Ion Activation in MS/MS - ccic.ohio-state.edu · Ion Activation in MS/MS Briefly introduce...

1

Ion Activation in MS/MS

Briefly introduce different activation methods

Provide framework for understanding differences

Vicki H. Wysocki

1

Ion Activation in MS/MS

If different activation parameters

e.g., different energy distribution,

different time frame

predict expected change in spectra

MS #1Isolate

MS #2Analyze

Activate

2

2

SORI-CIDADSGEGDFLAEGGGVR + H+

msec to sec, collision gas in FTICR

y5 y9

y14

y11

3

SID (Surface-Induced Dissociation) sec Time Frame

4

3

Q-SID-TOFADSGEGDFLAEGGGVR + H+

[M+H] +

y14y11

y9

y1

b3

y5

b6

200 400 600 800 1000 1200 1400 1600

80 eV SIDb2

[M+H] +

y14y11

y9

y1

b3

y5

b6

200 400 600 800 1000 1200 1400 1600200 400 600 800 1000 1200 1400 1600

80 eV SIDb2

5

Ion Activation in MS/MS Traditionally Collision with Gas

but Other Types of Activation Exist

H2NNH

NNH

NHOH

O

O

O

O

O

H CID

6

4

Ion Activation in MS/MS

WHY DO WE NEED ADDITIONAL ACTIVATION METHODS?

More complete fragmentation large biomoleculesmolecules that fragment too little or too much

Further develop understanding of activation and unimolecular dissociation

Improve ease of use small instruments, remote applications

H2NNH

NNH

NHOH

O

O

O

O

O

H CID

CID WORKS!

7

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) in traps or transmitted through

*Infrared Multiphoton Dissociation (IRMPD)

Electron Capture Dissociation (ECD)

*Electron Transfer Dissociation (ETD)

Surface-Induced Dissociation (SID) 8

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

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

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

10^9

10^8

10^7

10^6

10^5

10^4

ns 12

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 probability13

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

8

R. G. Kessel, E. Pollack, W. W. Smith, p. 164 in Collision Spectroscopy,Ed. R. G. Cooks, Plenum Press, New York, London, 1978

Internal Energy Distribution, P(E)

CID: For Whole Population, Range of Impact Parameters

15

Direct bond cleavage

Rearrangement

RRK (simplified)

k(E) = (1 E0/E)s-1

Internal Energy Distribution P(E) and k(E) Curves

16

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

• A = local (global) minimum

• B = local (global)maximum

• S = saddle point2P(x,y) / x2 > 02P(x,y) / y2 < 0

A

SB

xyz

Potential Energy Surface (PES)

Beynon, J. H.; Gilbert, J. R. “Application of Transition State Theory to UnimolecularReactions. An Introduction”, John Wiley & Sons, Chichester, New York, Brisbane,Toronto, Singapore, 1984

18

10

∆E, ∆H

E0

Eint

Kinetic shift

Er

Reverse critical

energy

Beynon, J. H.; Gilbert, J. R. “Application of Transition State Theory to UnimolecularReactions. An Introduction”, John Wiley & Sons, Chichester, New York, Brisbane,Toronto, Singapore, 1984

Potential Energy Profile of a Reaction

E0 (kinetic term) can be significantly different from ΔE (ΔH) (thermodynamicterm)

Kinetic shift is energyneeded to drive areaction with a givenrate constant, k.

19

Potential Energy Profile of a Reaction

Internal Energy

Critical Energy

Reaction Path

Jurgen Gross, Mass Spectrometry, Springer: Verlag, p27, 200420

11

•

What Determines k at a given Eint?

Beynon, J. H.; Gilbert, J. R. “Application of Transition State Theory to UnimolecularReactions. An Introduction”, John Wiley & Sons, Chichester, New York, Brisbane,Toronto, Singapore, 1984

The ratio of microstates above the transition state(n#) and at the reactant (m)

∆E, ∆H

E0

Eint

Kinetic shift

Er

Reverse critical

energym

n#

21

Loose and Tight TS

loose tight

More microstates areavailable above theloose TS than abovethe tight TS.

