used for ms short course at tsinghua by r. graham cooks ... · used for ms short course at tsinghua...
TRANSCRIPT
7/4/2011
1
Gas-Phase Ion/Ion and Ion/Electron Chemistry
Yu XiaDepartment of Chemistry, Purdue University
July 5th, 2011
Statistics from Web of Science®
Gas-Phase Bio-Ion/Ion Chemistry - History
Smith group, 1991, J. Phys. Chem.
+ESI
-ESI
MS
‐+
McLuckey group, 1995, JACSElectron transfer dissociation of oligo-nucleotide anions
LTQ HCTultra
Hunt group, 2004, PNAS
Used for MS Short Course at Tsinghua by R. Graham Cooks, Hao Chen, Zheng Ouyang, Andy Tao, Yu Xia and Lingjun Li
7/4/2011
2
• Uni-molecular Dissociation (e.g. dissociation after EI)• Ions and light – e.g., photodissociation• Ion/neutral interactions
– CID– ion/molecule reactions
• Ion/Ion reactions
1+ / 1-
n+ / 1-
1+ / n-
n+ / m-
Gas-Phase Ion Chemistry in Analytical Mass Spectrometry
• Ion/Electron reactions
n+/1- Proton Transfer Ion/Ion Rxns
• (M+nH)n+ + Y- (M+(n-1)H)(n-1)+ + HY• Most commonly observed reaction so far
Used for MS Short Course at Tsinghua by R. Graham Cooks, Hao Chen, Zheng Ouyang, Andy Tao, Yu Xia and Lingjun Li
7/4/2011
3
n+/1- Multiple Proton Transfer
• (M-H)- + (Y+nH)n+ (M+H)1+ + (Y+(n-2)H)(n-2)+
• Charge inversion in an ion trap
Anal. Chem. 2004, 76, 4189-4192.
[M-H]-
a
800 20001200 1600800 20001200 1600
Arb
. Un i
ts
1.2e4
8e3
4e3
1.2e4
8e3
4e3
5e4
3e4
1e4
5e4
3e4
1e4
[DAB+4H]4+
b
800 20001200 1600400 800 20001200 1600400
[DAB+3H]3+[DAB+5H]5+
Arb
. Un i
ts
m/z
c[M+H]+6e3
4e3
2e3
6e3
4e3
2e3
800 20001200 1600800 20001200 1600
Arb
. Uni
t s
+
M = GLSDGEWQQVLNVWGK
n+/1- Electron Transfer
• (M+nX)n+ + Y-• (M+nX)(n-1)+• + Y
• Dissociative electron transfer ion/ion reactions can produce ECD-like results for polypeptides
J. Am. Chem. Soc. 1996, 118, 7390-7397
Used for MS Short Course at Tsinghua by R. Graham Cooks, Hao Chen, Zheng Ouyang, Andy Tao, Yu Xia and Lingjun Li
7/4/2011
4
[M+Na]+
2500
7500
5000
m/z300 650 13501000
abun
danc
e
[PF6] -
m/z
abun
danc
e
300 750 1200
4000
8000
12000 [M+H+Na]2+
m/zab
unda
nce
100 300 500
2000
4000
6000
2000
4000
6000
[M+H+Na]2+
[M+H]+
[M+H+NaPF6]+
[M+2Na]2+
[M+2Na]2+
a) b)
c)
n+/1- Cation Transfer
• (M+nX)n+ + Y- (M+(n-1)X)(n-1)+ + XY
• Not commonly observed
Na+ transfer
Phys. Chem. Chem. Phys. 2004, 6, 2710-2717
n+/1- Cation Exchange
• (M+2X)n+ + (YZ2)- (M+Y)+ + 2XY
• Seen when reacting multiply charged peptide/protein cations with metal containing anions
J. Am. Chem. Soc. 2003, 125, 12404-12405
[M+H+Na]2+
6000
18000
12000
m/z500 675 1025 1200850
abu
ndan
ce [M+2H]2+
[M+3H]3+
m/z
abun
danc
e
550 700 850
20000
40000
60000 [M+3H]3+
++
m/z
abun
danc
e
200 400 600
400
800
1200 [C10H20N2S4N a] -a)
b)[M+H+Na]2+
6000
18000
12000
m/z500 675 1025 1200850
abu
ndan
ce [M+2H]2+
[M+3H]3+
m/z
abun
danc
e
550 700 850
20000
40000
60000 [M+3H]3+
++
m/z
abun
danc
e
200 400 600
400
800
1200 [C10H20N2S4N a] -a)
b)
Trp-11 neurotensin
sodium diethyldithiocarbamate
Used for MS Short Course at Tsinghua by R. Graham Cooks, Hao Chen, Zheng Ouyang, Andy Tao, Yu Xia and Lingjun Li
7/4/2011
5
n-/n+ Proton Transfer
• (M+nH)n+ + (Y-mH)m- (M+(n-x)H)(n-x)+ + (Y-(m-x)H)(m-x)-
• Both single and multiple proton transfers in a single encounter observed
• Proton transfer usually observed with complex formation
• Degree of proton transfer tends to increase with reactants total absolute charge
J. Am. Chem. Soc. 2003, 125, 7238-7249
8x103
1000 3000 900070005000
(U)7+
500 1500
1.5x104
450035002500
(U)11+
(U)3+/(2U)6+
(U)2+
(U)5+
(U)7+
(U)6+
(2U)5+(U)9+
a)
b)
4x103
Abu
ndan
ce (
arb.
