive 1/3 of low mass range lost in MS/MS mode, typical operation Low E collisions 37

38 Advantages and Disadvantages Mass Spectrometer Advantages Disadvantages QIT Sensitive Poor quantification Collision energy not well-defined in CID MS/MS Mass Accuracy To increase mass accuracy in a Trap LT, orbitrap, FT-ICR 38

39 RF ION TRAP ELECTRODE STRUCTURES LCQ-Type 3D Quadrupole Trap LTQ-Type (2D) Linear Quadrupole Trap ASMS Fall Workshop 2006 JEPS 2D vs. 3D Ion Traps Ion Transfer Tube Skimmer Tube Lens 39

40 Linear Trap Demo Ion Transfer Tube Skimmer Tube Lens Overall Performance Gains LTQ 3D Traps Increase Trapping efficiency Detection efficiency Trapping capacity ~ 55-70% ~5% ~ 11-14x ~ % ~50% ~ 1-2x ~ 20,000 ions ~500 ions ~ 40x 40

41 Motivating Factors Realized Increased Trapping Efficiency Increased Trapping Capacity Which means... Increased Sensitivity Increased Inherent Dynamic Range Increased S/N for full Scan MS Practical MS n Faster Scan Times - no µscans (only one) How are 3-D traps and linear ion traps used in MS/MS Stand alone 3-D traps LTQ Front or back end of tandem-in-space LT-TOF LT-FT, LT-Orbitrap, QTrap 41

42 LIT and QTrap; Different ways of using Linear Traps Linear Ion Trap ~ 100% of ions trapped are detected due to radial ejection Skimmer Axial ejection allows for hybridization without compromise (FT)! Hybrid Quadrupole Trap Majority of trapped ions cannot be scanned out ~ 15% detected* Axial ejection depends on fringe fields generated by grid this grid compromises triple quadrupole performance *Hager, J., RCM., 2003, 17, 1389 Activation: CID Low-energy (ev) Collisions with Gas IRMPD (Infrared Multiphoton Dissoc) ETD (electron transfer dissociation) 42

43 Analyzers TOF Quadrupole Quadrupole Ion trap FTICR Orbitrap 43

44 v F cp B F cp = q [vxb] Cyclotron motion of an ion in a magnetic field (B). The magnetic field points out of the plane. Note: ion motion is much more complex due to the presence of electrostatic field (magnetron/cyclotron motion) RF-excitation Ion Detection Plates v B Ion Detection Plates Note: for clarity, the front and back trapping plates are not shown 44

45 Cyclotron motion of an ion in a magnetic field (B). Note: ion motion is much more complex due to the presence of electrostatic field (magnetron/cyclotron motion, see below) 45

46 FTICR animation QuickTime and a TIFF (LZW) decompressor are needed to see this picture. Fourier Transform-Ion Cyclotron Resonance (FTICR) In the presence of a magnetic field, sample ions orbit according to cyclotron frequency, f c Cyclotron frequency related to charge of ion (z), magnetic field strength (B) and mass of ion (m). All ions of same m/z will have same cyclotron frequency at a fixed B and will move in a coherent ion packet. 46

47 FTICR Image is Fourier transformed to obtain the component frequencies and amplitudes (intensity) of the various ions. Cyclotron frequency value is converted into a m/z value to produce mass spectrum w/ the appropriate intensities. a b c C 9 H 16 N time (ms) C 7 H 12 N C 11 H 20 N

48 48

49 Fourier Transform-Ion Cyclotron Resonance (FTICR) Ion packets produce a detectable image current on the detector cell plates. As the ion(s) in a circular orbit approach the top plate, electrons are attracted to this plate from ground. Then as the ion(s) circulate towards the bottom plate, the electrons travel back down to the bottom plate. This motion of electrons moving back and forth between the two plates produces a detectable current. FTICR Typical Specs m/z Range: > u Resolution: High, 10 6 Mass Accuracy: 100 ppb Scan Speed: fast Vacuum: Torr Quantification: possible, not accurate Positive and Negative Ions 49

50 Charge state/mw determination for proteins m = z (m/z) Carbons isotopes so m=1 Da Calc (m/z) 50

51 Finnigan LTQ FT Linear Ion Trap MS MS, MS/MS and MS n Analysis AGC Control Secondary Electron Multiplier Detector FTICR MS Ion Image Current Detector Accurate Mass, High Resolution ECD, IRMPD FTMS Data Linear Ion Trap Data 7 T Actively Shielded Superconducting Magnet 60m 3 /hr 15L/s 300L/s 400L/s 210L/s Triple Ported Turbo Pump 210L/s ECD Assembly IRMPD Laser Assembly Introducing the New apex -ultra Hybrid Qq-FTMS Announced at PittCon 2007 in Chicago, IL The new apex-ultra combines ease-of-use with power and versatility Improved electronics for substantial gains in analytical performance and capabilities Comprehensive software tools that are specifically designed for protein characterization 51

