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Mass Spectrometry-based Methods for Phosphorylation Site Mapping of Hyperphosphorylated Proteins Applied to Net1, a Regulator of Exit from Mitosis in Yeast

Chen, Susan Loughery and Huddleston, Michael J. and Shou, Wenying and Deshaies, Raymond J. and Annan, Roland S. and Carr, Steven A. (2002) Mass Spectrometry-based Methods for Phosphorylation Site Mapping of Hyperphosphorylated Proteins Applied to Net1, a Regulator of Exit from Mitosis in Yeast. Molecular and Cellular Proteomics, 1 (3). pp. 186-196. ISSN 1535-9476. doi:10.1074/mcp.M100032-MCP200.

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Prior to anaphase in Saccharomyces cerevisiae, Cdc14 protein phosphatase is sequestered within the nucleolus and inhibited by Net1, a component of the RENT complex in budding yeast. During anaphase the RENT complex disassembles, allowing Cdc14 to migrate to the nucleus and cytoplasm where it catalyzes exit from mitosis. The mechanism of Cdc14 release appears to involve the polo-like kinase Cdc5, which is capable of promoting the dissociation of a recombinant Net1·Cdc14 complex in vitro by phosphorylation of Net1. We report here the phosphorylation site mapping of recombinant Net1 (Net1N) and a mutant Net1N allele (Net1N-19m) with 19 serines or threonines mutated to alanine. A variety of chromatographic and mass spectrometric-based strategies were used, including immobilized metal-affinity chromatography, alkaline phosphatase treatment, matrix-assisted laser-desorption post-source decay, and a multidimensional electrospray mass spectrometry-based approach. No one approach was able to identify all phosphopeptides in the tryptic digests of these proteins. Most notably, the presence of a basic residue near the phosphorylated residue significantly hampered the ability of alkaline phosphatase to hydrolyze the phosphate moiety. A major goal of research in proteomics is to identify all proteins and their interactions and post-translational modification states. The failure of any single method to identify all sites in highly phosphorylated Net1N, however, raises significant concerns about how feasible it is to map phosphorylation sites throughout the proteome using existing technologies.

Item Type:Article
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Deshaies, Raymond J.0000-0002-3671-9354
Carr, Steven A.0000-0002-7203-4299
Additional Information:© 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Received, December 3, 2001, and in revised form, January 18, 2002. Published, MCP Papers in Press, January 22, 2002, DOI 10.1074/mcp.M100032-MCP200 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Issue or Number:3
Record Number:CaltechAUTHORS:LOUmcp02
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ID Code:8762
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Deposited On:13 Sep 2007
Last Modified:08 Nov 2021 20:53

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