Ion Activation in MS/MS. Ion Activation in MS/MS

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Ion Activation in MS/MS Briefly introduce different activation methods Provide framework for understanding differences Árpád Somogyi OSU CCIC MSP Summer Workshop July 10, 2018 1 Ion

1 Ion Activation in MS/MS Briefly introduce different activation methods Provide framework for understanding differences Árpád Somogyi OSU CCIC MSP Summer Workshop July 10, Ion Activation in MS/MS MS #1 Isolate Activate MS #2 Analyze If different activation parameters e.g., different energy distribution, different time frame predict expected change in spectra 2 1

2 SORI-CID ADSGEGDFLAEGGGVR + H + msec to sec, collision gas in FTICR y 14 y 5 y 9 y 11 3 SID (Surface-Induced Dissociation) sec Time Frame 4 2

3 Q-SID-TOF ADSGEGDFLAEGGGVR + H + y 1 b 2 y 5 80 ev SID b 3 [M+H] + b 6 y 9 y 11 y Ion Activation in MS/MS Traditionally Collision with Gas but Other Types of Activation Exist H H 2 N NH O N NH O NH OH CID O O O 6 3

4 Ion Activation in MS/MS H H 2 N NH O N NH O NH OH CID O O O CID WORKS! WHY DO WE NEED ADDITIONAL ACTIVATION METHODS? More complete fragmentation large biomolecules molecules that fragment too little or too much Further develop understanding of activation and unimolecular dissociation Improve ease of use small instruments, remote applications 7 Activation Methods *Collision Induced Dissociation (CID) low energy (ev) high energy (kev) single collision multiple collisions Sustained Off-Resonance Irradiation (SORI) Resonance Excitation (RE) *Infrared Multiphoton Dissociation (IRMPD) Electron Capture Dissociation (ECD) *Electron Transfer Dissociation (ETD) Surface-Induced Dissociation (SID) Blackbody Infrared Radiative Dissociation (BIRD) 8 4

MS/MS Spectra Result of Two step Activation Process: activation + unimolecular dissociation Internal energy distribution Molecule: Different

5 MS/MS Spectra Result of Two step Activation Process: activation + unimolecular dissociation Internal energy distribution Molecule: Different unimolecular reaction pathways Energy dependent rate constants Instrument Configuration: Time for reaction 9 Ion Activation and Unimolecular Dissociation 10 5

6 Rate Theory: RRKM, QET (statistical) Theory of Unimolecular Reactions [M] = [M] 0 e -kt [M] = ion abundance at time t [M] 0 = ion abundance at t=0 k = unimolecular rate constant k depends on internal energy (E) 11 Unimolecular Decay ^9 10^8 10^7 10^6 10^5 10^ ns 12 6

7 Assumptions Separation of translational, rotational, vibrational, and electronic motion Motion of the nuclei classical mechanics (quantum corrections, if necessary) Postulates Transition state (TS) on potential energy surface (PES) boundary between reactants, R, and products, P Each microstate has equal a priori probability 13 Other Assumptions The time required for dissociation is long compared with the time of excitation The rate of dissociation is slow relative to the rate of internal energy randomization over all degrees of freedom The ion is an isolated system in a state of internal equilibrium Fragmentation products are formed by a series of competing and consecutive unimolecular reactions 14 7

8 CID: For Whole Population, Range of Impact Parameters Internal Energy Distribution, P(E) R. G. Kessel, E. Pollack, W. W. Smith, p. 164 in Collision Spectroscopy, Ed. R. G. Cooks, Plenum Press, New York, London, Internal Energy Distribution P(E) and k(e) Curves A B + C D. Direct bond cleavage Rearrangement RRK (simplified) k(e) = (1 E 0 /E) s

9 Kinetic Shift: excess energy required to observe detectable dissociation within a certain experimental time frame Competitive Shift: excess energy required to cause dissociation to be competitive with pathway of lower appearance energy 17 Potential Energy Surface (PES) S B A = local (global) minimum B = local (global) maximum y z x A S = saddle point δ 2 P(x,y) /δ x 2 > 0 δ 2 P(x,y) /δy 2 < 0 Beynon, J. H.; Gilbert, J. R. Application of Transition State Theory to Unimolecular Reactions. An Introduction, John Wiley & Sons, Chichester, New York, Brisbane, 18 Toronto, Singapore,

