Femtosecond laser-induced ionization/dissociation tandem mass spectrometry (fsLID-MS/MS) of deprotonated phosphopeptide anions.
BACKGROUND
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 ~10(14)  W/cm(2)) 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 n(th) 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 x(n)-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.
RTSF Mass Spectrometry & Metabolomics Core, Michigan State University, East Lansing, MI, 48824, USA.
Scott A
SA
Smith
Christine L
CL
Kalcic
Li
L
Cui
Gavin E
GE
Reid
D000595:Amino Acid Sequence; D000838:Anions / Q000032:analysis / Q000737:chemistry*; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000032:analysis / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D000838:Anions; D010748:Phosphopeptides