uni
ts)
m/z (Da/charge)
(U)4+
(U)3+ (U)2+
(U) +/(2U)2+
m/z (Da/charge)
9x103
3x103
Abu
ndan
ce (
arb.
uni
ts)
(U)4+
U7+ + U5-
U11+ + U5-
n-/n+ Complex Formation
• (M+nH)n+ + (Y-mH)m- (M+Y+(n-m)H)(n-m)+
• Extent of proton transfer vs. complex formation depends on:– Reactant charge states
– Reactant Identities
J. Am. Chem. Soc. 2003, 125, 7238-7249
U8+ + C5-
C8+ + U5-
3x104
1000
a)
b)
500 2500 4500 6500 8500m/z (Da/charge)
2x104
1x104
Abu
ndan
ce (
arb.
uni
ts)
(U)8+
(U)5+
(U)4+
(U)3+
(U)2+
(UC)3+
3000 5000 7000 9000 11000 13000m/z (Da/charge)
1.6x104
8x103
Abu
ndan
ce (
arb.
uni
ts)
(C)8+
(CU)3+
Used for MS Short Course at Tsinghua by R. Graham Cooks, Hao Chen, Zheng Ouyang, Andy Tao, Yu Xia and Lingjun Li
7/4/2011
6
n-/1+ Proton Transfer
• (M-nH)n- + YH+ (M-(n-1)H)(n-1)- + Y
• Studied in Y-Tube: 5’-d(pAAA)-3’ anions with protonated water clusters, methanol clusters, and solvent clusters proton transfer and some fragmentation
• 3D Ion Trap: – oligo and polypeptide anions with pyridine cations
• Fragmentation with [2-hydroxynaphthalene-3-6 disulfonic acid]2- and [Direct red 81]2-
– Benzoquinoline cations with protein anions and PAMAM dendrimer anions
• Mostly proton transfer
– Benzoquinoline cations with 5’-d(T)20-3’ and 5’-d(CGGG)5-3’• Extensive benzoquinoline attachment
– Amount of fragmentation and proton transfer depends on reagent
n-/1+ Proton Transfer
Int. J. Mass Spectrom. 2003, 228, 577-597
Abu
ndan
ce (
Arb
. Uni
ts)
m/z200 400 600 800 1000 1200
7000
3500
0
4000
2000
0
30000
15000
0
[M-3H]3-
x2
[M-3H]3-
[M-2H]2-
[M-H]-
[M-3H]3-
[M-2H]2-
[M-H]-
w2-
w3-
2-
w1-
w32-
w2-
w3-y3
-(a3-A3)
-
(a2-A2)-
(a2-A2)-
#
200 400 600 800 1000 1200
200 400 600 800 1000 1200
A
B
C
[5’-d(AAAA)-3’]3- with:
A) O2•+ (Electron transfer)
-No proton transfer-Fragmentation
B) C4H9+
-Mostly proton transfer-Some fragmentation
C) BQH+
-Proton transfer
Reaction exothermicities with various reagentsO2
•+ > C4H9+ > BQH+
Used for MS Short Course at Tsinghua by R. Graham Cooks, Hao Chen, Zheng Ouyang, Andy Tao, Yu Xia and Lingjun Li
7/4/2011
7
n-/1+ Electron Transfer
• (M-nH)n- + Y+ (M-nH)(n-1)- + Y
• Select cations that cannot readily proton or other cation transfer mostly use rare gas cations
• Extensive dissociation commonly observed as a result of electron transfer
– High reaction exothermicities and relatively small anion size are conducive to fragmentation
• Demonstrated with oligonucleotides and xenon and oxygen cations
• Demonstrated with peptides and xenon cations
n-/1+ Electron Transfer
J. Am. Soc. Mass Spectrom. 2005, 16, 880-882
ETD of peptide anions
Used for MS Short Course at Tsinghua by R. Graham Cooks, Hao Chen, Zheng Ouyang, Andy Tao, Yu Xia and Lingjun Li
7/4/2011
8
n-/1+ Cation Attachment• (M-nH)n- + Y+ (M-nH+Y)(n-1)-
• Seen in several cases:– Bovine insulin anions and pyridine cations
– Fe+ and insulin anions
– Leucine enkephalin cations and oligonucleotide anions
10000
7500
5000
2500
0400 700 1000 1300 1600 1900 2200 2500
[M-4H]4-
[M-3H]3-
[M-2H]2-
[M+LE-3H]3-
[M+LE-2H]2-
[M+2LE-2H]2-
A
[12-mer]4- with LE+
Int. J. Mass Spectrom. 2003, 228, 577-597
Thermodynamics
J. Am. Chem. Soc. 1996, 118, 7390-7397
Ion/Ion Reaction: (M+nH)n+ + Y- (M+(n-1)H)(n-1)+ + HY
