SELECTIVE DETECTION AND SEQUENCING OF PHOSPHOPEPTIDES AT THE FEMTOMOLE LEVEL BY MASS-SPECTROMETRY

We describe a new procedure that enables selective detection and seque ncing of Ser-, Thr-, and Tyr-phosphopeptides at the low femtomole leve l in protein digests. Radiolabeling with P-32 is not required, nor is prior chromatographic separation of the peptide mixture. One to two mi croliters of the unfractionated protein digest is infused at basic pH into an electrospray mass spectrometer at a how rate of 20-40 nl/min u sing an ultra-low flow sprayer. A precursor-ion scan of m/z 79 (PO3-) produces a mass spectrum containing only the molecular ions of the pho sphopeptides that are present in the sample. In eases where the protei n sequence is known, the peptide molecular weights obtained are often sufficient to identify the specific sequences that are phosphorylated. If the protein sequence is not known, tandem MS with collision-induce d dissociation of phosphopeptide precursor-ions may be used to obtain the amino acid sequences including the site(s) of phosphorylation. We demonstrate that phosphopeptides may be selectively detected using as little as 3 fmol of a 10 fmol/mu l solution and that sequence informat ion for a phosphopeptide in the mixture may be obtained using as littl e as 3 femtomole of the same solution. In addition, we show that the s toichiometry of phosphorylation at specific sites may be estimated fro m the ratio of the ion signals for the respective forms of the peptide s observed in the normal full-scan mass spectra of the digest. These p rocedures are illustrated here to identify and sequence phosphopeptide s from alpha-casein, a milk-derived protein possessing up to nine phos phorylation-sites. Numerous MS and tandem MS experiments were carried out on a single, 250 fmol mu l loading of the phosphoprotein digest. P hosphopeptides derived from an unexpected variant of the protein were also observed. (C) 1996 Academic Press, Inc.