Alkaline liquid chromatography/electrospray ionization skimmer collision-induced dissociation mass spectrometry for phosphopeptide screening

A rapid on-line method for the identification of phosphorylated peptides in enzymatic protein digests by specific marker ion signals is described. In our study we investigated the use of alkaline conditions together with a pr eviously described method for selective and sensitive detection of phosphop eptide ions combining high-performance capillary liquid chromatography (LC) and electrospray ionization mass spectrometry (ESI-MS).(1,2) Phosphorylati on-specific marker ions (m/z 79, PO3-, and m/z 97, H2PO4-) were generated b y skimmer collision-induced dissociation (sCID) in the negative-ion mode. T he method was evaluated and validated for mono-phosphorylated synthetic pep tides using different alkaline pH values and CID offsets. Alkaline conditio ns (pH 10.5) enhance the generation of phosphopeptide-specific fragment ion s from serine- and tyrosine-phosphorylated peptides, and enable the use of m/z 79 (PO3-) and m/z 97 (H2PO4-) as phosphorylation-specific marker traces . Note that HPLC separation in trifluoroacetic acid containing solvents imp airs the use of m/z 97 (C2F3O- fragment ion at m/z 97) as a phosphorylation -specific marker. The optimized method was applied for the detection of pho sphorylated peptides in a tryptic beta -casein digest. The expected mono-an d tetra-phosphorylated peptides were detected and rapidly identified by mu LC/ESI-sCID-MS and mu LC/ESI-MS analysis. Copyright (C) 2001 John Wiley & S ons, Ltd.