Ion/Molecule Reaction: (M+nH)n+ + B (M+(n-1)H)(n-1)+ + BH+
• Entrance channel dominated by short attractive polarization forces
• Exit channel dominated by Coulombic repulsive forces
• Entrance channel dominated by long-range attractive –Z1Z2e2/r potential (Coulomb attraction)
• Exit channel dominated by shorter range ion-dipole and ion-induced dipole potentials
Used for MS Short Course at Tsinghua by R. Graham Cooks, Hao Chen, Zheng Ouyang, Andy Tao, Yu Xia and Lingjun Li
7/4/2011
9
Thermodynamics
(M+nH)n+ + Y- (M+(n-1)H)(n-1)+ + HY Ion/Ion Reaction(M+nH)n+ + B (M+(n-1)H)(n-1)+ + BH+ Ion/Molecule Reaction
• Free Energy of Ion/Ion Reaction:Grxn = GB[(M+(n-1)H)(n-1)+] – GB[Y-]
• Enthalpy of Ion/Ion ReactionHrxn = PA[(M+(n-1)H)(n-1)+] – PA[Y-]
• Enthalpy of Ion/Molecule ReactionHrxn = PA[(M+(n-1)H)(n-1)+] – PA[B]
• GB = gas-phase basicity; PA = proton affinity
• Deprotonation via ion/ion reactions is expected to be exothermic for every proton (value of n) usually by 100 kcal/mol or greater
• PA[Y-] >> PA[B] (B=strong base for ion/molec. rxn)• Ion/ion reactions expected to be far more exothermic than ion/molecule
reactions
Kinetics
Anal. Chem. 2009, 81, 8669–8676
Used for MS Short Course at Tsinghua by R. Graham Cooks, Hao Chen, Zheng Ouyang, Andy Tao, Yu Xia and Lingjun Li
7/4/2011
10
o Rate-determining step: the formation of a stable orbit for the two reactants.
Kinetics
Z1, Z2 :the unit charges of the ionsε0: the vacuum permittivityv: is the relative velocityμ: reduced mass.
o “Tidal mechanism”, i.e. convert translation energy into internal modes to reduce ion-ion distance so that chemistry can happen.
o As a result of long range 1/r attraction, an oppositely charged ion pair can form a stable orbiting complexes at values of r that are significantly larger than typical distances over which a proton or electron can jump.
o Small charged particle (H+, e, Na+…) can hop in distance or chemical complex can form.
• Rate constant for ion/ion reactions is assumed equal to the rate constant for forming a stable orbiting complex (kc)
Kinetics
2
2
221
v
eZZvkc
• Specific charge states of ubiquitin isolated and subjected to reactions w/ large excess of PDCH anions pseudo-first order kinetics
• Ion/ion reaction rates follow a charge-squared dependence
J. Am. Chem. Soc. 1996, 118, 7390-7397.
0 25 50 75 100 125
Charge State of Ubiquitin Squared
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
Rat
e( s
-1)
Used for MS Short Course at Tsinghua by R. Graham Cooks, Hao Chen, Zheng Ouyang, Andy Tao, Yu Xia and Lingjun Li
7/4/2011
11
• Ions of different initial charge states are completely neutralized over very similar time frames
• Wide range of charges can be converted to low charge states by using a single ion/ion reaction period
Implications of Charge-Squared Dependence of Ion/Ion Reaction Rates
Anal. Chem. 1998, 70, 1198-1202
+16 Myoglobin and Products +2 Bradykinin and Products
• Proton (or electron) Hopping at a distance
– The electrical fields are strong enough to pull protons off at distances of around 100Å
*
• Formation of Coulombically bound orbital complexes
– Orbit may bring reactants into close enough proximity for reaction.Orbits can collapse due to tidal effect and/or collisions.