It is more difficult to build a road (or roads)in the Grand Canyon thanin the Shenandoah Valley.

22

12

• k(E) = (/h) {G#(EE0) / (E)}

= reaction path degeneracy

h = Planck constant

G#(EE0) = 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

Counting the Microstates

G#(EE0)

23

24

13

Predict Shapes of k(E) vs. E

which pathway highest E0?

which pathway steepest rise?

Busch, Glish, McLuckey, MS/MS VCH Publishers, 198825

Busch, Glish, McLuckey, MS/MS VCH Publishers, 1988

k vs. E curve

breakdown curve

(abundance vs. E)

26

14

Need Internal Energy Distribution P(E) to calculate spectrum

How can internal energy distribution be determined?

EI : PES

MS/MS: ?

27

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

Internal Energy Distributions for MS/MS

28

15

W(CO)2+

W(CO)3+

W(CO)4+

W(CO)5+

W(CO) W(CO)6+

Energy Deposition from a Thermometer Molecule

Wysocki V H, Kenttäma H I, Cooks R G, Int. J. Mass Spectrom. Ion Proc., 1987, 75, 181-20829

Spectra Depend on Reaction Pathways, Energy Distribution Deposited, and Observation Time Window

CH3CO(Pro)OCH3

Predict fragments ofProtonated molecule:

30

16

MH

+

[MHM

eOH

]+

[MHH

CO

OM

e]+

172

140

112

IT 16%

31

MH

+

[MHM

eOH

]+

[MHC

H2=

C=

O]+

[MHH

2O-C

O]+

P

QQQ 13 eV

70

130

140

112

32

17

Q-TOF

SID, 13eV

MH

+

[MHC

H3O

H]+

[MHC

H2=

C=

O]+

[MHC

H3O

HC

O]+

P70

112

130

140

33

Proton Transfer to Ring (amide) N

34

18

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

Factors to Compare Qualitatively

35

Elab = 7 keV

Wysocki V H, Kenttäma H I, Cooks R G, Int. J. Mass Spectrom. Ion Proc., 1987, 75, 181-208

Typical P(E) Curves for keV and eV CID

Elab = 28 eV

36

19

Modified from Laskin J, Futrell J H, Mass Spectrom Rev., 2003, 22, 158-181

Activation Can be “Energy-Sudden” or “Slow Heating”

37

Consider

CH3CH2CH2OH+

CH2CH2CH2OH2+

In ion trap CID, both lose water

What happens in triple quad?

Single Step (Sudden) vsStepwise (Slow Heating) Activation

38

20

Isomeric ion structures

10 eV CID, QQQ

Wysocki V H, Henttämaa H I, J. Am. Chem. Soc., 1990, 112, 5110-5116

QQQ

IT

IT shows loss of H2O for both39

Vekey, K. “Internal Energy Effects in Mass Spectrometry”, J. Mass Spectrom. 1996, 31, 445-463

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”, VerlagChemie, 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, 1978

Suggested Readings

40

21

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) 41

Spengler B. J. Mass Spectrom., 1997, 32, 1019-1036

Ef=(mf/mp)Ep

Linear reflectrons

must be adjusted

for Ef

not a “real” MS/MS

without gas (metastable) or with gas (keV CID)

Post Source Decay (PSD)

42

22

•

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)

Orbitrap

•

43

Orbitrap: Collisional activation OUTSIDE orbitrap

Higher-energy C-trap dissociation (HCD), previously performed in the C-trap (a), is now performed in an octopolecollision cell (b) (reference Olsen, 2007)

44

23

45

Maximum energy available

Depends on target mass and kinetic energy:

center of mass energy (Ecm)

Ecm = Elab [Mt / (Mt + Mp)]

Example: [YGGFL]H+ = 556 u = Mp

Internal energy, P(E ≤ Ecm)

