Journal
ANALYTICAL CHEMISTRY
Volume 76, Issue 13, Pages 3590-3598Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ac0497104
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Funding
- NCRR NIH HHS [F32 RR 018688-01] Funding Source: Medline
- NIGMS NIH HHS [GM 37537] Funding Source: Medline
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Recent advances in phosphopeptide enrichment prior to mass spectrometric analysis show genuine promise for characterization of phosphoproteomes. Tandem mass spectrometry of phosphopeptide ions, using collision-activated dissociation (CAD), often produces product ions dominated by the neutral loss of phosphoric acid. Here we describe a novel method, termed Pseudo MSn for phosphopeptide ion dissociation in quadrupole ion trap mass spectrometers. The method induces collisional activation of product ions, those resulting from neutral loss(es) of phosphoric acid, following activation of the precursor ion. Thus, the principal neutral loss product ions are converted into a variety of structurally informative species. Since product ions from both the original precursor activation and all subsequent neutral loss product activations are simulataneously stored, the method generates a composite spectrum containing fragments derived from multiple precursors. In comparison to analysis by conventional MS/MS (CAD), Pseudo MSn shows improved phosphopeptide ion dissociation for 7 out of 10 synthetic phosphopeptides, as judged by an automated search algorithm (TurboSEQUEST). A similar overall improvement was observed upon application of Pseudo MSn to peptides generated by enzymatic digestion of a single phosphoprotein. Finally, when applied to a complex phosphopeptide mixture, several phosphopeptides misassigned by TurboSEQUEST under the conventional CAD approach were successfully identified after analysis by Pseudo MSn.
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