• Direct Hard-Sphere Collision
Types of Single Ion/Ion Encounters
-
- --
-
H+H+
H+
H+
H+
H+H+
H+H+
H+
H+
H+H+H+
-
- ---
H+H+
H+
H+
H+
H+H+
-
- ---
H+
*: electron, proton, metal ions
Used for MS Short Course at Tsinghua by R. Graham Cooks, Hao Chen, Zheng Ouyang, Andy Tao, Yu Xia and Lingjun Li
7/4/2011
12
(M+nH)n+ + A-
(M+(n-1)H)(n-1)+ + HA
∆Hrxn,PT
(M+A+(n-1)H)(n-1)+
r((M+nH)n+--- A-)
V(r)rPT
rET∆VrET
(M+nH)n+ + A-
MHnn+ + A-
(M+nH)(n-1)+• + A•(M+nH)(n-1)+• + A•
(M+nH)(n-1)+• + A•
∆Hrxn,ET
(M+A+nH)(n-1)+•
Courtesy of McLuckey
b
rh-srPT
rET
rorbit
Courtesy of McLuckey
Used for MS Short Course at Tsinghua by R. Graham Cooks, Hao Chen, Zheng Ouyang, Andy Tao, Yu Xia and Lingjun Li
7/4/2011
13
• The formation of a bound orbiting complex accounts for charge dependence and magnitudes of ion/ion reaction rates
• Elliptical orbits bring reactants in close proximity at high velocities allowing protons to transfer
• If reactants have sufficient relative translational energy to escape their orbits proton transfer
• Ions that do not escapte their mutual attraction collide complex formation
• Factors that influence whether proton transfer or complex formation is observed:
• Orbit shape• Physical size of ions• Ion charge states• Binding strengths of charge sites• …
Proton Transfer vs. Complex Formation
1000 3000 5000 7000 9000 11000 13000
1x104
2x104
3x104
Abu
ndan
ce (
arb.
uni
ts)
m/z (Da/charge)
(C)6+
(C)5+
(C)4+
(C)3+
(C)2+/(2C)4+
(2C)3+
(C)+/(2C)2+
(2C)5+
1000 3000 5000 7000 9000 11000 13000m/z (Da/charge)
Abu
ndan
ce (
arb.
uni
ts)
1x103
2x103
(C)4-
(C)3-
(C)2-
(C)-
(C)5-
a)
b)
Cytochrome c (C) 8+/5-
(all products shown to arisefrom single interactions –no sequential products)
H+H+
H+
H+
H+
H+H+
-
- ---
H+
J. AM. CHEM. SOC. 9 VOL. 125, NO. 24, 2003
Used for MS Short Course at Tsinghua by R. Graham Cooks, Hao Chen, Zheng Ouyang, Andy Tao, Yu Xia and Lingjun Li
7/4/2011
14
J. AM. CHEM. SOC. 9 VOL. 125, NO. 24, 2003
Ubiquitin (M+9H)9+/I- (25 ms)Promote complex formationo Ions with large physical sizeo Low charge state
Used for MS Short Course at Tsinghua by R. Graham Cooks, Hao Chen, Zheng Ouyang, Andy Tao, Yu Xia and Lingjun Li
7/4/2011
15
H+
H+
H+
H+
n+
Anion-•
H+
eH+
H+
H+
H+
(n-1)+●
H+
H+
H+(n-1)+
ETD(neutral losses)
ETD(c-/z• ion
formation)
ETD(c-/z• ion
formation)
H+
H+
H+
H+
(n-1)+●
ET, no D
ElectronTransfer
Proton transfer
Peptide cations
Major Competing Channels in an ETD Expt.Major Competing Channels in an ETD Expt.
•-
fluoranthene
•-
azobenzene
Electron Transfer vs. Proton Transfer
Gunawardena et al. JACS, 2005, 127, 12627-12639.
Used for MS Short Course at Tsinghua by R. Graham Cooks, Hao Chen, Zheng Ouyang, Andy Tao, Yu Xia and Lingjun Li
7/4/2011
16
0.0
0.2
0.3
0.4
0.5
0.1
Ne
t ele
ctro
n tr
ansf
er
prob
abili
ty, P
ET
Region of high PLZ Region of low PLZ
100.387.975.462.750.337.625.112.5
Electron affinity (kcal/mol)
Gunawardena et al. JACS, 2005, 127, 12627-12639.