He (4 u) ≤ 0.714%, Elab (71.4 [10,000]; 0.36 [50])

Ar (40 u) ≤ 6.7% Elab

Xe (131 u) ≤ 19% Elab 46

[YGGFL]H+ = 556 u = Mp

24

400 560 720 880 1040 1200

Mass (m/z)

0

176.9

01020

30

405060

708090

100

% I

nte

nsi

ty

TOF TOF MS/MS Precursor 1347 Spec #1=>AdvBC(50,0.5,0.1)=>NF0.9=>NF0.9=>TR[BP = 837.4, 177]

a7 -

17a7

a5 -

17,P

KP

QQ

-17

a8 -

17

b5

PQ

QF

a6 -

17

b6 -

17

a10

-17

b6d6a

b7 -

17

d5a

b8 -

17

PQ

QF

FG

L -

28

a9 -

17

QF

F,P

KP

Q -

28

b4 d10a

400 560 720 880 1040 1200

Mass (m/z)

0

263.9

0

10

20

30

40

50

60

70

80

90

100%

In

ten

sity

TOF TOF MS/MS Precursor 1347 Spec #1=>AdvBC(36,0.5,0.1)=>NF0.7=>NF0.7=>TR[BP = 650.4, 264]

d6a

a8a7

d5a

a7 -

17

d10a

a5 -

17,P

KP

QQ

-17

a6 -

17

a8 -

17

PQ

QF

b5

a10

-17

QF

F -

17

QF

FG

-17

797.

48

925.

45

b6 -

17

a9 -

17

Collision Gas Kr P=1x10-5

Collision Gas He P=2x10-6

400 560 720 880 1040 1200

Mass (m/z)

0

176.9

01020

30

405060

708090

100

% I

nte

nsi

ty

TOF TOF MS/MS Precursor 1347 Spec #1=>AdvBC(50,0.5,0.1)=>NF0.9=>NF0.9=>TR[BP = 837.4, 177]

a7 -

17a7

a5 -

17,P

KP

QQ

-17

a8 -

17

b5

PQ

QF

a6 -

17

b6 -

17

a10

-17

b6d6a

b7 -

17

d5a

b8 -

17

PQ

QF

FG

L -

28

a9 -

17

QF

F,P

KP

Q -

28

b4 d10a

400 560 720 880 1040 1200

Mass (m/z)

0

263.9

0

10

20

30

40

50

60

70

80

90

100%

In

ten

sity

TOF TOF MS/MS Precursor 1347 Spec #1=>AdvBC(36,0.5,0.1)=>NF0.7=>NF0.7=>TR[BP = 650.4, 264]

d6a

a8a7

d5a

a7 -

17

d10a

a5 -

17,P

KP

QQ

-17

a6 -

17

a8 -

17

PQ

QF

b5

a10

-17

QF

F -

17

QF

FG

-17

797.

48

925.

45

b6 -

17

a9 -

17

Different Collision Gas TOF TOF

47

Onset Energy Lower for SID

SIDCID

48

25

Carol Robinson, Cambridge

CID of Large Molecules works but not Understood

49

50

26

51

52

27

Fragmentation of GroEL (14-mer)

53

Care needed in ionization

reduced charge

“normal” charge NH4OAc

supercharged

28

GroEL: Influence of charge on fragmentation pattern

+70 vs +50 ??

GroEL +50 CID and SID

14-mer

29

GroEL +50 CID/SID-IM

GroEL (+50) SID-IM

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

HDX in the ICR cell

60

31

SORI: low energy processes, not always good

SID

eV CID(SORI)

keV CID

Tsaprailis et al.,

J. Am. Chem. Soc.,

1999, 121, 5142-515461

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

32

Infrared Multiphoton Dissociation of O-Linked Mucin-Type

Oligosaccharides

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

Anal. Chem., 2005, 77, 208-214

63

J. Beauchamp et al., J. Am. Chem. Soc., 1978, 100, 3248-3250

F. McLafferty et al., Anal. Chem., 1994, 66, 2809-2815

Infrared Multiphoton Dissociation (IRMPD)

