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 MS/MSMS/MS/MS, or MS 3 Benefits: 1.Extremely high specificity 2.More structural information Limitations: 1.Isolation window 2.Fragmentation efficiency 3.Ion Losses Isolation Fragmentation Isolation Fragmentation

Collisionally Activated Dissociation also called Collision Induced Dissociation (CID) + N2N2 N2N2 N2N2 N2N2 N2N2 N2N2 N2N2 N2N2 + 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, ITMS n, Triple quad, TOF/TOF, etcetera

Photo-Dissociation 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 +*+* hυhυ

Surface induced dissociation 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 model Valid for “high energy” collisions

Langevin collision model Valid for “low energy” collisions z=charge state r = ion-neutral distance α = polarizability

Energy deposition During an ion-molecule collision, the fraction of kinetic energy that is lost by the ion is: Θ = scattering angle For the usual case of m ion >> m neutral, and for the “worst case” scenario of a head-on collision (θ=0), this reduces to: 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 ( )i)i

“standard” CAD spectrum of a peptide

Hemoglobin alpha chain: What’s in a sequence? m/z 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 structureProtonated primary amine

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 acidProton affinity (kcal/mol) Proton affinity (eV’s) Lysine Histidine Arginine Backbone amide ~401.7 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

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):

CAD/SID/IRMPD of Phosphopeptides -98 = H 3 PO = HPO 3 Dehydroalanine Serine Phosphoserine

CAD/SID/IRMPD of Phosphopeptides -98 = H 3 PO = HPO 3 Phosphotyrosine 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):

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 spectrometry Lecture 1

The Fragmenting Structure of a Protonated Peptide