10 Potential Energy Profile of a Reaction E int E 0 Kinetic shift E r Reverse critical energy E 0 (kinetic term) can be significantly different from ΔE (ΔH) (thermodynamic term) ΔE, ΔH Kinetic shift is energy needed to drive a reaction with a given rate constant, k. Beynon, J. H.; Gilbert, J. R. Application of Transition State Theory to Unimolecular Reactions. An Introduction, John Wiley & Sons, Chichester, New York, Brisbane, 19 Toronto, Singapore, 1984 Kinetic Shift: excess energy required to observe detectable dissociation within a certain experimental time frame Competitive Shift: excess energy required to cause dissociation to be competitive with pathway of lower appearance energy 20 10

11 Potential Energy Profile of a Reaction Internal Energy Critical Energy Reaction Path Jurgen Gross, Mass Spectrometry, Springer: Verlag, p27, Spectra Depend on Reaction Pathways, Energy Distribution Deposited, and Observation Time Window CH 3 CO(Pro)OCH 3 Predict fragments of Protonated molecule: 22 11

(m) Beynon, J. H.; Gilbert, J. R. Application of Transition State Theory to Unimolecular Reactions.

12 What Determines k at a given E int? E int m E 0 n # Kinetic shift ΔE, ΔH E r Reverse critical energy The ratio of microstates above the transition state (n # ) and at the reactant (m) Beynon, J. H.; Gilbert, J. R. Application of Transition State Theory to Unimolecular Reactions. An Introduction, John Wiley & Sons, Chichester, New York, Brisbane, 23 Toronto, Singapore, 1984 Loose and Tight TS It is more difficult to build a road (or roads) in the Grand Canyon than in the Shenandoah Valley. loose tight More microstates are available above the loose TS than above the tight TS

13 Counting the Microstates G # (E E 0 ) k(e) = ( /h) {G # (E E 0 ) / (E)} = reaction path degeneracy h = Planck constant G # (E E 0 ) = number of states above TS (E) = density of states in the reactant ion of internal energy E Illustration of the subset of systems which includes all reacting species

14 Predict Shapes of k(e) vs. E which pathway highest E 0? which pathway steepest rise? Busch, Glish, McLuckey, MS/MS VCH Publishers, k vs. E curve breakdown curve (abundance vs. E) Busch, Glish, McLuckey, MS/MS VCH Publishers,

15 Need Internal Energy Distribution P(E) to calculate spectrum How can internal energy distribution be determined? EI : PES MS/MS:? 29 Internal Energy Distributions for MS/MS Thermometer molecule method: Fragment molecule with known thermochemistry Work backwards from spectrum to figure out P(E) that must have been present to lead to measured spectrum 30 15

16 Energy Deposition from a Thermometer Molecule W(CO) + 4 W(CO) + 3 W(CO) + W(CO) W(CO) W(CO) Wysocki V H, Kenttäma H I, Cooks R G, Int. J. Mass Spectrom. Ion Proc., 1987, 75, Spectra Depend on Reaction Pathways, Energy Distribution Deposited, and Observation Time Window CH 3 CO(Pro)OCH 3 Predict fragments of Protonated molecule: Komȧromi, I.; Somogyi, Ȧ.; Wysocki, V. H. Int. J. Mass Spectrom. 2005, 241,

17 [MH CH 3 OH CO]+ [MH CH 3 OH]+ 140 IT 16% MH P 70 [MH CH 3 OH CO]+ 112 [MH CH 2 =C=O]+ 130 QQQ 13 ev [MH CH 3 OH]+ MH

18 Q-TOF SID, 13eV [MH CH 3 OH CO]+ 112 [MH CH 3 OH]+ [MH CH 2 =C=O]+ 140 MH+ P Proton Transfer to Ring (amide) N 36 18

19 Factors to Compare Qualitatively Average amount of energy deposited Distribution of energy deposited Ease of variation of energy deposition (e.g., change a voltage, pressure) Form in which energy is deposited (electronic vs. vibrational) Time frame activation vs. dissociation How readily activation can be driven 37 Typical P(E) Curves for kev and ev CID E lab = 7 kev E lab = 28 ev 38 Wysocki V H, Kenttäma H I, Cooks R G, Int. J. Mass Spectrom. Ion Proc., 1987, 75,

