Rs. Annan et Sa. Carr, PHOSPHOPEPTIDE ANALYSIS BY MATRIX-ASSISTED LASER-DESORPTION TIME-OF-FLIGHT MASS-SPECTROMETRY, Analytical chemistry, 68(19), 1996, pp. 3413-3421
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.