Sa. Carr et al., SELECTIVE DETECTION AND SEQUENCING OF PHOSPHOPEPTIDES AT THE FEMTOMOLE LEVEL BY MASS-SPECTROMETRY, Analytical biochemistry, 239(2), 1996, pp. 180-192
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.