Effect of Anion Nature on ETD Efficiency
2 eV
ET becomes competitive with PT when EA of neutral reagent is not too high, typically < 3 eV and when Frank-Condon factor (FCF) is not too low ( > 10-2)
b)
d)
200
100
300
400
m/z
[M+H]+
(x2)~
100
50
150
200
Abu
ndan
ce
[M+H]+
(x1.1)~
[M+H]+
(x5)~
300
200
400
500
100
Abu
ndan
ce
400
300
200
+1(x3.5)
~
100
c6+
z6+
c7+
c8+
z7+
c9+
z8+
z9+
c10+
z10+
600 800 1000m/z
600 800 1000m/z
600 800 1000 600 800 1000m/z
-(H
2N) 2
C=
NH
-(H
2N) 2
C•
-2N
H3
-NH
3
Abu
ndan
ce
(KGAILKGAILR + 3H+)/A-
A = nitrobenzene-•
FCF Σ<0|≤10>2 = 0.14EA (A) = 1.0 eV
A = SF6-•
FCF Σ<0|≤10>2 = 6.7x10-11
EA (A) = 1.05 eV
A = I-
FCF Σ<0|≤10>2 = NAEA (A) = 3.06 eV
A = (PDCH-F)-
FCF Σ<0|≤10>2 = 1.3x10-3
EA (A) = 4.17 eV
Gunawardena et al. JACS, 2005, 127, 12627-12639.
Used for MS Short Course at Tsinghua by R. Graham Cooks, Hao Chen, Zheng Ouyang, Andy Tao, Yu Xia and Lingjun Li
7/4/2011
17
Gunawardena et al. JACS, 2005, 127, 12627-12639.
+
[M+13H]13+
[M+15H]15+
[M+17H]17+
[M+19H]19+
600 800 1000 1200 1400 1600
1000
Abu
ndan
ce (
arb.
uni
ts)
m/z
Latter part of the 1980sLatter part of the 1980s
Early to mid 1980s:Early to mid 1980s:
Possibility for Bio-Ion/Ion Chemistry
negative ions
positive ions
Wolfgang Paul
John B. Fenn
Ion Trap MS
ESI MS
Used for MS Short Course at Tsinghua by R. Graham Cooks, Hao Chen, Zheng Ouyang, Andy Tao, Yu Xia and Lingjun Li
7/4/2011
18
Quadrupole Ion Traps as Reaction Vessels
Guard Ring
Electron Multiplier
Conversion Dynode
GateLens
Electrospray solution
ElectrosprayNeedle
Ele
ctro
spra
y Io
niza
tion
Sou
rce
Positive ions
PDCH Vapor inlet
Application of High VoltageDC Pulse
Atmospheric Sampling Glow Discharge Ionization Source
Negative ions
-•
PT reagents
perfluoro -1,3-dimthylcyclohexane
Mutual Storage of +/- Ions: 3D vs. 2D Ion Traps
Add Aux. RF to End LensesRF trapping
~~3-D Ion Trap
++ ---
2-D Ion Trap
+ + ++ ++
Challenge for ion/ion rxns Mutual storage of +/- ions
Xia, Wu, Londry, Hager, McLuckey, J. Am. Soc. Mass Spectrom., 2005, 16, 71-81
DC Trapping
0 V
-- -
-----
- ---
+ + ++ ++
+ + ++ ++
-- -
-----
- ---
Advantage of 2-D trap: 50x ion storage capacity 20x higher ion injection high transmission efficiency
Used for MS Short Course at Tsinghua by R. Graham Cooks, Hao Chen, Zheng Ouyang, Andy Tao, Yu Xia and Lingjun Li
7/4/2011
19
Q0 Q1 Q2 Q3
pulsed + HV
pulsed- HV
Sequential injection of +/- ions with same ion path
Q1 for mass isolationQ2 or Q3 for ion/ion reactionQ3 for mass analysis
ETD on Sciex linear ion trap configuration for ETD
RF RF
For APCI
For ESI
McLuckey lab at Purdue, Anal.Chem., 78 (2006) 3208-3212
Q0 Q2
Q1
+HV
- HV
+
-
-
--
-++
++
++ +
++-
---
--- --
--
----
LIT Ion/Ion Reactor TOF AnalyzerDual Ion Source
Ion/Ion Rxns in Q-q-TOF Mass Spectrometer
QSTAR XL
~~
Anal. Chem., 78 (2006) 4146-4154.
