Do ECD and ETD proceed by the same mechanism in peptide cations?

Carl Weisbecker

Research Advisor: Professor Athula AttygalleCenter for Mass Spectrometry

Stevens institute of Technology, Hoboken, Castle Point on Hudson, NJ 07030

Abstract

Electron capture dissociation (ECD) and electron transfer dissociation (ETD) are two new techniques in mass spectrometry to dissociate peptide ions, yielding extensive, analytically useful information about the amino acid sequence of peptides.

•There is an older, more established method for determination of primary structure in peptide cations called collision induced dissociation (CID). CID results in heterolytic cleavage of amide bonds. The mechanism of peptide dissociation by CID is relatively well understood.

•ECD and ETD operate by different mechanisms that result in homolytic cleavage of N-C bonds. The exact nature of the process by which this happens has been debated.

This critique will describe several mechanisms proposed to explain ECD and ETD. Researchers assume the mechanism applying to both processes is the same. The research proposal will highlight differences between ECD and ETD and suggest a way to use differences to understand the mechanism better.

Dissociation Products of Protonated Peptides

NH2NH

NHNH

O

OH

R4

R3

R2

R1

O

O

O

N Terminus C Terminusa

x

b

yz

cThere are three different bond types in the peptide backbone.

• CID produces b and y type anions by heterolytic amide bond cleavage.• ETD and ECD produce c and z type ions by homolytic bond cleavage.

+ n H+

m+

am

ide

C -

C

N -

C

MS/MS Peptide Structure Determination

ExampleThe CID spectrum of a doubly-charged parent ion containing this amino acid sequence:

DYEEFLEIAK

Mass differences between adjacent y or b ions in the spectrum reveal the amino acid sequence.

Differences between c and z ions in ECD or ETD spectra also convey this information.

D115

Y163

E129

E129

F147

L/I147

E129

L/I147

A71

Journal of Proteonomics 2008, 71, 46.

Distinguishing Features of ECD and ETD

• Fragmentation is more extensive and less selective than CID, resulting in fewer missing fragments.

• Secondary fragmentation processes help to distinguish between isobars (such as leucine versus isoleucine.)

• Disulfide bonds in peptides are readily cleaved.

• Non-covalent bonding within the peptide may be preserved while covalent bonds are broken.

• So-called post-translational modifications of peptides (PTMs) are also preserved.

• ECD and ETD are often cited in literature as examples of non-ergodic processes; although, this classification has been a subject of debate.

Ergodicity

Definition: An ergodic process tends in probability toward a limiting final state that is independent of initial conditions.

CID is an example of an ergodic process:

A non-ergodic process:

+

ion – neutral collision

+excitation +

excitation randomly

redistributed

++

dissociation

+ + + +fast

slow (> 1 ps)

The Overall Process in ECD

e-

+ k H

k+ (k-1)+

m+

+ + n H

n+

v

O

NHCHR

O

NH

CHR

O

NHCHR

w

+ k H

v

O

NHCHR

O

NH

CHR

O

NHCHR

H w

+ m H

v

O

NHCHR

CH

R

wO

NH2

O

NHCHR

protonated peptide radical cation

cvm+ ion zw

n+● ion

Electron Capture Dissociation

The Overall Process in ETDElectron Transfer Dissociation

Competing Proton Transfer Process

+ k H

k+ (k-1)+

v

O

NHCHR

O

NH

CHR

O

NHCHR

w

+ ( k-1) H

v

O

NHCHR

O

NH

CHR

O

NHCHR

H w

A-

+ HA

+ k H

k+ (k-1)+

m+

+ + n H

n+

v

O

NHCHR

O

NH

CHR

O

NHCHR

w

+ k H

v

O

NHCHR

O

NH

CHR

O

NHCHR

H w

+ m H

v

O

NHCHR

CH

R

wO

NH2

O

NHCHR

A-

+ A

A- is an electron donor molecule

Comparison of ECD and ETD

• ETD and ECD processes generate the same types of dissociation products, which are primarily c+ and z+ ions.

