used for ms short course at tsinghua by r. graham cooks ... · used for ms short course at tsinghua...

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


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


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


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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+


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+


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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


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


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


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)


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


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


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


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


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.


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)


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



7/4/2011

17


+

[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


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


7/4/2011

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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.


7/4/2011

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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


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


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


7/4/2011

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


7/4/2011

24

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


7/4/2011

25

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.


7/4/2011

26

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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“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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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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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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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


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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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• 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


• 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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