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

Creese and Cooper

of phosphorylation is identified. Again, higher electron energy is required to obtain maximum
sequence coverage for the phosphopeptide.

Conclusions

We have shown that the site and frequency of phosphorylation has a marked effect on a peptide's
ECD behavior. For the synthetic peptide, addition of a single phosphorylation reduced the
sequence coverage from 100% (omitting proline residues) to 67% (best case) or 42% (worst
case). Our results suggest that the phospho-groups exist in their deprotonated form and
therefore phosphopeptide ions require additional protons to achieve a 2+ charge state. Sequence
coverage of phosphopeptides can be improved by conducting ECD with higher electron
energies. It is postulated that deposition of additional energy on electron capture results in
cleavage of noncovalent interactions (salt bridges) between deprotonated phospho-groups and
protonated amino acid side chains, accompanied in some cases by hydrogen rearrangement
and reprotonation of the phospho-group. This is akin to activated ion ECD [43]. Sequence
coverage can also be improved by performing ECD on the triply-charged peptide ions. This is
perhaps unsurprising as ECD efficiency is known to improve with increasing charge state
[50]. The presence of phosphorylation on the β-casein tryptic peptide also reduced the sequence
coverage from 47% to 33%. The poor coverage for the unmodified peptide can be explained
by the presence of salt bridges between deprotonated glutamic acid residues and protonated
glutamine residues. The presence of the phosphorylation alters the intramolecular bonding and
consequently the observed ECD fragmentation. Increasing the ECD electron energy improved
sequence coverage for both peptides (100% for the unmodified peptide and 87% for the
phosphopeptide). For the α-S1-casein peptide, the unmodified peptide and phosphopeptide had
sequence coverages of 30% and 23% (omitting proline residues), respectively, at the standard
electron energy but this increased to a maximum of 92% (unmodified) and 85%
(phosphorylated) with increased electron energy. Again, this result suggests that salt bridges
between deprotonated glutamic acid residues and protonated proline or glutamine prevent
detection of ECD fragments at lower electron energies.

This work is important for the understanding of gas-phase fragmentation of phosphopeptides
and other peptides in which relatively strong noncovalent interactions are present. The peptides
studied here are either very basic (synthetic phosphopeptides) or acidic (tryptic
phosphopeptides), and represent extremes of the spectrum in terms of a protein tryptic digest.
Nevertheless, this work has relevance to the inclusion of ECD in global proteomic strategies.
Global analyses require that optimum parameters suiting the majority of analytes to be
investigated are applied. Our results suggest that LC-ECD MS/MS of an enriched
phosphopeptide mixture, or an acidic strong cation exchange fraction, may require higher
electron energies, or an alternative form of activation. Work in this area is ongoing.

Supplementary data

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

The authors acknowledge the Wellcome Trust (074131) (H.J.C.) and BBSRC (A.J.C.) for funding.

References

1. Zubarev R.A. Kelleher N.L. McLafferty F.W. Electron Capture Dissociation of Multiply Charged
Protein Cations. J. Am. Chem. Soc 1998;120(13):3265-3266.

2. Baba T. Hashimoto Y. Hasegawa H. Hirabayashi A. Waki I. Electron Capture Dissociation in a Radio
Frequency Ion Trap. Anal. Chem 2004;76(15):4263-4266. [PubMed: 15283558]

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

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