Used for MS Short Course at Tsinghua by R. Graham Cooks, Hao Chen, Zheng Ouyang, Andy Tao, Yu Xia and Lingjun Li
7/4/2011
20
NCI
ETD in Thermo LTQ
ETD in Bruker HCTultra
ETD in Thermo LTQ-Orbitrap
NCI
ETD in Bruker FT-ICR
Rapid Commun. Mass Spectrom. 2008; 22: 271–278
Used for MS Short Course at Tsinghua by R. Graham Cooks, Hao Chen, Zheng Ouyang, Andy Tao, Yu Xia and Lingjun Li
7/4/2011
21
+r
-
proton transfer
electron transfer complex formation
+10+20+30
+1+1
+1
m/z
simplify
m/z
+16
+13
concentrate
[M+H]+
[M-H]-
[M+2H]2+
-2H+
+3H+
manipulate
synthesize
NH
NR1H
O R2HOH
OC
Z
dissociateCu2+
+
+
+
+
+
+
+
+
+
+
+
(g)
Cu2+
+
+
+
+
Cu2+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+Cu(NO3)3
-3)3
- 2 HNO 3
+
metal transfer
Gas-phase Bio-Ion/Ion Chemistry
Complex Mixture Analysis
Abu
n da n
ceA
bun d
a nce
m/z5000
1000500
200001500010000
250
125
12000
6000
20001500
0
0
insulinaprotininubiquitinrat cytochrome cbovine cytochrome cequine cytochrome cribonuclease A-lactalbuminlysozymemyoglobin-lactoglobulin A
+1+1+1
+1
+1
+1
+1+1
+1
+1+1
Pre ion-ion
Post ion-ion
Used for MS Short Course at Tsinghua by R. Graham Cooks, Hao Chen, Zheng Ouyang, Andy Tao, Yu Xia and Lingjun Li
7/4/2011
22
Anal. Chem. 2009, 81, 8669–8676
Ion trap CID of (M+19H)19+
ion of porcine elastase.
Simplify Product Ion Spectrum
[M+11H]11+
[M+15H]15+
[M+15]15+
Fine Control of Reaction Extent: Ion Parking Technique
Chrisman et al., Anal. Chem., 2006, 310‐316 ; McLuckey et al., Anal. Chem., 2002, 336‐346.
increase velocitydecrease rxn rate
ion parking3kion/ion
Excitation
f
High m/zHigh frequency
Used for MS Short Course at Tsinghua by R. Graham Cooks, Hao Chen, Zheng Ouyang, Andy Tao, Yu Xia and Lingjun Li
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23
Strategy: Two-step Charge Inversion via Ion/ion Rxns
N
NH
O
OHO
HO
N
HN
O
OHO
HO
N
NH
O
OOH
OH
N
HN
O
OOH
OHO O
OO
N N
+ - 2+
+ H+ - H+ + 2H+
n-
m+
N
N
NH2N
H2N
NH2N
H2N
NH2N
H2N
N
H2N
H2N
N
N
N
N
NH2N
H2N
NH2N
H2N
NH2N
H2N H2NH2N
N
N
N
NNH2
NH2
NNH2
NH2
NNH2
NH2
N
NH2
NH2
N
N
N
NNH2
NH2
N NH2
NH2
NNH2
NH2
N
NH2
NH2
N
NN
Charge IncreaseRobust and efficient means for increasing ion z in the gas-phase
He et al., JACS, 125 (2003) 7756-7757.
b1
y2 y1
b2CIDc1 c2
z2 z1 ECD or ETD
H2N—CH—C—NH—CH—C—NH—CH—COH
— — ——
— — — —
R3
——
O OR2O
———
R1
Electron Transfer Ion/Ion Rxns
ETD is similar to ECD in inducing fragmentation
compared to CID wider sequence coverage preserve labile post translational modifications
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Predicting Fragment Ions for ETD or ECDIn-silico fragmentationhttps://prosightptm.northwestern.edu/
Developed by Kelleher group
Coon, Ueberheide, Syka, Dreyser, Ausio, Shabanowitz, Hunt PNAS (2005) 102, 9463-9468.
Ion Trap ETD + PT with No Mass Extension on LTQ
+13 ubiquitin
•-
-
ETD
proton transfer
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7/4/2011
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Ion/ion Reactions in Electrodynamic Ion Traps:Big Picture
Useful reaction rates – 1-1000 s-1
“Universal” kinetics – (Z, n, overlap)
Software control over admission/removal of breactants
Enormous chemical dimensionality
Analytically useful applications…
Electron Capture Dissociation Reactions
• Originated in McLafferty’s Lab in 1998 while attempting to do 193 nm photodissociation
• Provides information complementary to traditional activation methods.
• Claimed to be non-ergodic: unimolecular dissociation that occurs before the excitation energy is randomized *– Laser techniques can make such studies in vibrational time
periods* – energy randomization can require 200 ps *– This claim is disputed
[M+nH]n+ + e-→([M+nH](n-1)+●)transient → fragments
*K. Breuker et al. PNAS 2004, 101, 14011-14016.
