CH 908: Mass Spectrometry
Lecture 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
Isolation
Fragmentation
Isolation
Fragmentation
Benefits:
1.Extremely high
MS/MS/MS,
MS/MS
or MS3
specificity
2.More structural
information
Limitations:
1.Isolation window
2.Fragmentation
efficiency
3.Ion Losses
Collisionally Activated Dissociation
also called Collision Induced Dissociation (CID)
N2
N2
+
N2
N2
N2
N2
+
N2
0
N2
• Ion’s smack into neutral gas • By far the most common
molecules and break up
MS/MS technique
• Energy of the collision is
controlled by changing the
kinetic energy of the ion.
• Fragments scatter radially
• slow fragmentation method,
deposits vibrational energy
throughout the molecule prior to
fragmentation.
•SORI-CAD, ITMSn, Triple
quad, TOF/TOF, etcetera
Photo-Dissociation
+*
+
+
0
hυ
• Ion absorbs photon(s) and
break
•slow fragmentation method,
deposits vibrational energy
throughout the molecule prior to
• Energy of the fragmentation
fragmentation (depends on
is controlled by changing the
wavelength).
photon’s wavelength.
•IRMPD, UVPD, BIRD
• No scattering, except for
multiply charged ions
Surface induced dissociation
+
0
+
• Ion smack into a surface,
break, and rebound
•slow fragmentation method,
deposits vibrational energy
throughout the molecule prior to
• Energy of the fragmentation
fragmentation.
is controlled by changing the
ion kinetic energy.
•Ions are lost by neutralization
at the surface (much better with
• Fragments scatter radially
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 model
2
Area   AB
Valid for “high energy” collisions
Langevin collision model
2
Area   AB
 Ion Neutral
z  neutral
 4
rIon  Neutral
2
z=charge state
r = ion-neutral distance
α = polarizability
Valid for “low energy” collisions
Energy deposition
During an ion-molecule collision, the fraction of kinetic energy that is lost
by the ion is:
4mneutral mion
2
Ek 
E
cos

k
2
(mneutral  mion )
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:
4mneutral
Ek 
Ek
mion
Note: increasing the neutral’s
mass, increases energy deposition
This is the maximum amount of collision energy available, which will be
distributed into translational, vibrational, and rotational modes.
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
(
)
i
“standard” CAD spectrum of a peptide
What’s in a sequence?
Hemoglobin alpha chain: 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
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, 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
n
mass   ai  H 2O  protons
i 1
Protonated primary amine
m
mass   ai  H 2O  protons
in
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
Dehydroalanine
Phosphoserine
Serine
CAD/SID/IRMPD of
Phosphopeptides
Phenylalanine
Phosphotyrosine
Tyrosine
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.
Relative frequency of Xxx-Zzz cleavage
Xxx
Zzz
High energy CAD
Immonium Ions:
Glycans
N-linked glycan
O-linked glycan
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?
CH908: Mass spectrometry
Lecture 1
Fini…
The Fragmenting Structure of a Protonated
Peptide
NH2
HN
+
NH
H
R1
O
H
N
H2N
O
R2
N
H
OH
O