he Effect of Phosphorylation on the Electron Capture Dissociation of Peptide Ions

by University of Birmingham Research Archive, E-prints Repository

PubMed

central Journal of the American Society for

Mass Spectrometry

Published as: JAm SocMass Spectrom. 2008 September ; 19(9): 1263-1274.

The Effect of Phosphorylation on the Electron Capture

Dissociation of Peptide Ions

Andrew J. Creese and Helen J. Cooper*

School of Biosciences, University of Birmingham, UK Birmingham, United Kingdom.

Abstract

The effect of site and frequency of phosphorylation on the electron capture dissociation of peptide
ions has been investigated. The ECD of a suite of synthetic peptides (APLSFRGSLPKSYVK; one
unmodified, three singly-phosphorylated, three-doubly phosphorylated, and one triply-
phosphorylated); two tryptic phosphopeptides (YKVPQLEIVPN_pSAEER, α-casein and
FQ_pSEEQQQTEDELQDK, β-casein) and their unmodified counterparts, were determined over a
range of ECD cathode potentials. The results show that, for doubly-charged precursor ions, the
presence of phosphorylation has a deleterious effect on ECD sequence coverage. The fragmentation
patterns observed suggest that for peptides with multiple basic residues, the phospho-groups exist in
their deprotonated form and form salt-bridges with protonated amino acid side chains. The
fragmentation observed for the acidic tryptic peptides suggested the presence of noncovalent
interactions, which were perturbed on phosphorylation. Increasing the ECD electron energy
significantly improves sequence coverage. Alternatively, improved sequence coverage can be
achieved by performing ECD on triply-charged precursor ions. The findings are important for the
understanding of gas-phase fragmentation of phosphopeptides.

Graphical Abstract

Phosphorylation has a deleterious effect on the ECD of doubly-charged basic peptides due to salt
bridge formation between deprotonated phospho-groups and protonated lysine/arginine side chains.

© 2008 Elsevier Inc..

*Address reprint requests to Dr. H. J. Cooper, School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT,
UK [email protected].

This document was posted here by permission of the publisher. At the time of deposit, it included all changes made during peer review,
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is available for free, on ScienceDirect.

Published online May 28, 2008

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