Nature Biotechnology23, 94 - 101 (2004)
Published online: 12 December 2004; | doi:10.1038/nbt1046
Immunoaffinity profiling of tyrosine phosphorylation in cancer cells
John Rush1, Albrecht Moritz1, Kimberly A Lee1, Ailan Guo1, Valerie L Goss1, Erik J Spek1, Hui Zhang1, 2, Xiang-Ming Zha1, 2, Roberto D Polakiewicz1
& Michael J Comb1
1
Cell Signaling Technology Inc., 166B Cummings Center, Beverly, Massachusetts 01915, USA.
2
Present addresses: Institute for Systems Biology, 1441 N. 34th St., Seattle, Washington 98103, USA (H.Z.) and Department of Internal Medicine, Howard Hughes Medical Institute, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242, USA (X.Z.).
Tyrosine kinases play a prominent role in human cancer, yet the oncogenic signaling pathways driving cell proliferation and survival have been difficult to identify, in part because of the complexity of the pathways and in part because of low cellular levels of tyrosine phosphorylation. In general, global phosphoproteomic approaches reveal small numbers of peptides containing phosphotyrosine. We have developed a strategy that emphasizes the phosphotyrosine component of the phosphoproteome and identifies large numbers of tyrosine phosphorylation sites. Peptides containing phosphotyrosine are isolated directly from protease-digested cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. Applying this approach to several cell systems, including cancer cell lines, shows it can be used to identify activated protein kinases and their phosphorylated substrates without prior knowledge of the signaling networks that are activated, a first step in profiling normal and oncogenic signaling networks.
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