PHOSPHOPEPTIDE ANALYSIS BY MATRIX-ASSISTED LASER-DESORPTION TIME-OF-FLIGHT MASS-SPECTROMETRY

In this paper we present methods for identifying and sequencing phosph opeptides in simple mixtures, such as HPLC fractions, at the subpicomo le level by (+) ion matrix-assisted laser desorption time-of-flight ma ss spectrometry (MALDI-MS). Data are presented which indicate that whe n a reflectron time-of-flight mass spectrometer is used, MALDI can dis tinguish tyrosine phosphorylation from serine and threonine phosphoryl ation for peptides containing a single phosphate group. Phosphopeptide s are identified in the (+) ion MALDI reflector spectrum by the presen ce of [MH - H3PO4](+) and [MH - HPO3](+) fragment ions formed by metas table decomposition. An abundant [MH - H3PO4](+) ion, accompanied by a weaker [MH - HPO3](+) ion indicates that the peptide is most likely p hosphorylated on serine or threonine. In contrast, phosphotyrosine-con taining peptides generally exhibit [MH - HPO3](+) fragment ions and li ttle, if any [MH - H-3-PO4](+). Ambiguities do arise, most often with phosphopeptides that contain residues which readily lose water (such a s unmodified serine), but these can often be resolved by recording a c omplete metastable fragment ion (postsource decay) spectrum. Postsourc e decay is shown here to be a viable technique for sequencing phosphop eptides. It can be used to distinguish between serine/threonine and ty rosine phosphorylation and in many cases can be used to determine the exact site of phosphorylation in a peptide sequence. Nearly complete s equence coverage and phosphorylation site mapping is generally possibl e using similar to 300 fmol of peptide.