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 pathway.
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