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7/4/2011
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Electron Capture Dissociation (ECD) instrument config
EI source
Cleavages Associated With ETD/ECD
H2N—CH—C—NH—CH—C—NH—CH—COH
R1 O R2 O R3 O
— — ——— —— ——
y2
b1
z2
c1
x2
a1
y1
b2
z1
c2
x1
a2
• Cleaves peptides at N-Cα bonds
• Smaller dependence on peptide sequence than CID
• Labile post-translational modifications are preserved
• Preferential cleavage at S-S bond
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7/4/2011
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“Slow Heating” Techniques
• Many fragmentation techniques result in the ions being effectively heated to a higher Boltzmann temperature. Fragments form as the ions exceed the minimum energy required to break bonds via the lowest energy pathways.
• Site specific fragmentation prevalent.
• Examples:– CID
– Sustained off resonance collision induced dissociation (SORI-CID)
– Infrared Multi-Photon Dissociation (IRMPD)
– Blackbody Infrared Dissociation (BIRD)
Threshold Dissociations (collisions, infrared photons)
Electron Capture Dissociation
Two General Ways to Fragment Gas Phase Ions
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7/4/2011
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Cleavage of Backbone Bonds
• ECD typically produces c’ and z• ions
L.M. Mikesh et al. / Biochimica et Biophysica Acta 1764 (2006) 1811–1822
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L.M. Mikesh et al. / Biochimica et Biophysica Acta 1764 (2006) 1811–1822
Cleavage of Backbone Bonds
• ECD minor fragmentation pathway produces a• and y ions
ECD on Ubiquitin (~8.6 kDa) [M+9H]9+
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7/4/2011
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Preferential Disulfide Bond Cleavage
• Disulfide bonds are preferentially cleaved in ECD/ETD.
• Polarization of the disulfide bond results in more highly charged portion being the R-SH portion (rather than R-S• portion).
J. Am. Soc. Mass Spectrom. 16 (2005) 1020–1030.
Cleavage of –S-S– Bonds with ETD:
J. Am. Soc. Mass Spectrom. 16 (2005) 1020–1030.
[M+4H]4+ with SO2-•
-Lactalbumin Digest Reacted with SO2-• shows preferential cleavage of –
S-S– linkages
[M+3H]3+ with SO2-•
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7/4/2011
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Common Side Chain Losses
• Loss of 17 – Lysine or N-Terminal Amine (or Arginine)
• Loss of 18 – Serine or Threonine
• Loss of 17, 44, 59, and 101 – Arginine
• Loss of 45 and 59 – Asparagine
• Loss of 46 and 60 – Aspartic Acid
• Loss of entire side chain plus H – Histidine, Tryptophan, Tyrosine, Threonine
What is the Mechanism of ECD/ETD?
• Widely debated
• Important experimental observations for a proposed mechanism to explain:– preference for amine bond cleavage over amide bond
cleavage– preference for disulfide cleavage over backbone cleavage– wide variety of backbone bonds cleaved
• Is it similar to dissociative electron attachment (DEA) or dissociative recombination (DR)?
• First proposed mechanism – electron captured at protonationsite which is solvated to backbone carbonyl, and quickly leads to dissociation of N-C bond before energy can be redistributed
• Flaw – disulfide bond cleavages
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“Cornell Mechanism”
• Disulfide bonds have hydrogen atom affinities ~24 kcal/mol greater than amide backbone
• Capture of electron at protonation site generates a “hot hydrogen atom”, which is released
• “hot hydrogen atom” recaptured by to molecule to initiate dissociation
Cornell Mechanism Cont’d
• Hydrogen capture cross section for gas-phase peptide ions is low, as shown by studies where ions were exposed to H atoms in ICR cells– Hot hydrogen atoms may overcome this limitation
• modeling study of complex of hot hydrogen atom and amide bond showed low probability for N-C cleavage (H loss most common)
• H atom addition to carbonyl should occur equally at C and at O. If at C, should lead to oxide radical which would result in a and y type fragments, yet – But, a and y ions are usually less than 10% of observed
products• In model peptides with charges in the form of quaternary
ammonium groups (no hydrogens available), ECD, and in particular disulfide bond cleavage still observed.
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Washington/Utah Mechanism --Coulomb-stabilized direct electron attachment
• Calculations showed that capturing electrons can result in long-lived excited electronic states have been shown to exist– Captured electron mainly located on the carbonyl carbon (J. Am.