• In ECD the cation is reduced by an electron emitted from a filament. This electron has low kinetic energy (< 0.2 eV).

• In ETD the cation is reduced by a collision with an electron donor (A-). This donor is often a radical anion of an aromatic hydrocarbon.

• ECD was first reported in 1998 by McLafferty. ECD experiments are conducted in an FT-ICR mass spectrometer. The time-scale of ECD experiments ranges from milliseconds to seconds.

• ETD was first reported in 2004 by Hunt. ETD experiments can be conducted using quadrupole ion-trap mass spectrometers, and the time-scale is typically faster than ECD.

ECD and ETD Mechanisms

The following proposed mechanisms will be discussed in this critique:

1. Hot Hydrogen Atom Transfer chronologically the first proposed mechanism.

2. Coulomb-Assisted Direct Electron Attachment

3. Proton Transfer to a “Superbase”

4. Intramolecular Electron Transfer

The latter mechanisms were proposed more recently in an effort to explain new experimental results.

Attempts have also been made to model ECD and ETD processes using a combination of two or more competing mechanisms.

Hot Hydrogen Atom Transfer Mechanism

O

NH

HCR

N+

H

H H

e-

O

NH

HCR

N

H

H H

OH

NH

HCR

NH H

OH

NH

NH H

CR

H

O

NH

C

N+

H

H H

S

S

e-

O

NH

C

N

H

H H

S

S

O

NH

C

NH H

S

S

HSH

S

Zubarev; Kruger; Fridriksson; Lewis; Horn; Carpenter; McLafferty J. Am. Chem. Soc., 1999, 121, 2857.

Peptide Bond Cleavage :

Disulfide Bond Cleavage :

hypervalent ammonium

radical

aminoketyl radical

thiasulfonium radical

Hot Hydrogen Atom Transfer Mechanism

• An electron is captured by a positively charged site, such as a protonated amine terminus of lysine.

• The reduction of the positive site is exothermic by an estimated 400 – 700 kJ/mol, which is called recombination energy. (Typical bond dissociation energies in peptides are 300 – 400 kJ/mol.)

• A hydrogen atom is lost from the resulting ammonium radical. The hydrogen atom is sometimes captured by nearby amide or disulfide groups. The “hot” hydrogen atom is thus supposed to transport recombination energy from the capture site to the cleavage site.

• Some hydrogen atoms are lost.

• A N-C bond dissociates adjacent to the resulting aminoketyl radical, or a disulfide bond dissociates adjacent to a thiasulfonium radical.

Zubarev, et al. European Journal of Mass Spectrom., 2002, 8, 337.

Energy Level Diagram

Zubarev, et al. European Journal of Mass Spectrom., 2002, 8, 337.

+ e-

hydrogen bonded complex

recombination

CH3 NH2

H

CH3

O

NHCH3

CH3 NH2+

H

CH3

O

NHCH3

60CH3

OH

NHCH3

CH3 NH2

+

H loss aminoketylradical

CH3

O

NHCH3

CH3 NH2 + H● 123

CH3

OH

NH CH3+

dissociationproducts

330 kJ/mol

Ener

gy

N-C bond dissociation is predicted to be the rate-limiting step. It competes with loss of the captured H●.

Cys-Alan-Lys

Lys-Alan-Cys

Ac

Ac

+ 2H

2+

Problem 1: Hydrogen Transfer Distance

Sawicka, A.; Skurski, P.; Hudgins, R. R.; Simons, J. J. Phys. Chem. B, 2003, 107, 13505.

n = 10, 15, 20

H● transfer distance up to 32 Å

The Marshall group synthesized peptides with capture sites separated from a disulfide bond by alanine groups. Their results were interpreted by Simons et al.

• Products of disulfide bond cleavage were observed in ECD protonated peptides with alanine spacers.

