HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: M800307-MCP200v1 8/11/2418    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Glossary Google Scholar Articles by Nguyen, V. Articles by Salomon, A. R. PubMed PubMed Citation Articles by Nguyen, V. Articles by Salomon, A. R. Social Bookmarking                      What's this?

Molecular & Cellular Proteomics 8:2418-2431, 2009.
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

---

Research

A New Approach for Quantitative Phosphoproteomic Dissection of Signaling Pathways Applied to T Cell Receptor Activation^*,

Vinh Nguyen^,, Lulu Cao^¶,, Jonathan T. Lin^,, Norris Hung, Anna Ritz^||,**, Kebing Yu^¶, Radu Jianu^||, Samuel P. Ulin, Benjamin J. Raphael^||,**, David H. Laidlaw^||, Laurent Brossay and Arthur R. Salomon^,¶,,¶¶

From the Department of Molecular Biology, Cell Biology, and Biochemistry,
¶Department of Chemistry,
||Department of Computer Sciences,
**Center for Computational Molecular Biology,
Department of Molecular Microbiology and Immunology, and
Center for Genomics and Proteomics, Brown University, Providence, Rhode Island 02912

Reversible protein phosphorylation plays a pivotal role in the regulation of cellular signaling pathways. Current approaches in phosphoproteomics focus on analysis of the global phosphoproteome in a single cellular state or of receptor stimulation time course experiments, often with a restricted number of time points. Although these studies have provided some insights into newly discovered phosphorylation sites that may be involved in pathways, they alone do not provide enough information to make precise predictions of the placement of individual phosphorylation events within a signaling pathway. Protein disruption and site-directed mutagenesis are essential to clearly define the precise biological roles of the hundreds of newly discovered phosphorylation sites uncovered in modern proteomics experiments. We have combined genetic analysis with quantitative proteomic methods and recently developed visual analysis tools to dissect the tyrosine phosphoproteome of isogenic Zap-70 tyrosine kinase null and reconstituted Jurkat T cells. In our approach, label-free quantitation using normalization to copurified phosphopeptide standards is applied to assemble high density temporal data within a single cell type, either Zap-70 null or reconstituted cells, providing a list of candidate phosphorylation sites that change in abundance after T cell stimulation. Stable isotopic labeling of amino acids in cell culture (SILAC) ratios are then used to compare Zap-70 null and reconstituted cells across a time course of receptor stimulation, providing direct information about the placement of newly observed phosphorylation sites relative to Zap-70. These methods are adaptable to any cell culture signaling system in which isogenic wild type and mutant cells have been or can be derived using any available phosphopeptide enrichment strategy.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?