Soc. Mass Spectrom. 2005, 16, 208-224)
• In these forms, the carbonyl oxygen becomes very basic – Proton affinity is large enough to remove a proton from a
charged ammonium or guanidinium group
• Aminoketyl radical is produced by capture of a proton – Expected to fragment by cleavage of the N- C bond
• Hydrogen atom transfer from an ammonium group to a backbone carbonyl is exothermic in the ground state, – But it competes with hydrogen loss– Will be favored at internally solvated sites
Direct electron attachment to a Coulomb stabilized OCN * orbital to cleave a N-C bond.
D. Neff et al. / International Journal of Mass Spectrometry 283 (2009) 122–134
Washington/Utah Mechanism --Coulomb-stabilized direct electron attachment
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Washington/Utah MechanismDirect electron attachment to a Coulomb stabilized S–S * orbital to cleave a disulfide bond.
D. Neff et al. / International Journal of Mass Spectrometry 283 (2009) 122–134
Colum stabilization = 14.4 eV A/R > 1 eV to overcome e- attachment to S-S σ*.For +1, R < 14 angstroms, for 2+ , R < 14 angstroms
Other Mechanisms
• Non-ergodic e- capture fragmentation generates radical species • Radical migration initiates multiple free radical rearrangements,
• multiple backbone cleavages• additional side-chain cleavages
o Radical Cascade in Cyclic Peptides
J. AM. CHEM. SOC. 2003, 125, 8949-8958
o Electron shuttling in ETD or ECD
• Electron transfer or capture is more likely to happen at a Rydberg orbital of a positive site.
• The Rydberg orbital subsequently shuttle an electron to an SS * or amide * orbital via. a surface crossing.
• Requirement for a Rydberg orbital for e shuttling:• close to S-S or amide site• Similar energy level• having sufficient radial extent
International Journal of Mass Spectrometry 283 (2009) 122–134
Used for MS Short Course at Tsinghua by R. Graham Cooks, Hao Chen, Zheng Ouyang, Andy Tao, Yu Xia and Lingjun Li
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Thermodynamics of ECD and ETD
E
Reaction coordinateMHn
n+ + A-•
ΔH
et
MHn(n-1)+ • + A
ΔHet = EA(A) – RE(MHnn+)
Curve crossingExit ChannelExit Channel
Entrance ChannelEntrance Channel
(EA: electron affinity, RE: recombination energy, HA: hydrogen atom affinity)
ECD
Eur. J. Mass Spectrom. 2002, 8, 337-349
ΔHec= -RE(MHnn+) = 13.6 eV-PA(MHn
(n-1)+) - HA(MHn(n-1)+)
ETD: less exothermic by the amount of EA (A)
RE ~ 4-7 eVRE ~ 4-7 eV
Other Ion/Electron Interactions
• Irradiate negative ions with >10 eV electrons
o Electron Detachment Dissociation
• C -C backbone cleavage, forming a• and x fragment ions
Chem. Eur. J. 2005, 11, 1803 – 1812, Chem. Phys. Lett, 2001, 342 299-302
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Other Ion/Electron Interactions
• Irradiate negative ions with >10 eV electrons
o Electron Detachment Dissociation (EDD)
• C -C backbone cleavage, forming a• and x fragment ions
Chem. Eur. J. 2005, 11, 1803 – 1812, Chem. Phys. Lett, 2001, 342 299-302
Other Ion/Electron Interactions
• Irradiate positive ions with >10 eV electrons
o Electron Induced Dissociation (EID) similar to EIEIO by Cody and Freiser (1979) Anal Chem 51:547–551
• Similar fragmentation patterns to hydrogen deficient peptide cations
frag
MHnn+ + e- (fast) MHn
(n+1)•+ + 2e- (slow)
Anal Bioanal Chem (2007) 389:1429–1437, Rapid Commun. Mass Spectrom. 2009; 23: 2099–2101Chemical Physics Letters 330 (2000) 558 -562
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Suggested Reading
• S.J. Pitteri, S.A. McLuckey, Mass Spectrom. Rev., 24 (2005) 931-958."Recent Developments in the Ion/Ion Chemistry of High-Mass
Multiply Charged Ions.“
• S. A. McLuckey and T. Huang. Anal. Chem. 81 (2009), 8669-8676. Ion/Ion Reaction: New Chemistry for Analytical MS.“
• T. Huang, S. A. McLuckey Ann. Rev. in Anal. Chem. 2010, 3, 365-385:"Gas-Phase Chemistry of Multiply-Charged Bio-ions in Analytical Mass Spectrometry.“
• R. A. Zubarev, K. F. Haselmann, B. Budnik, F. Kjeldsen, F. Jensen. Eur. J. Mass Spectrom. 2002, 8, 337-349
• H.J. Cooper, K. Håkansson, A.G. Marshall. The Role of Electron Capture Dissociation in Biomolecular Analysis. Mass Spectrometry Reviews, 2005, 24, 201– 222.
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