20 Activation Can be Energy-Sudden or Slow Heating Modified from Laskin J, Futrell J H, Mass Spectrom Rev., 2003, 22, Single Step (Sudden) vs Stepwise (Slow Heating) Activation Consider CH 3 CH 2 CH 2 OH + CH 2 CH 2 CH 2 OH 2 + In ion trap CID, both lose water What happens in triple quad? 40 20

21 Isomeric ion structures QQQ IT 10 ev CID, QQQ IT shows loss of H 2 O for both Wysocki V H, Henttämaa H I, J. Am. Chem. Soc., 1990, 112, Suggested Readings Vekey, K. Internal Energy Effects in Mass Spectrometry, J. Mass Spectrom. 1996, 31, Beynon, J. H.; Gilbert, J. R. Application of Transition State Theory to Unimolecular Reactions. An Introduction, John Wiley & Sons, Chichester, New York, Brisbane, Toronto, Singapore, 1984 Levsen K. Fundamental Aspects of Organic Mass Spectrometry, Verlag Chemie, Weinheim, 1978 Cooks, R. G.; Beynon, J. H.; Caprioli, R. M.; Lester, G. R. Metastable Ions, Elsevier, Amsterdam, 1973 Forst, W. Theory of Unimolecular Reactions, Academic Press, New York and London, 1973 (replaced by Unimolecular Reactions 2003) Cooks, R. G. Collision Spectroscopy, Ed. R. G. Cooks, Plenum Press, New York, London,

22 Activation Methods Collision Induced Dissociation (CID) low energy (ev) high energy (kev) single collision multiple collisions Sustained Off-Resonance Irradiation (SORI) Resonance Excitation (RE) Infrared Multiphoton Dissociation (IRMPD) Ultraviolet Multiphoton Dissociation (UVMPD) Electron Capture Dissociation (ECD) Electron Transfer Dissociation (ETD) Surface-Induced Dissociation (SID) Blackbody Infrared Radiative Dissociation (BIRD) 43 Collision-Induced Dissociation (CID) High (kev) energy (electronic excitation possible) Sector ( s time scale, single collision) TOF/TOF ( s time scale, single collision) Low (ev) energy QQQ, sector/q (BEQQ), Q-TOF ( s, multiple collision) IT (ms, multiple collision) ICR (ms-min, multiple collision) 44 Orbitrap 22

23 Orbitrap: Collisional activation OUTSIDE orbitrap Higher-energy C-trap dissociation (HCD), previously performed in the C-trap (a), is now performed in an octopole collision cell (b) (reference Olsen, 2007)

24 Maximum energy available Depends on target mass and kinetic energy: center of mass energy (E cm ) E cm = E lab [M t / (M t + M p )] Example: [YGGFL]H + = 556 u = M p Internal energy, P(E E cm ) He (4 u) 0.714%, E lab (71.4 [10,000]; 0.36 [50]) Ar (40 u) 6.7% E lab Xe (131 u) 19% E lab 47 Different Collision Gas TOF TOF % Intensity % Intensity QFF Mass (m/z) TOF TOF MS/MS Precursor 1347 Spec #1=>AdvBC(50,0.5,0.1)=>NF0.9=>NF0.9=>TR[BP = 837.4, 177] a5-17,pkpqq Mass (m/z) QFF,PKPQ - 28 QFFG - 17 b4 PQQF PQQF d5a d5a TOF TOF MS/MS Precursor 1347 Spec #1=>AdvBC(36,0.5,0.1)=>NF0.7=>NF0.7=>TR[BP = 650.4, 264] a5-17,pkpqq - 17 b5 b5 d6a d6a a6-17 a6-17 b6-17 b6-17 b6 Collision Gas Kr P=1x PQQFFGL - 28 a7-17 a7-17 a7 a7 b Collision Gas He P=2x10-6 a8-17 a8-17 a8 b8-17 a9-17 a9-17 d10a d10a a10-17 a

