CH 908: Mass Spectrometry Lecture 8 Collisionally Activated Dissociation (of proteins and peptides)...

CH 908: Mass SpectrometryLecture 8

Collisionally Activated Dissociation

(of proteins and peptides)

Prof. Peter B. O’Connor

EI Mass Spectrum of an acetylated and reduced peptide

Tandem Mass Spectrometry or MS/MS

MS/MSMS/MS/MS, or MS3

Benefits: 1.Extremely high specificity2.More structural information

Limitations:1.Isolation window2.Fragmentation efficiency3.Ion Losses

Isolation

FragmentationIsolation

Fragmentation

Collisionally Activated Dissociationalso called Collision Induced Dissociation (CID)

+

N2N2

N2

N2 N2 N2

N2 N2

+

0

• Ion’s smack into neutral gas molecules and break up

• Energy of the collision is controlled by changing the kinetic energy of the ion.

• Fragments scatter radially

• By far the most common MS/MS technique

• slow fragmentation method, deposits vibrational energy throughout the molecule prior to fragmentation.

•SORI-CAD, ITMSn, Triple quad, TOF/TOF, etcetera

Photo-Dissociation

++

0

• Ion absorbs photon(s) and break

• Energy of the fragmentation is controlled by changing the photon’s wavelength.

• No scattering, except for multiply charged ions

•slow fragmentation method, deposits vibrational energy throughout the molecule prior to fragmentation (depends on wavelength).

•IRMPD, UVPD, BIRD

+*

hυ

Surface induced dissociation

+

+0

• Ion smack into a surface, break, and rebound

• Energy of the fragmentation is controlled by changing the ion kinetic energy.

• Fragments scatter radially

•slow fragmentation method, deposits vibrational energy throughout the molecule prior to fragmentation.

•Ions are lost by neutralization at the surface (much better with perfluorinated surfaces)

Outline: Collision models• Collision theory

– Hard-sphere– Soft-sphere– Collision forces– Langevin Cross-section– Measuring cross-section– Use of cross-sections – ion mobility– Reactive collisions – ion molecule reactions– Internal energy deposition– Many, low energy collisions versus single high

energy collisions

• Peptide fragmentation nomenclature– Roepstorff– Biemann

• examples• Preferential cleavage sites

– Asp/glu– Pro

•Structure of b,y ions

•B2 ion

•More examples

•Breakdown diagrams

•Proteins versus peptides

•Oddball spectra – a/x ions in ubiquitin or CA

Hard Sphere collision model2ABArea

Valid for “high energy” collisions

Langevin collision model2ABArea

Valid for “low energy” collisions

2

4neutral

Ion NeutralIon Neutral

z

r

z=charge state

r = ion-neutral distance

α = polarizability

Energy depositionDuring an ion-molecule collision, the fraction of kinetic energy that is lost by the ion is:

22

4cos

( )neutral ion

k kneutral ion

m mE E

m m

Θ = scattering angle

For the usual case of mion >> mneutral, and for the “worst case” scenario of a head-on collision (θ=0), this reduces to:

4 neutralk k

ion

mE E

m

This is the maximum amount of collision energy available, which will be distributed into translational, vibrational, and rotational modes.

Note: increasing the neutral’s mass, increases energy deposition

Internal Energy

Conversion

Typically, 20-50% of the ΔE is converted to internal vibrational energy.

This ratio is a function of temperature, number of states, transition state energies of each reaction channel, etc.

CAD/IRMPD/SID of Peptides and proteins

H

HN

HC

C

OH

O

Ri

( )i

“standard” CAD spectrum of a peptide

Hemoglobin alpha chain:

What’s in a sequence?

m/z 1529.7384

Roepstorff, P. and J. Fohlman (1984). "Proposal for a Common Nomenclature for Sequence Ions in Mass Spectra of Peptides." Biomed. Mass Spectrom. 11: 601.