• Dissociation of the disulfide bond was unaffected by the number of alanine amino acids (n = 10, 15, or 20.)

• Disulfide bonds of peptides with sodiated lysine groups also dissociated in ECD.

These observations seem inconsistent with the hot hydrogen atom mechanism.

The Simons group proposed that the disulfide bond dissociation is better explained by direct electron attachment to the * orbital of the disulfide bond.

Problem 1: Hydrogen Transfer Distance

Coulomb-Assisted Direct Attachment Mechanism

O

NH

C

N+

H H

S

SH

e-

O

NH

C

N+

H H

S

SH

S-

S

SH

S

Sobczyk, M.; Neff, D.;  Simons, J.  Int. J.  Mass Spectrom., 2008, 269, 149.

Anusiewicz, I.; Berdys-Kochanska, J.; Simons, J. J. Phys. Chem. A, 2005, 109, 5801.

Disulfide Bond Cleavage :

O

NH

HCR

N+

H

H H

e-

OH

NH

NH H

CR

H

O-

NH

HCR

N+

H H

H

O-

NH

N+

H H

CR

H

H

N-C Bond Cleavage :

Coulomb-Assisted Direct Attachment Mechanism

• Direct electron attachment to a disulfide * orbital is endothermic by 96 kJ/mol

• Attachment to an amide * orbital is also endothermic by 240 kJ/mol.

• Some assistance is required to make electron attachment less costly energetically.

The Simons group proposed that electron attachment is assisted by Coulombic interaction with neighboring positive charges in the peptide.

A point-charge model was used to describe the energy lowering of an attached electron due to a nearby positive charge at a distance (r):

(1/40) q1 q2 -1390 r r

VC = = (kJ/mol) ; r (Å)

Problem 2: Electron Attachment to Amides

O

NH

HCR

N+

H

H H

e-

OH

NH

NH H

CR

H

O-

NH

HCR

N+

H H

H

OH

NH

HCR

NH H

superbase

Syrstad, E. A.; Tureček, F.  J.  Am. Soc.  Mass Spectrom., 2005, 16, 208.

Calculations suggest that electron attachment to an amide group does not lead directly to N-C bond cleavage. This dissociation is too endothermic. Tureček and Systad proposed an alternative dissociation mechanism. They calculated that the reduced amide bond has a high proton affinity and acts as a “superbase” to abstract a proton from nearby functional groups.

Problem 3: Multiple Capture Sites

AGCK

TFTSC+ 3TMAB

3+

Gunawardena; Gorenstein; Erickson; Xia; McLuckey, S. A.  Int. J. Mass Spectrom., 2007, 265, 130.

The McLuckey group studied branched disulfide linked peptides with multiple capture sites using ETD.

They modified peptides, converting some protonated amines into trimethylammonium ions.

They found dissociation products inconsistent with proposed mechanisms.

R NH2

N

O

O

O

ON+CH3

CH3

CH3

RNH

ON

+CH3

CH3

CH3

pH 9 / 2 hrs.

Dissociation Products of Branched Peptides

Peptide Total Dissociations (%) S-S Dissociations (%)

no TMAB 83 71one TMAB 82 68two TMABs 52 36three TMABs 68 80

Intramolecular Electron Transfer Mechanism

SS

N+

CH3

CH3

N+H

H

H

CH3

e- SS

N CH3

CH3

N+

HH

H

CH3

e-

SS

N+

CH3

CH3

NH

H

H

CH3

SS

N+

CH3

CH3

N+H

H

H

CH3

S-

N+H

H

H

S

N+

CH3

CH3

CH3

Sobczyk, M.; Neff, D.;  Simons, J.  Int. J.  Mass Spectrom., 2008, 269, 149.

The Simons group proposedan intramolecular electron transfer mechanism to explain dissociation of branched peptides.

The Simons group reported an important theoretical simulation of ETD.

