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Methods for the Detection of Paxillin Post-translational Modifications and Interacting Proteins by Mass Spectrometry

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Melanie J. Schroeder,*^† Donna J. Webb,^‡ Jeffrey Shabanowitz,^† Alan F. Horwitz,^‡ and Donald F. Hunt^†^§

Department of Chemistry, McCormick Road, University of Virginia, Charlottesville, Virginia 22904, Department of Cell Biology, P.O. Box 800732, Charlottesville, Virginia 22908-0732, and Department of Chemistry and Pathology, University of Virginia, Charlottesville, Virginia 22904

J. Proteome Res., 2005, 4 (5), pp 1832–1841

DOI: 10.1021/pr0502020

Publication Date (Web): September 13, 2005

To whom correspondence should be addressed. University of Chicago, Department of Chemistry, 5735 S. Ellis Ave., Chicago, IL 60637. Tel: (773) 834-0994. Fax: (773) 702-0805. E-mail: mjschroe@uchicago.edu.

†

Departmentof Chemistry.

These authors contributed equally to this work.

‡

Department of Cell Biology.

Department of Chemistry and Pathology.

Abstract

Methods for the simultaneous identification of interacting proteins and post-translational modifications of the focal adhesion adapter protein, paxillin, are presented. The strategy includes (1) lower-level, transient transfection of FLAG-GFP-Paxillin into HEK293 cells, (2) incubation of cells with phosphatase inhibitors prior to lysis, (3) purification of paxillin by anti-FLAG immunoprecipitation, (4) analysis of peptides generated from on-beads digestion using LTQ−FT or LTQ−ETD mass spectrometry, and (5) enrichment of phosphopeptide methyl esters with IMAC. Using the above strategies, we identify 29 phosphorylation sites (19 novel and 10 previously reported) and a novel glycosylation site on Ser 74. Furthermore, with this method, we simultaneously detect 10 co-purifying proteins which are present in focal adhesion complexes. Extension of these methods to other substrates should facilitate generation of global phosphorylation maps and protein−protein interactions for any protein of interest.

Keywords: paxillin • IMAC • phosphorylation • glycosylation • GlcNAc • methyl esters • peroxovanadate • calyculin A • ETD

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Published In Issue October 10, 2005
Received July 1, 2005

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Article

Methods for the Detection of Paxillin Post-translational Modifications and Interacting Proteins by Mass Spectrometry

Abstract

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SciFinder Links

History

Recommend & Share

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Exploiting Differential Dissociation Chemistries of O-Linked Glycopeptide Ions for the Localization of Mucin-Type Protein Glycosylation

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