Femtosecond laser-induced ionization/dissociation tandem mass spectrometry (fsLID-MS/MS) of deprotonated phosphopeptide anions.

RATIONALE Radical-directed dissociation techniques provide structural information which is complementary to that from conventional collision-induced dissociation (CID). The analysis of phosphopeptide anions is warranted due to their relatively acidic character. As femtosecond laser-induced ionization/dissociation tandem mass spectrometry (fsLID-MS/MS) is uniquely initiated by field ionization, an investigation is warranted to determine whether fsLID may provide novel analytical utility for phosphopeptide anions. METHODS Twenty-three synthetic deprotonated phosphopeptide anions were introduced into a three-dimensional quadrupole ion trap mass spectrometer via electrospray ionization. The ion trap was interfaced with a near-IR (802 nm) ultrashort-pulsed (35 fs FWHM) ultrahigh-powered (peak power ~1014 W/cm2) laser system. Performance comparisons are made with other techniques applied to phosphopeptide anion analysis, including CID, electron detachment dissociation (EDD), negative electron transfer dissociation (NETD), activated electron photodetachment dissociation (activated-EPD), and ultraviolet photodissociation (UVPD). RESULTS FsLID-MS/MS of multiply deprotonated phosphopeptide anions provides sequence information via phosphorylation-intact a/ x ions in addition to other sequence ions, satellite ions, and side-chain losses. Novel fragmentation processes include selective c-ion formation N-terminal to Ser/Thr and a phosphorylation-specific correlation between xn-98 ion abundances and phosphorylation at the nth residue. Sequencing-quality data required about 30 s of signal averaging. fsLID-MS/MS of singly deprotonated phosphopeptides did not yield product anions with stable trajectories, despite significant depletion of the precursor. CONCLUSIONS Multiply deprotonated phosphopeptide anions were sequenced via negative-mode fsLID-MS/MS, with phosphosite localization facilitated by a/x ion series in addition to diagnostic xn-98 ions. fsLID-MS/MS is qualitatively competitive with other techniques. Further efficiency enhancements (e.g., implementation on a linear trap or/and higher pulse frequencies) may permit sequence analyses on chromatographic timescales. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]