52 Versatility matched with Performance The apex -ultra h1 Q1 h2 to analyzer ESI source region ECD / IRMPD Masses can be filtered here in a results-driven, targeted approach CASI provides the means to enrich or amplify low abundant species for subsequent fragmentation and analysis Ions accumulated here to build significant populations an in-cell event (e.g. ECD) Continuous Accumulation of Selected Ions CASI Bruker 9.4 T FT-ICR Apex-Qh Tandem Mass Spectrometer MALDI Dual source, MS only, no ion selection ESI QCID (ev) MS/MS 52

53 Ion selection and activation in the ICR IRMPD, SORI, ECD HDX in the ICR cell Activation: SORI CID Low-energy (ev) Collisions with Gas (off resonance) RE (resonance excitation) IRMPD (Infrared Multiphoton Dissoc) ECD (electron transfer dissociation) 53

54 MS/MS spectra depend on i) Charge state ii) Ion activation method iii) Collision energy (more details in the Ion Activation Methods by Vicki Wysocki) Intens. x x MH + QCID 22 ev [M+2H] 2+ QCID 6 ev YIGSR_QCID_22eV_ d: +MS2(595.0) YIGSR_doubly_QCID_6eV_ d: +MS2(298.0) 0.5 y x [M+2H] 2+ IRMPD 30s, 30% YIGSR_doubly_IRMPD_40P_0_30s_ d: +MS2(298.0) m/z Advantages and Disadvantages Mass Spectrometer Advantages Disadvantages FTICR Highest recorded mass resolution of all mass spectrometers MS n Non-destructive ion detection Accurate mass measurement Powerful capabilities for ion chemistry experiments (ionmolecule rxns) Limited Dynamic Range Low E collisions High vacuum Low presure (difficult GC/LC coupling) Big size Not a high throughput technique 54

55 TOF Quadrupole Quadrupole Ion trap FTICR Magnetic sector Orbitrap 55

56 Orbitrap Orbitrap 56

57 LTQ Orbitrap Hybrid MS Finnigan LTQ Linear Ion Trap API Ion source Linear Ion Trap C-Trap Differential pumping Orbitrap Differential pumping LTQ Orbitrap Hybrid MS System Integration Finnigan LTQ Linear Ion Trap V Gas <1 mtorr x -2 k V Central Electrode Voltage -3.5 k V 57

58 Lord of the ion rings: Retainment of the rings Image current detection on split outer electrodes ω = k m / z 1. Frequencies are determined using a Fourier Transformation 2. For higher sensitivity AND resolution, transients should not decay too fast Ultra-high vacuum Ultra-high precision Thermo slide Orbitrap Animation 58

59 Ion Motion in the Orbitrap Simulation of ion trajectory with SIMION Characteristic frequencies Frequency of rotation ω φ Frequency of radial oscillations ω r Frequency of axial oscillations ω z ω z 2 R ω = m ϕ 1 2 R 2 R ω = m r ω z 2 R ω = z k m / q Orbitrap --Resolving Power +5 Insulin: m/z = 5733 Charge state: +5, +4 and m/ m = 70, ,147 1,148 m/z 1, ,200 m/ m = 45,000 1,400 m/z 1, ,800 2,000 m/ m = 40,000 1,434 1,435 m/z 1,436 R. Noll, H. Li, ,911 1,912 m/z 1,913 1,914 59

60 Question: Consider an ion source block and an extraction lens. How would you bias the block and lens if you want the ions to be accelerated by a) 8000 ev (appropriate for magnetic sector) b) 5 ev (appropriate for entering a quadrupole) c) 20,000 ev (appropriate for entering a TOF) 60

61 Learning Check Draw the appearance of the mass spectrum in which both singly and doubly charged YGGFLR (molecular weight ) are produced and analyzed with a quadrupole, TOF and FT-ICR (Hint: peaks widths important) 712 in Quad, TOF, ICR TOF QUAD Intensity Intensity m/z Intensity m/z ICR m/z 61

62 Instrument Performance for Proteomic Applications Han et al., Curr. Opin. Chem. Biol. 2008; 12(5):

63 Many instruments are complementary More Activation methods, the better Assess your needs Assess your Research level, may want to build your own Suggested Reading List GENERAL MS AND MS/MS Kinter, M. and Sherman, N. E., Protein Sequencing and Identification Using Tandem Mass Spectrometry, Wiley and Sons, New York, NY, De Hoffmann E., Mass Spectrometry Principles and Applications (Second Edition), Wiley and Sons, New York, NY, Busch, K.L., et al., Mass spectrometry/ mass spectrometry: techniques and applications of tandem mass spectrometry, VCH Publishing, New York, NY, McLafferty, F.W. (Ed) Tandem Mass Spectrometry, Wiley and Sons, New York, NY, McLafferty, F.W. and Turecek, F. Interpretation of Mass Spectra, University Science Books, New York, NY, Siuzdak, G. Mass Spectrometry for Biotechnology, Academic Press, San Diego, CA, Gygi, S.P. and Aebersold, R., Curr. Opin. Chem. Biol., 4 (5): 489, Roepstorff, P., Curr. Opin. Biotech., 8: 6, De Hoffmann, JMS, 31: 129, Mann, M. et al., Annu. Rev. Biochem., 70: 437, McLuckey, S and Wells, J.M., Chem. Rev., 101: 571,