25 CID of Large Molecules works but not Understood Carol Robinson, Cambridge

27 Fragmentation of GroEL (14-mer) 53 Care needed in ionization reduced charge normal charge NH 4 OAc supercharged 27

28 GroEL: Influence of charge on fragmentation pattern +70 vs +50?? GroEL +50 CID and SID 14-mer 28

29 GroEL +50 CID/SID-IM GroEL (+50) SID-IM 29

Activation Methods Post Source Decay (PSD) - MALDI instruments Collision Induced Dissociation (CID) low energy (ev) high energy (kev) single collision multiple collisions Sustained Off-Resonance

30 Activation Methods Post Source Decay (PSD) - MALDI instruments Collision Induced Dissociation (CID) low energy (ev) high energy (kev) single collision multiple collisions Sustained Off-Resonance Irradiation (SORI) Resonance Excitation (RE) Infrared Multiphoton Dissociation (IRMPD) Electron Capture Dissociation (ECD) Electron Transfer Dissociation (ETD) Surface-Induced Dissociation (SID) Blackbody Infrared Radiative Dissociation (BIRD) 59 Ion selection and activation in the ICR IRMPD, SORI, ECD IR IRMPD and HDX in the ICR cell 60 30

31 SORI: low energy processes, not always good SID ev CID (SORI) kev CID Tsaprailis et al., J. Am. Chem. Soc., 1999, 121, Comparison of Collision-Induced Dissociation and Infrared Multiphoton Dissociation in the Structural Determination of Oligosaccharides Jinhua Zhang, Katherine Schubothe and Carlito B. Lebrilla Department of Chemistry University of California at Davis 62 31

Infrared Multiphoton Dissociation of O-Linked Mucin-Type Oligosaccharides Anal. Chem., 2005, 77, 208-214 Jinhua Zhang, Katherine Schubothe, Bensheng Li, Scott Russell, and Carlito B.

32 Infrared Multiphoton Dissociation of O-Linked Mucin-Type Oligosaccharides Anal. Chem., 2005, 77, Jinhua Zhang, Katherine Schubothe, Bensheng Li, Scott Russell, and Carlito B. Lebrilla Department of Chemistry and School of Medicine, Biochemistry and Molecular Medicine University of California at Davis 63 Infrared Multiphoton Dissociation (IRMPD) BaF 2 window J. Beauchamp et al., J. Am. Chem. Soc., 1978, 100, F. McLafferty et al., Anal. Chem., 1994, 66,

33 Carbohydrate Fragmentations Domon B., Costello C.E., Glycoconjugate J., 1988, 5, Comparisons between CID and IRMPD of mucin-type oligosaccharides CID of neutral oligosaccharide XT-1415 (positive mode) MS/MS only produced high abundance for high mass fragmets Zhang et al., Anal. Chem., 2005, 77,

34 IRMPD of neutral oligosaccharide XT-1415 The fragment ions ranging from the parent ion to the last fragment (m/z 228) are readily observed Zhang et al., Anal. Chem., 2005, 77, Why does IRMPD yield more extensive fragments? CID deposits energy only into the precursor ions IRMPD excites both precursor and fragment ions 68 34

35 Conclusion Compared to CID, IRMPD has advantages: No collision gas needed, faster No multistage (MS n ) needed Easy and good control of energy deposited The fragmentation efficiency of IRMPD increases with the increasing size of oligosaccharides IRMPD provides an attractive alternative to CID in the structural elucidation of oligosaccharides 69 SORI-RE of oligosaccharide Alditol Herrmann et al., Anal. Chem., 2005, 77,

Mass Spectrom. 22, 2011. 1105 1108. Photodissociation energy deposition Brodbelt.

