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Published February 1, 2001 | public
Journal Article

A Multidimensional Electrospray MS-Based Approach to Phosphopeptide Mapping


A new, multidimensional electrospray MS-based strategy for phosphopeptide mapping is described which eliminates the need to radiolabel protein with ^(32)P or ^(33)P. The approach utilizes two orthogonal MS scanning techniques, both of which are based on the production of phosphopeptide-specific marker ions at m/z 63 and/or 79 in the negative ion mode. These scan methods are combined with liquid chromatography‚ąíelectrospray mass spectrometry and nanoelectrospray MS/MS to selectively detect and identify phosphopeptides in complex proteolytic digests. Low-abundance, low-stoichiometry phosphorylation sites can be selectively determined in the presence of an excess of nonphosphorylated peptides, even in cases where the signal from the phosphopeptide is indistinguishable from background in the conventional MS scan. The strategy, which has been developed and refined in our laboratory over the past few years, is particularly well suited to phosphoproteins that are phosphorylated 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 is illustrated by the complete in vitro and in vivo phosphopeptide mapping of Sic1p, a regulator of the G1/S transition in budding yeast.

Additional Information

© 2001 American Chemical Society. Received 20 September 2000; accepted 7 December 2000; published online 29 December 2000; published in print 1 February 2001.

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