FOURIER TRANSFORM MASS SPECTROMETRY

Transcription

1 FOURIER TRANSFORM MASS SPECTROMETRY FT-ICR Theory Ion Cyclotron Motion Inward directed Lorentz force causes ions to move in circular orbits about the magnetic field axis Alan G. Marshall, Christopher L. Hendrickson, and George S. Jackson Encyclopedia of Analytical Chemistry, R.A. Meyers (Ed.), John Wiley & Sons Ltd, Chichester, 2, pp

2 FT-ICR Theory Ion Cyclotron Motion Ion cyclotron motion. Ions rotate in a plane perpendicular to the direction of a spatially uniform magnetic field, Note that positive and negative ions orbit in opposite senses. FT-ICR Theory Ion Cyclotron Motion X Y c = qb m Z 2

3 Once we make an ion, we move it into the center of the Magnet. Then, we trap it before it can escape. Electrostatic Barrier ION + From Primer 1998 Marshall. Once we make an ion, we move it into the center of the Magnet. Then, we trap it before it can escape. Electrostatic Barrier ION + Ion sees barrier and is turned back From Primer 1998 Marshall. 3

4 Once we make an ion, we move it into the center of the Magnet. Then, we trap it before it can escape. Electrostatic Barrier ION + Gate shut before the ion escapes From Primer 1998 Marshall. Once we make an ion, we move it into the center of the Magnet. Then, we trap it before it can escape. Ion is now trapped in the magnet. ION + From Primer 1998 Marshall. 4

5 FT-ICR Theory - Ion Trapping X Y Z T T Magnetic Field (B) E Axial Position FT-ICR Theory - Ion Trapping X Y Z T T Magnetic Field (B) E Axial Position 5

6 FT-ICR Theory - Ion Trapping X Z T T Magnetic Field (B) Y E Axial Position FT-ICR Theory Combined Ion Motion v m = magnetron motion v c = cyclotron motion v t = trapping oscillations Alan G. Marshall, Christopher L. Hendrickson, and George S. Jackson Encyclopedia of Analytical Chemistry, R.A. Meyers (Ed.), John Wiley & Sons Ltd, Chichester, 2, pp

7 FT-ICR Theory - Excitation Image Time (ms) FT-ICR Theory - Excitation X Z or B o Y Amplitude Excitation Electrodes Time 7

8 FT-ICR Theory Excitation Alan G. Marshall, Christopher L. Hendrickson, and George S. Jackson Encyclopedia of Analytical Chemistry, R.A. Meyers (Ed.), John Wiley & Sons Ltd, Chichester, 2, pp SWIFT Excitation Single Notch SWIFT Event (MS/MS) Data Count Affects Resolution (Limited to < 512K) Freq Cutoff Bandwidth 1% Power Start Frequency End Frequency % Frequency 8

9 SWIFT Excitation 1% Power % Frequency IFT FT-ICR Theory - Excitation X Z or B o Y Amplitude Time Excitation Electrodes 9

10 SWIFT Excitation m/z On -the -fly SWIFT isolation of a single Isotope of Bovine Ubiquitin M+4 Isotope m/z m/z FT-ICR Theory - Detection X Z or B o Y 1

11 Multiplex Detection in FT-ICR.5 Time Domain Transient.4.3 Differential Amplifier Image Current Time (ms) Multiplex Detection in FT-ICR.5.4 Time Domain Transient.3 Differential Amplifier Image Current Time (ms) 11

12 Signal Apodization in FT MS Fourier Transforms in FT-ICR Differential Amplifier Image Current Time Domain Transient Time (ms) Fourier Transform Frequency Spectrum Frequency (khz)

13 Mass Calibration in FT-ICR Frequency Spectrum Mass Spectrum m z = A + BV t f f Frequency (khz) m/z Space Charge and Resolution in FT-ICR T T LOW ION DENSITY FT Detection Time (msec) m/z FT T T Detection Time (msec) 8 HIGH ION DENSITY m/z

14 Effect of Magnetic Field Strength Resolving Power Highest Non-Coalesced Mass Scan Speed (LC/MS) Axialization Efficiency Number of Ions Trapped Ion Upper Mass Limit 2D-FT Resolving Power Ion Trapping Time Ion Energy Field Strength (Tesla) FT-ICR Experiment - Event Sequences - Use a single mass analyzer but separate the mass analysis and ion isolation events in time - Can perform many successive stages of MS (MS n ) Event Sequence Ion Transfer / Ion Trapping Parent Ion Fragmentation Ionization Parent Ion Isolation Daughter Ion Detection 14

15 Ultra-high Resolving Power Peak Capacity = (m/z) max -(m/z) min m 5% m 5% (m/z) min m/z (m/z) max Separation Method Maximum # of Components Maximum Peak Capacity Theoretical Plates HP-TLC , Isocratic LC , Gradient LC , HPLC 37 1, 1,5, CE 37 1, 1,5, Open Tubular GC 37 1, 1,5, ESI FT-ICR MS 525 2, 6,,, m/ m 5% > 2, 2 < m/z < 1, m average +/-.25 Da Skip Prior Chemical Separation and Identify Components by MS! 15

