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A Multidimensional Electrospray MS-Based Approach to Phosphopeptide Mapping

Annan, Roland S. and Huddleston, Michael J. and Verma, Rati and Deshaies, Raymond J. and Carr, Steven A. (2001) A Multidimensional Electrospray MS-Based Approach to Phosphopeptide Mapping. Analytical Chemistry, 73 (3). pp. 393-404. ISSN 0003-2700. http://resolver.caltech.edu/CaltechAUTHORS:20160803-144337603

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Abstract

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−electrospray 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.


Item Type:Article
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URLURL TypeDescription
http://dx.doi.org/10.1021/ac001130tDOIArticle
http://pubs.acs.org/doi/abs/10.1021/ac001130tPublisherArticle
ORCID:
AuthorORCID
Deshaies, Raymond J.0000-0002-3671-9354
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.
Record Number:CaltechAUTHORS:20160803-144337603
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20160803-144337603
Official Citation:Annan, R. S., Huddleston, M. J., Verma, R., Deshaies, R. J., & Carr, S. A. (2001). A Multidimensional Electrospray MS-Based Approach to Phosphopeptide Mapping. Analytical Chemistry, 73(3), 393-404. doi:10.1021/ac001130t
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:69419
Collection:CaltechAUTHORS
Deposited By: Melissa Ray
Deposited On:03 Aug 2016 23:28
Last Modified:03 Aug 2016 23:28

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