A multidimensional electrospray MS-based approach to phosphopeptide mapping

A new, multidimensional electrospray MS-based strategy for phosphopeptide m apping is described which eliminates the need to radiolabel protein with P- 32 or P-33. The approach utilizes two orthogonal MS scanning techniques, bo th of which are based on the production of phosphopeptide-specific marker i ons at m/z 63 and/or 79 in the negative ion mode. These scan methods are co mbined with liquid chromatography-electrospray mass spectrometry and nanoel ectrospray MS/MS to selectively detect and identify phosphopeptides in comp lex proteolytic digests. Low-abundance, low-stoichiometry phosphorylation s ites can be selectively determined in the presence dan excess of nonphospho rylated peptides, even in cases where the signal from the phosphopeptide is indistinguishable from background in the conventional MS scan, The strateg y, which has been developed and refined in our laboratory over the past few years, is particularly well suited to phosphoproteins that are phosphoryla ted to varying degrees of stoichiometry on multiple sites. Sensitivity and selectivity of the method are demonstrated here using model peptides and a commercially available phosphoprotein standard. In addition, the strategy i s illustrated by the complete in vitro and in vivo phosphopeptide mapping o f Sic1p, a regulator of the G1/S transition in budding yeast.