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
-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
-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
-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
-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