64 Suggested Reading List Q-TOF Morris, H.R. et al., J. Protein Chem., 16: 469, Shevchencko A., et al., Rapid Commun. Mass Spectrom., 11: 1015, Shevchencko A., et al., Anal. Chem., 72: 2132, Medzihradszky, K.F., et al., Anal. Chem., 72: 552, Glish, G.L. and Goeringer, D.E., Anal. Chem., 56: 2291, 1984 Morris, H.R. et al., Rapid Commun. Mass Spectrom., 10: 889, Wattenberg, A. et al., JASMS, 13 (7): 772, Lododa, A.V. et al., Rapid Commun. Mass Spectrom., 14(12): 1047, TOF Bush, K., Spectroscopy, 12: 22, Cotter, R.J., Time-of-Flight: Instrumentation and Applications in Biological Research, ACS, Washington, DC, TOF-TOF Suggested Reading List Yergey, A.L. et al., JASMS, 13 (7): 784, Go, E.P. et al., Anal. Chem., 75: 2504, FTICR Buchanan, M.V. (Ed), Fourier Transform MS, ACS, Washington, DC, Comisarow, M.B. and Marshall, A. G., Chem. Phys. Lett., 25: 282, McIver, R.T. et al., Int. J. Mass Spectrom. Ion Processes, 64: 67, Kofel, P. et al., Int. J. Mass Spectrom. Ion Processes, 65: 97, Williams, E. R. Anal. Chem., 70: 179A, McLafferty, F. W. Acc. Chem. Res., 27: 379, Fridriksson, E. K. et al., Biochemistry, 39: 3369, Fridriksson, E. K. et al., Biochemistry, 38: 8056, Kelleher, N. L.et al., Protein Science, 7: 1796, McLafferty, F. W. et al., Int. J. Mass Spectrom., 165: 457,

65 Suggested Reading List Green, M. K. and Lebrilla, C. B. Mass Spectrom. Rev., 16: 53, Guan, S. and Marshall, A. G. Chem. Rev., 94: 2161, 1994 QIT Marsh, R.E. et al., Quadrupole Storage Mass Spectrometry, vol.102, Wiley and Sons, New York, NY, Cooks, R.G. et al., Ion trap mass spectrometry. Chem. Eng. News, 69(12): 26, Jonscher, K.R. et al., Anal Biochem, 244(1): 1, Louris, J.N. et al., Anal. Chem., 59(13): 1677, Louris, J.N. et al., Int. J. Mass Spectrom. Ion Processes, 96(2): Cooks, R.G. et al., Int. J. Mass Spectrom. Ion Processes, : McLuckey, S. A. et al., Int. J. Mass Spectrom. Ion Processes, 106: 213, Ngoka, L. C. M. and Gross, M. L. Int. J. Mass Spectrom. 194: 247, Suggested Reading List QQQ Yost, R. A. and Enke, C. G. J. Am. Chem. Soc., 100: 2274, Arnott, D. in Proteome Research: Mass Spectrometry, james, P (Ed.), pp 11-31, Springer-Verlag, Germany, Steen H. et al., JMS, 36: 782, 2001 Miller, P.E. and Denton, M.B., J. Chem. Educ., 7: 617, Yang, l. et al., Rapid Commun. Mass Spectrom., 16(21): 2060, Mohammed, S. et al., JMS, 36: 1260, Dongre, A.R. et al., JMS, 31: 339, Orbitrap Hu, Q, Noll, RJ, Li, H., Makarov, A., Hardman, M., Cooks, R.G. JMS, , Makarov A. Anal. Chem. 2000; 72(6): Makarov A, Denisov E, Kholomeev A, Baischun W, Lange O, Strupat K, Anal. Chem. 2006; 78(7): Makarov A, Denisov E, Lange O, Horning S. JASMS. 2006; 17(7): Macek B, Waanders LF, Olsen JV, Mann M. Mol. Cell. Proteom. 2006; 5(5): Perry RH, Cooks RG, Noll RJ. Mass Spectrom. Rev. 2008; 27(6): Scigelova M, Makarov A. Orbitrap mass analyzer - Overview and applications in proteomics. Proteomics. 2006: Olsen JV, Macek B, Lange O, Makarov A, Horning S, Mann M. Nature Methods. 2007; 4(9):