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

Andrew J Creese,Helen J Cooper

doi:10.1016/j.jasms.2008.05.015

Andrew J Creese, Helen J Cooper

Open Access

https://doi.org/10.1016/j.jasms.2008.05.015

Copy DOI

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 (YKVPQLEIVPNpSAEER, α-casein and FQpSEEQQQTEDELQDK, 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.

Highlights

The maximum sequence coverage was obtained at ECD cathode potentials between Ϫ14 V and Ϫ15 V
Our results suggest that the phosphogroups exist in their deprotonated form and phosphopeptide ions require additional protons to achieve a 2ϩ charge state
Sequence coverage of phosphopeptides can be improved by conducting ECD with higher electron energies

Summary

Capture Dissociation of Peptide Ions

The introduction of electron capture dissociation (ECD) [1] in 1998 provided a unique fragmentation technique for biomolecular analysis. Zubarev and coworkers have developed a method performing both ECD and CID on all eluting peptides [34 –38] This combined approach provides information about the relationship between b/y and c/z· fragments, i.e.; the “golden rules” [39]. An LC-ECD MS/MS analysis is performed in which only potential phosphopeptides are investigated These methods combine the high scan rate of CID with the improved peptide sequence coverage and the retention of PTMs of ECD. The results show that for doubly-charged peptide precursor ions the presence of phosphorylation has a deleterious effect on ECD sequence coverage. The tryptic peptides showed improved sequence coverage with increasing electron energy for both phosphopeptides and their unmodified counterparts These results suggest that for these peptides the phosphogroup was not directly involved in noncovalent bonding but that its presence altered the peptide conformation and intramolecular bonding

Preparation of Synthetic Peptides

Results and Discussion

Conclusions