Amino Acid masses

Roepstorff, P. and J. Fohlman (1984). "Proposal for a Common Nomenclature for Sequence Ions in Mass Spectra of Peptides." Biomed. Mass Spectrom. 11: 601.

protonation sites

Roepstorff, P. and J. Fohlman (1984). "Proposal for a Common Nomenclature for Sequence Ions in Mass Spectra of Peptides." Biomed. Mass Spectrom. 11: 601.

Roepstorff nomenclature for peptide fragmentation.

Roepstorff, P. and J. Fohlman (1984). "Proposal for a Common Nomenclature for Sequence Ions in Mass Spectra of Peptides." Biomed. Mass Spectrom. 11: 601.

CAD, SID, IRMPD all produce b/y-type ions from peptides and proteins (primarily)

Acylium structure Protonated primary amine

21

n

ii

mass a H O protons

2

m

ii n

mass a H O protons

A Mobile Proton Theory of Peptide Fragmentation

• The most stable protonated form may not be the fragmenting structure

• Fragmentation (backbone) occurs due to the weakening of the amide bond, i.e. decrease of the bond order

• Calculations showed that this will happen in the case of the protonation of the amide N

• The more “mobile” (not localised) the proton, the more fragments in a MS/MS spectrum =>the more information from the spectrum

Proton affinity

Amino acid Proton affinity (kcal/mol)

Proton affinity (eV’s)

Lysine 110 4.8

Histidine 290 12.6

Arginine 315 13.7

Backbone amide

~40 1.7

Thus, the amino acids are protonated at Arginine first, then Histidine, then Lysine, then the backbone.

Mobile proton model

The Proline Effect in CAD/SID/IRMPD

The Proline Effect in CAD/SID/IRMPD

The Proline Effect in CAD/SID/IRMPD

Selective Aspartic acid cleavage

Tsaprailis, G., H. Nair, et al. (1999). "Influence of secondary structure on the fragmentation of protonated peptides." Journal of the American Chemical Society 121(22): 5142-5154.

Tsaprailis, G., H. Nair, et al. (1999). "Influence of secondary structure on the fragmentation of protonated peptides." Journal of the American Chemical Society 121(22): 5142-5154.

CAD/SID/IRMPD of Phosphopeptides

H

HN

HC

C

OH

O

O

P

OH

OHOH

HN

HC

C

OH

O

HO

H

HN

C

C

OH

O

-98 = H3PO4

-80 = HPO3

Dehydroalanine

SerinePhosphoserine

CAD/SID/IRMPD of Phosphopeptides

H

HN

HC

C

OH

O

O

P

OH

OH

O

H

HN

HC

C

OH

O

OH

-98 = H3PO4

-80 = HPO3

Phosphotyrosine

H

HN

HC

C

OH

O

Tyrosine

Phenylalanine

Strong

Weak

Comparison of CAD spectra on different instruments

Tsaprailis, G., H. Nair, et al. (1999). "Influence of secondary structure on the fragmentation of protonated peptides." Journal of the American Chemical Society 121(22): 5142-5154.

Xxx

Zzz

Relative frequency of Xxx-Zzz cleavage

High energy CAD

Immonium Ions:

N-linked glycan

O-linked glycan

Glycans

Self Assessment questions• What’s the main cleavage type for peptides/proteins under

CAD/SID/IRMPD condition? Draw the structures of the fragments.• What additional fragment ions come from higher energy

fragmentation? Draw the structures of the fragments.• Name two preferential cleavage points in peptide sequences.• What happens when a phosphoserine containing peptide undergoes

CAD?• Memorize the structures of all 20 natural amino acids. (this is a very

common viva question…)• Would a hard-sphere collision model or a langevin collision model

yield a higher cross section for collision with Argon?

Fini…

CH908: Mass spectrometryLecture 1

NH2

O

NH

O

NH

O

OH

R2

R1

NH

NH2NH

H+

The Fragmenting Structure of a Protonated Peptide