36 UV Photodissociation instrument set-up Dual linear ion trap Orbitrap with photodissociation in the HCD cell L. A. Vasicek, et. al. J. Am. Soc. Mass Spectrom. 22, Photodissociation energy deposition Brodbelt. Chem. Soc. Rev 43, B. J. Ko and J. S. Brodbelt, Anal. Chem. 83,

37 Electron Capture Dissociation [M + nh] n+ + e - [[ M + nh] (n-1)+ ]* fragments 73 or or z ion c ion 74 Cooper H J, Håkansson H, Marshall A G, Mass Spectrom. Rev., 2005, 24,

38 Electron Capture Dissociation Multiply charged (ESI) ions and also their fragments can be reduced by low energy electrons Electrons produced by a conventional heated filament outside the FTMS magnet opposite to the ESI source (10-5 torr Ar for cooling, < 0.2 ev) Mostly c and z type ions are formed Gentle fragmentation, good for detecting post-translational modification sites (e.g., phosphorylation, glycosylation, sulfation, gamma-carboxylation) Disadvantage: reduced charge state may require extended m/z ion analyzer range 75 Zubarev R A, Kelleher N, McLafferty F W, J. Am. Chem. Soc. 1998, 120, Comparison of ECD and SORI-CAD in FTICR c and z ion series c 3 + c 2 + c 4 + c 5 + c 6 + c + c c 10 + no loss of water, phosphate groups or phosphoric acid y 2 + y 4 + b 7 + b 9 + b 10 + Stensballe, et al. Rapid Commun. Mass Spectrom., , 14,

39 Cleave Peptide Bonds without Cleaving Sugar 77 Cooper H J, Håkansson H, Marshall A G, Mass Spectrom. Rev., 2005, 24, ECD for Peptide Bonds, IRMPD for Sugar ECD IRMPD 78 Cooper H J, Håkansson H, Marshall A G, Mass Spectrom. Rev., 2005, 24,

41 Instrument Configuration for ETD ASMS Fall Workshop 2006 John E. P. Syka 81 Linear Trap With End Segments: Axial Confinement With DC Potentials V Axial Confinement With DC Potentials Trapping is Charge Sign Dependent ASMS Fall Workshop 2006 JEPS 41

42 Linear Trap With End Segments: Removes Ions From Proximity To Trapping and Excitation Fringe Fields Fringe Field Region + - Axial Confinement With DC Potentials Trapping is Charge Sign Dependent Fringe Field Region 0 V ASMS Fall Workshop 2006 JEPS Simion Simulation of Dipole Excitation Field Segmented Trap Unsegmented Trap ASMS Fall Workshop 2006 JEPS 42

43 Procedure for ETD ASMS Fall Workshop 2006 John E. P. Syka 85 Syka J E P et al., Proc. Natl. Acad. Sci. USA, 2004, 101,

44 Ion Activation in MS/MS 87 Linear Change in Internal Energy Characteristic of SID [Ala-Ala]+H + Laskin J, Futrell J H, Mass Spectrom. Rev., 2003, 22,

45 Activation Can be Energy-Sudden or Slow Heating Modified from Laskin J, Futrell J H, Mass Spectrom. Rev., 2003, 22, Energy Deposition in SID vs. CID CID Wysocki V H et al., J. Am. Soc. Mass Spectrom., 1992, 3,

46 Energy Deposition in SID vs. CID SID CID Wysocki V H et al., J. Am. Soc. Mass Spectrom., 1992, 3, Onset Energy Lower for SID SID CID 92 46

47 Instrument Modification for SID Collision Cell Detector Quadrupole Analyzer Surface Transfer Optics Galhena A et al., Anal. Chem., 2008, 80, SID Design Galhena A et al., Anal. Chem., 2008, 80,

48 SID Design Galhena A et al., Anal. Chem., 2008, 80, GroEL +50 CID and SID (AA and TEAA) 14-mer 48

49 7mers Released from SID of GroEL (+50) Zhou, M; Jones, C; Wysocki, V. Anal. Chem. GroEL PDB:1OEL Substructure! 7mers Released by SID Zhou, M; Jones, C; Wysocki, V. Anal. Chem. GroEL PDB:1OEL 49

