A neutral loss activation method for improved phosphopeptide sequence analysis by quadrupole ion trap mass spectrometry.

Abstract

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 MS(n), 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 simultaneously stored, the method generates a "composite" spectrum containing fragments derived from multiple precursors. In comparison to analysis by conventional MS/MS (CAD), Pseudo MS(n) 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 MS(n) to peptides generated by enzymatic digestion of a single phosphoprotein. Finally, when applied to a complex phosphopeptide mixture, several phosphopeptides mis-assigned by TurboSEQUEST under the conventional CAD approach were successfully identified after analysis by Pseudo MS(n).