A Neutral Loss Activation Method for Improved Phosphopeptide Sequence Analysis by Quadrupole Ion Trap Mass Spectrometry

TLDR

Recent advances in phosphopeptide enrichment and tandem mass spectrometry have improved phosphoproteome characterization, but collision‑activated dissociation often yields product ions dominated by neutral loss of phosphoric acid. The study introduces Pseudo MSn, a novel method for phosphopeptide ion dissociation in quadrupole ion trap mass spectrometers. Pseudo MSn activates neutral loss product ions after precursor activation, producing a composite spectrum that incorporates fragments from both the original precursor and subsequent neutral loss activations. Compared to conventional CAD, Pseudo MSn improved phosphopeptide dissociation for 7 of 10 synthetic peptides, yielded similar gains for enzymatically digested peptides, and enabled correct identification of phosphopeptides that were misassigned by TurboSEQUEST in a complex mixture.

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 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.