64

33

Carbohydrate Fragmentations

Domon B., Costello C.E., Glycoconjugate J., 1988, 5, 397-40965

Comparisons between CID and IRMPD of mucin-type oligosaccharides

Zhang et al.,

Anal. Chem.,

2005, 77, 208-214

CID of neutral oligosaccharide XT-1415

(positive mode)

MS/MS only produced high abundance for high mass fragmets

66

34

IRMPD of neutral oligosaccharide XT-1415

Zhang et al., Anal. Chem., 2005, 77, 208-214

The fragment ions ranging from the parent ion to the last fragment (m/z 228) are readily observed

67

Why does IRMPD yield more extensive fragments?

CID deposits energy only into the precursor ions

IRMPD excites both precursor and fragment ions

68

35

Conclusion

Compared to CID, IRMPD has advantages:

No collision gas needed, faster

No multistage (MSn) 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, 7626-763870

36

L. A. Vasicek, et. al. J. Am. Soc. Mass Spectrom. 22, 2011. 1105–1108.

Dual linear ion trap

Orbitrap with photodissociationin the HCD cell

UV Photodissociation instrument set-up

Brodbelt. Chem. Soc. Rev 43, 2014. 2757-2783.

B. J. Ko and J. S. Brodbelt, Anal. Chem. 83, 2011. 8192–8200

Photodissociation energy deposition

37

Electron Capture Dissociation

[M + nH]n+ + e- → [[ M + nH](n-1)+]* → fragments

www.hitachi-hta.com73

or

c ion

or

z ion

Cooper H J, Håkansson H, Marshall A G, Mass Spectrom. Rev., 2005, 24, 201-22274

38

• Multiply charged (ESI) ions and also their fragments can be reduced by low energy electrons

• Electrons produced by a conventional heated filamentoutside 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-translationalmodification sites (e.g., phosphorylation, glycosylation, sulfation, gamma-carboxylation)

• Disadvantage: reduced charge state may require extendedm/z ion analyzer range

Electron Capture Dissociation

Zubarev R A, Kelleher N, McLafferty F W, J. Am. Chem. Soc. 1998, 120, 3265-326675

Comparison of ECD and SORI-CAD in FTICR

no loss of water,

phosphate groups or

phosphoric acid

c and z ion series

Stensballe, et al.

Rapid Commun. Mass Spectrom.,

2000, 14, 1793-1800

c5+

c6+

c9+

c10+

c8+

c4+

c3+

c2+

b7+

b9+

b10+

y4+

y2+

76

39

Cleave Peptide Bonds without Cleaving Sugar

Cooper H J, Håkansson H, Marshall A G, Mass Spectrom. Rev., 2005, 24, 201-22277

ECD for Peptide Bonds, IRMPD for Sugar

Cooper H J, Håkansson H, Marshall A G, Mass Spectrom. Rev., 2005, 24, 201-222

ECD

IRMPD

78

40

www.chem.wisc.edu/~coon/coonionion.html79

80

41

ASMS Fall Workshop 2006 John E. P. Syka

Instrument Configuration for ETD

81

+

0 V

-

Axial Confinement With DC PotentialsTrapping is Charge Sign Dependent

Linear Trap With End Segments:Axial Confinement With DC Potentials

ASMS Fall Workshop2006 JEPS

42

+

0 V

-

Axial Confinement With DC PotentialsTrapping is Charge Sign Dependent

Linear Trap With End Segments:Removes Ions From Proximity To

Trapping and Excitation Fringe Fields

Fringe FieldRegion

Fringe FieldRegion

ASMS Fall Workshop2006 JEPS

Simion Simulation of Dipole Excitation Field

SegmentedTrap

UnsegmentedTrap

ASMS Fall Workshop2006 JEPS

43

ASMS Fall Workshop 2006 John E. P. Syka

Procedure for ETD

85

Syka J E P et al., Proc. Natl. Acad. Sci. USA, 2004, 101, 9528-9533

86

44

Ion Activation in MS/MS

87

Laskin J, Futrell J H, Mass Spectrom. Rev., 2003, 22, 158-181

Linear Change in Internal Energy

Characteristic of SID

[Ala-Ala]+H+

88

45

Modified from Laskin J, Futrell J H, Mass Spectrom. Rev., 2003, 22, 158-181

Activation Can be “Energy-Sudden” or “Slow Heating”