They examined competing mechanisms of electron transfer to either a protonated amine or to a disulfide * orbital, both present in the same molecule. The objective was to determine whether the hydrogen atom transfer mechanism or the Coulomb-assisted electron attachment mechanism is more probable.

They calculated potential energy diagrams of the reactants and products of electron transfer in a model system.

They calculated the probability of electron transfer at different crossing points on the potential energy curves.

Competing Electron Transfer Processes

Competing Electron Transfer Processes

+ A- + A

+ A- + A

II.

I.

S S

CH2

CH3

CH2

NH3+

S S

CH2

CH3

CH2

NH3

S S

CH2

CH3

CH2

NH3+

S S

CH2

CH3

CH2

NH3+

e-

I.

II.

Inte

r-Io

n P

ote

ntia

l

Decreasing A- ---- NH3+ Distance

(Increasing Time)

RNH3

RSS

0

Transfer Site Probability (%)

-S-S- 0.0454

-NH3+ 2.67

Simons et al. J. Phys. Chem. A, 2005, 109, 5801.

+ CH3

-

+ CH3●

+ CH3

-

+ CH3●

The probability of electron transfer at each crossing point was calculated using Landau-Zener theory. The probability of a transition between two potential energy curves at a crossing point depends on:

• the velocity of approach to the crossing point • the slopes of the potential energy curves• the strength of coupling between electronic states

Different electron transfer processes occur at a different inter-ion distances:

-S-S- 10.75 Å-NH3

+ 4.31 Å

Simon’s model supposes a chronology for the approaching CH3-. The ion has

an opportunity to transfer an electron to the disulfide bond first.

Competing Electron Transfer Processes

Problem 4: Identifying the Rate Limiting Step

O

NH

HCR

N+

H

H H

e-

O-

NH

HCR

N+

H H

H

O

NH

HCR

N+

H H

H

HH+

N+

H H

CR

H

HOH

NH

rate

limiting

step

N+

H

H

C

RH

H

OHNH

Tureček, F.  J. Am. Chem. Soc., 2003, 125, 5954.

Calculations by Tureček predict that breaking of the N-C bond is not the rate-limiting step, if other hydrogen bonding functional groups are present.

Evaluation of Literature Findings

There is a lot of ambiguity about the mechanism in ECD and ETD.

Experiments of the Marshall and McLuckey groups and theoretical work by the Simons group suggest that the hot hydrogen atom transfer mechanism is inconsistent with observed dissociation products.

Three alternative mechanisms have been proposed:

• Coulomb assisted direct electron attachment • Intramolecular electron transfer • Proton transfer to a superbase

The overall kinetics of ECD and ETD are not well understood. The rate-limiting step in dissociation of N-C bonds was thought to be cleavage of the covalent bond, but work by Tureček was questioned that assumption.

The mechanism in ECD and ETD of peptide cations is considered by most researchers to be essentially the same, regardless of the whether the peptide cation is reduced by a free electron or by an electron transferred from a donor molecule.

Some isolated comments suggest mechanisms of ECD and ETD might differ in special cases, but (surprisingly) no experimental work has been found directly comparing ECD and ETD products from the same peptide.

There are important differences between a free electron and an electron transferred from a donor molecule:

1. Steric Effects: Donor molecules have finite size.

2. Electron Affinity: Donor molecules transfer less recombination energy to the cation than free electrons.

Are ECD and ETD mechanisms the same?

Research Proposal

Specific structural and electronic properties of electron donor molecules can be used as probes to distinguish among the proposed mechanisms of ECD and ETD.

Theoretical work of the Simons group established that different electron transfer sites on a molecule (i.e. disulfide groups, amide groups, protonated amines) correspond to different intermolecular distances where electron transfer can occur.

Hypothesis

• The finite size of different electron donor molecules can select one electron transfer mechanism over another by steric hindrance.

• The electron affinity of different donor molecules can select one electron transfer mechanism over another by energetics.

