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) All Versions of this Article: T500032-MCP200v1 5/4/758    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 Glossary Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Khan, I. H. Articles by Luciw, P. A. Search for Related Content PubMed PubMed Citation Articles by Khan, I. H. Articles by Luciw, P. A. Social Bookmarking                      What's this?

Molecular & Cellular Proteomics 5:758-768, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

---

Technology

Multiplex Analysis of Intracellular Signaling Pathways in Lymphoid Cells by Microbead Suspension Arrays^*

Imran H. Khan^,,¶, Sara Mendoza, Paul Rhyne^||, Melanie Ziman, Joseph Tuscano^**, Dominic Eisinger^||, Hsing-Jien Kung and Paul A. Luciw^,

From the Center for Comparative Medicine, Department of Medical Pathology and Laboratory Medicine, ^** Division of Hematology and Oncology, Department of Biological Chemistry and UC Davis Cancer Center, University of California, Davis, California 95616 and ^|| Upstate USA, Charlottesville, Virginia 22903

Phosphorylation analysis of signaling proteins is key for examining intracellular signaling pathways. Conventional biochemical approaches, e.g. immunoprecipitation, Western blot, and ELISA, have played a major role in elucidation of individual signaling events. However, these methods are laborious, time-consuming, and difficult to adapt for high throughput analysis. A multiplex approach to measure phosphorylation state of multiple signaling proteins simultaneously would significantly enhance the efficiency and scope of signaling pathway analysis for mechanistic studies and clinical application. This report describes a novel multiplex microbead suspension array approach to examine phosphoproteomic profiles in lymphoid cells. In the Jurkat T-cell leukemia line, the multiplex assay enabled targeted investigation of phosphorylation kinetics of signal transduction from receptor proximal events (tyrosine phosphoproteins CD3, Lck, Zap-70, and linker for T-cell activation) to cytosolic events (serine/threonine phosphoproteins Erk and Akt) to transcription factors (serine/threonine phosphorylated Rsk, cyclic AMP-response element-binding protein, and STAT3). To broaden the application of the multiplex analysis, signaling pathways were also studied in B-cell lymphoid tumor lines that included chronic lymphocytic leukemia lines. In these cell lines, multiplex suspension array enabled phosphoproteomic analysis of signaling cascade mediated by Syk, a homolog of Zap-70. Results obtained by multiplex analysis were confirmed by immunoprecipitation and Western blot methods. The examples of T-cell and B-cell signaling pathway analyses in this report demonstrate the utility of the multiplex suspension arrays to investigate phosphorylation dynamics and kinetics of several signaling proteins simultaneously in signal transduction pathways.

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

This article has been cited by other articles:

				I. H. Khan, R. Ravindran, J. Yee, M. Ziman, D. M. Lewinsohn, M. L. Gennaro, J. L. Flynn, C. W. Goulding, K. DeRiemer, N. W. Lerche, et al. Profiling Antibodies to Mycobacterium tuberculosis by Multiplex Microbead Suspension Arrays for Serodiagnosis of Tuberculosis Clin. Vaccine Immunol., March 1, 2008; 15(3): 433 - 438. [Abstract] [Full Text] [PDF]