50 Asymmetric Charge & Mass Partitioning 99 Multi-Step CID vs. Single Step SID B B A A R. L. Beardsley et al., Anal. Chem. 81, 2009,

51 Overall Approach Song, Nelp, Bandarian, Wysocki ACS Central Science. SID in High Resolution Platforms: FTICR 51

52 SID in Bruker solarix XR 15 T FTICR 35 V SID of 12+ streptavidin tetramer Intens. x Q12+ +MS 0.75 PDB: 1SWB Q11+ Q m/z 52

53 35 V SID of 12+ streptavidin tetramer Intens. x10 7 D6+ Intens. x Q12+ +MS(CASI ) +MS PDB: 1SWB D Q11+ Q m/z 2 D5+ 1 M4+ Q12+ Q11+ M5+ M3+ D8+ T8+ T7+ D m/z SID of 12+ streptavidin tetramer Zoom in SID 35 V D6+ D7+ D5+ M5+ M4+ M3 Q12+ T7+ D8+ + T8+ D_pos_4M_EDDA_BE2_2_4450_SID45_TOF1p8_ d: +MS(CASI M3+ D6+ Q12+ E2_2_4450_SID45_TOF1p8_ d: +MS(CASI SID 45 V D6+ M5+ M4+ D7+ M3+ D5+ T8+ T7+ noesi_sid_pos_4m_edda_4450_sid55_tof1p8_ d: +MS(CASI EDDA_4450_SID55_tof1p8_ d: +MS(CASI SID 55 V M4+ D7+ D6+ M3+ D m/z m/z 53

Intens. x10 7 8 +13 CTB SID 35 V M4+ +MS(CASI 4520.

54 Intens. x CTB SID 35 V M4+ +MS(CASI ) 6 D M5+ D7+ D6+ M3+ Q8+ T6+ P13+ M2+ T8+ T7+ D4+ Q9+ P m/z Zoom in m/z Resolution ,298 Intens. x10 7 M2+ +MS(CASI ) D4+ Q8+ Q D4+ Q Q8+ M2+ D4+ Q8+ 1 T6+ T6+ T6+ T m/z 54

Acknowledgements Anne Blackwell, Royston Quintyn, Yang Song, Akiko Tanimoto; Yue Ju, Nilini Ranbaduge, Sophie Harvey, Jing Yan, Ani Sahasrabuddhe, Jikang Wu, Pepsi Holmquist,

Elizabeth Vierling, Eman Basha, Indu Santhanagopalan. (shsp) Prof. Venkat Gopalan, Prof. Mark Foster, L. Lai, S. Lai (Rnase P) Prof. Michael G. Poirier and Morgan W.

55 Acknowledgements Anne Blackwell, Royston Quintyn, Yang Song, Akiko Tanimoto; Yue Ju, Nilini Ranbaduge, Sophie Harvey, Jing Yan, Ani Sahasrabuddhe, Jikang Wu, Pepsi Holmquist, Mengxuan Jia, Josh Gilbert, Florian Busch, and past members Collaborators for some recent complexes: Prof. Vahe Banderian, Micah Nelp, (TNH) Prof. Elizabeth Vierling, Eman Basha, Indu Santhanagopalan. (shsp) Prof. Venkat Gopalan, Prof. Mark Foster, L. Lai, S. Lai (Rnase P) Prof. Michael G. Poirier and Morgan W. Bernier (Nucleosomes) Prof. Jennifer Ottesen and Cecil J. Howard (Nucleosomes) Prof. Zucai Suo and group Prof. Karin Musier-Forsyth & L Chen Prof. Dan Wozniak and B. Xu Dmitri, Marcos, Ross, Tom, Dan SID Funding: NSF, NIH Bruker Thermo 55

What is Tandem Mass Spectrometry? (MS/MS)

What is Tandem Mass Spectrometry? (MS/MS) Activate MS1 MS2 Gas Surface Photons Electrons http://www.iupac.org/goldbook/t06250.pdf Major difference between MS and MS/MS: two analysis steps Ionization Analysis