11.6 Å

electron donor :[azobenzene]-●

The Simons group had estimated that electron transfer occurs when the donor and acceptor are 4 (-NH4

+) or 11 Å (-S-S-) distant.

What happens if the sizes of the donor and the peptide prevent approach to that distance?

Electron Transfer Directed by Steric Factors

~ 21 Å

Electron Transfer Directed by Steric Factors

III. RNH3●

I.

RSSII.

RC=O

approach stericallyhindered

Decreasing A- ---- NH3+ Distance 0

(Increasing Time)

Inte

r-Io

n P

oten

tial

peptide + A- products

Hypothesis:If the donor molecule is large, some electron transfer sites will be inaccessible.

NN

O

O

CH3CH3

The effect of steric hindrance on ETD has not previously been investigated.

Steric effects in ETD could be investigated by comparing the dissociation products using electron donor molecules with different size.

A variety of 4, 4’- and 3, 3’- substituted azobenzene derivatives can be readily synthesized.

Example of a 4.4’-Disubstituted Azobenzene Derivative:

ETD has also recently been demonstrated using neutral cesium atoms as electron donors. Use of neutral atoms eliminates steric factors in ETD as much as possible and represents another useful point of comparison.

Electron Transfer Directed by Steric Factors

Electron Transfer Directed by Electron Affinity

Electron affinity is the energy required to remove a bound electron from an anion.

(donor molecules ~ 50 250 kJ/mol)

Electron affinity diminishes the recombination energy acquired by the acceptor site. This recombination energy is used for bond dissociation.

As electron affinity increases, the energy level of the transfer products increases. Distances associated with curve crossing increase.

In the limit of sufficiently high electron affinity the potential energy curve of reactants and electron transfer products may no longer intersect; thus, no electron transfer will occur to that site.

A-A + e-

II.

RNH3

I.

Inte

r-Io

n P

ote

ntia

l

Decreasing A- ---- NH3+ Distance 0

(Increasing Time)

SS: no curve-crossing

Electron Transfer Directed by Electron Affinity

peptide + A- products

Hypothesis:If the donor molecule has high EA, some electron transfer sites will be inaccessible.

0

10

20

30

40

50

60

0 100 200 300 400 500

Electron Affinity (kJ/mol)

% E

TD

Some Precursor Molecules for ETD

azobenze

ne

fluora

nthene

perylene

m-dinitro

benzene

o-dinitro

benzene

p-dinitro

benzene

1,3,5-trinitro

benzene

I• CH 3COO

•

picric

acid

Gunawardena, H. P.; McLuckey, S. A. et al., J. Am. Chem. Soc., 2005, 127(36), 12627.

Electron Transfer Directed by Electron Affinity

Returning to theoretical predictions of the Simons group for electron transfer to a small molecule with two types of attachment site:

•Simons predicted that the anion of a hypothetical molecule with an electron affinity of near 193 kJ/mol will selectively transfer an electron to a protonated amine site only, not to the disulfide bond.

•The previous graph has p-dinitrobenzene as an example of a molecule with electron affinity that approximately matches their predicted value.

• p-dinitrobenzene could be used to confirm the hypothesis that donor molecules in ETD can be used to exclude specific electron transfer sites based on electron affinity.

•It might be tested versus other electron donor molecules in reactions with a model peptide.

Conclusions• ECD and ETD are two closely related new dissociation methods that can be

used to derive information about the chemical structure of peptides.

• Several mechanisms have been proposed to explain the products of ECD and ETD. The best interpretation seems to be that more than one competing reaction path is followed.

• ECD and ETD are typically described in literature as if their mechanisms must be essentially identical, but careful examination of the dissociation products versus structure and electronic properties of different ETD reagents is likely to demonstrate differences.

• The choice of some alternative ETD reagents, such as sterically hindered molecules or reagents with targeted electron affinity could potentially be used to direct the ETD process to favor one reaction pathway over another.

• Comparison of alternative ETD reagents with selected peptides would add to understanding of alternative reaction mechanisms occurring.