89

Energy Deposition in SID vs. CID

Wysocki V H et al., J. Am. Soc. Mass Spectrom., 1992, 3, 27-32

CID

90

46

Energy Deposition in SID vs. CID

Wysocki V H et al., J. Am. Soc. Mass Spectrom., 1992, 3, 27-32

CID

SID

91

Galhena A et al., Anal. Chem., 2008, 80, 1425-1436

Detector

Collision Cell

Transfer OpticsQuadrupole

AnalyzerSurface

Instrument Modification for SID

92

47

Galhena A et al., Anal. Chem., 2008, 80, 1425-1436

SID Design

93

Galhena A et al., Anal. Chem., 2008, 80, 1425-1436

SID Design

94

48

Asymmetric Charge & Mass Partitioning

95

Problem

CID (collision with gas) gives

monomer plus (n-1)-mer

doesn’t provide full sub-structure96

49

SID

Single collisionSudden energy deposition

CID

Multiple collisionsMulti-step activation

Why use SID vs CID for large complexes?

Cooks and coworkers97

SID

Access pathways not available by CID?

CID

Why use SID vs CID for large complexes?

98

50

R. L. Beardsley et al., Anal. Chem. 81, 2009, 1347-1356.

A

B

A

B

Multi-Step CID vs. Single Step SID

(CsI)nCs+ and (CsI)nCs2++

10 or 100 mg/mL CsI in 50% MeOH or 50% ACN

100

51

(CsI)43Cs22

+

CID SID

101

Cytochrome C MS/MS CIS and SID

Jones C M et al., J. Am. Chem. Soc., 2006, 128, 15044-15045102

52

Cytochrome C MS/MS CIS and SID

Jones C M et al., J. Am. Chem. Soc., 2006, 128, 15044-15045103

500 2000 3500 5000 6500 8000

m/z

M8+

M7+M9+

M6+

M5+/D10+

D9+

Q15+(precursor)

Q14+ T8+

T7+

T9+ T6+

CID

CE = 1350 eV

M = monomer

D = dimer

T = trimer

Q = tetramer

Asymmetric mass and charge partitioning

500 2000 3500 5000 6500 8000

m/z

M8+

M7+

M6+

M5+/D10+ M4+/D8+

Q14+/D7+Q15+(precursor)

D6+/T9+

T10+D9+

D11+ T8+T7+D5+

T11+

SID

CE = 750 eV

Mass and charge more equally distributed

500 2000 3500 5000 6500 8000

m/z

M8+

M7+

M6+

M5+/D10+ M4+/D8+

Q14+/D7+Q15+(precursor)

D6+/T9+

T10+D9+

D11+ T8+T7+D5+

T11+

SID

CE = 750 eV

Mass and charge more equally distributed

MS/MS of Transthyretin Homotetramers

104

53

MS/MS of Transthyretin Homotetramers

105

SID: Variation with Energy

M = monomer

D = dimer

T = trimer

Q = tetramer

Q15+ (precursor)

M6

M5 M4

D11D9

Q15+Q14/ D7

T10M3/ T9

T7 T6D5

M7

M6

M5 M4M3

Q14/ D7

∆V = 30 VCE = 450 eV

∆V = 50 VCE = 750 eV

∆V = 90 VCE = 1350 eV

M7

Monomer Q14

T7

Trimer

M7

A

B

SID

106

54

Onset Energy Lower for SID

SIDCID

107

NIH, NSF, Waters, UK EPSRC, OSU

108