16 )Ryan P. Rodgers, Alan G. Marshall, Petroleomics: Advanced Characterization of Petroleum-Derived Materials by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICRMS, Asphaltenes, Heavy Oils, and Petroleomics 27, pp )Ryan P. Rodgers, Alan G. Marshall, Petroleomics: Advanced Characterization of Petroleum-Derived Materials by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICRMS, Asphaltenes, Heavy Oils, and Petroleomics 27, pp

17 Isotopic Fine Structure Resolving power M = 8,, M 5% S. D.-H. Shi, C. L. Hendrickson, and A. G. Marshall Proc. Natl. Acad. Sci. USA, 1998, 95, Unit Mass Baseline Resolution for an Intact 148 kda Therapeutic Monoclonal Antibody by FT-ICR Mass Spectrometry Anal Chem. 211 November 15; 83(22): doi:1.121/ac22429c. 17

18 Unit Mass Baseline Resolution for an Intact 148 kda Therapeutic Monoclonal Antibody by FT-ICR Mass Spectrometry Anal Chem. 211 November 15; 83(22): doi:1.121/ac22429c. Unit Mass Baseline Resolution for an Intact 148 kda Therapeutic Monoclonal Antibody by FT-ICR Mass Spectrometry Anal Chem. 211 November 15; 83(22): doi:1.121/ac22429c. 18

19 Single-scan electrospray FT-ICR mass spectrum of the isolated 48+ charge state of bovine serum albumin J. Am. Soc. Mass Spectrom. (215) 26:1626Y1632 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) 19

20 Orbitrap - Electrostatic Field Based Mass Analyser U ( r, z) k z 2 2 r 2 / 2 R r ln( r / 2 m R m ) z φ Korsunskii M.I., Basakutsa V.A. Sov. Physics-Tech. Phys. 1958; 3: 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 , 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 k m / z 2

21 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 the Orbitrap? 21

22 Getting Ions into the Orbitrap The ideal Kingdon field has been known since 195 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. 2, 72: A.A. Makarov, US Pat. 5,886,346, A.A. Makarov et al., US Pat. 6,872,938, 25. Curved Linear Trap (C-trap) for Fast Injection Gate Deflector Push Trap Pull Lenses Orbitrap 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, 25. A. Kholomeev et al., WO5/124821,

23 Comparison of Resolving Power as a function of mass for Orbitrap and ICR Dependence of resolving power on m/z for the following analyzers (all data are shown for a.76 s scan): (i) standard trap (magnitude mode, 3.5 kv on central electrode), (ii) compact high-field trap (eft, 3.5 kv on central electrode), (iii) FTICR (magnitude mode, 15 T), (iv) FTICR (absorption mode, 15 T). Anal. Chem. 213, 85, Hybrid Orbitrap XL 23

24 Hybrid Orbitrap Elite Hybrid Orbitrap Fusion 24

25 ControlB3a #487 RT: AV: 1 NL: 7.16E3 T: ITMS + c NSI d Full ms @cid3. [ ] m/z ControlB3a #4871 RT: AV: 1 NL: 4.17E3 T: ITMS + c NSI d Full ms @cid3. [ ] ControlB3a #4873 RT: AV: 1 NL: 1.54E3 T: ITMS + c NSI d Full ms @cid3. [ ] m/z 15 ControlB3a #4872 RT: AV: 1 NL: 3.27E3 T: ITMS + c NSI d Full ms @cid3. [2.-79.] m/z ControlB3a #4873 RT: AV: 1 NL: 1.54E3 T: ITMS + c NSI d Full ms @cid3. [ ] m/z m/z ControlB3a #4869 RT: AV: 1 NL: 7.39E6 T: FTMS + p NSI Full ms [ ] m/z Highly Parallel Data Acquisition Parallel Detection in Orbitrap and Linear Ion Trap Relative Abundance RT: MS/MS of m/z Scan # 487 Relative Abundance RT: High resolution Full scan # 4869 Relative Abundance Relative Abundance RT: MS/MS of m/z Scan # 4871 RT: MS/MS of m/z Scan # 4872 Relative Abundance Relative Abundance RT: 41.6 MS/MS of m/z Scan # 4874 RT: MS/MS of m/z Scan # 4873 Total cycle is 2.4 seconds 1 High resolution scan 5 ion trap MS/MS in parallel 25

26 January 1, 214 Molecular & Cellular Proteomics, 13, January 1, 214 Molecular & Cellular Proteomics, 13,

27 Rate of protein identifications as a function of mass spectrometer scan rate for selected large-scale yeast proteome analyses over the past decade. Each data point is annotated with the year, corresponding author, type of MS system used, and reference number. January 1, 214 Molecular & Cellular Proteomics, 13,