Phosphopeptide analysis by matrix-assisted laser desorption time-of-flight mass spectrometry

In this paper we present methods for identifying and sequencing phosphopeptides in simple mixtures, such as HPLC fractions, at the subpicomole level by (+) ion matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-MS). Data are presented which indicate that when a reflectron time-of-flight mass spectrometer is used, MALDI can distinguish tyrosine phosphorylation from serine and threonine phosphorylation for peptides containing a single phosphate group. Phosphopeptides are identified in the (+) ion MALDI reflector spectrum by the presence of [MH - H3PO4](+) and [MH - HPO3](+) fragment ions formed by metastable decomposition. An abundant [MH - H3PO4](+) ion, accompanied by a weaker [MH - HPO3](+) ion indicates that the peptide is most likely phosphorylated on serine or threonine. In contrast, phosphotyrosine-containing peptides generally exhibit [MH - HPO3](+) fragment ions and little, if any [MH - H-3-PO4](+). Ambiguities do arise, most often with phosphopeptides that contain residues which readily lose water (such as unmodified serine), but these can often be resolved by recording a complete metastable fragment ion (postsource decay) spectrum. Postsource decay is shown here to be a viable technique for sequencing phosphopeptides. It can be used to distinguish between serine/threonine and tyrosine phosphorylation and in many cases can be used to determine the exact site of phosphorylation in a peptide sequence. Nearly complete sequence coverage and phosphorylation site mapping is generally possible using similar to 300 fmol of peptide.