Article

Gas-Phase Rearrangements Do Not Affect Site Localization Reliability in Phosphoproteomics Data Sets

Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Journal of Proteome Research (Impact Factor: 5). 04/2010; 9(6):3103-7. DOI: 10.1021/pr1000225
Source: PubMed

ABSTRACT Intramolecular transfer of phosphate during collision-induced dissociation (CID) in ion-trap mass spectrometers has recently been described. Because phosphorylation events are assigned to discrete serine, threonine, and tyrosine residues based on the presence of site-determining ions in MS/MS spectra, phosphate transfer may invalidate or confound site localization in published large-scale phosphorylation data sets. Here, we present evidence for the occurrence of this phenomenon using synthetic phosphopeptide libraries, specifically for doubly charged species. We found, however, that the extent of the transfer reaction was insufficient to cause localization of phosphorylation sites to incorrect residues. We further compared CID to electron-transfer dissociation (ETD) for site localization using synthetic libraries and a large-scale yeast phosphoproteome experiment. The agreement in site localization was >99.5 and 93%, respectively, suggesting that ETD-based site localization is no more reliable than CID. We conclude that intramolecular phosphate transfer does not affect the reliability of current or past phosphorylation data sets.

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