Immunoaffinity profiling of tyrosine phosphorylation in cancer cells.
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.
Rush
John
J
Cell Signaling Technology Inc., 166B Cummings Center, Beverly, Massachusetts 01915, USA.
Moritz
Albrecht
A
Lee
Kimberly A
KA
Guo
Ailan
A
Goss
Valerie L
VL
Spek
Erik J
EJ
Zhang
Hui
H
Zha
Xiang-Ming
XM
Polakiewicz
Roberto D
RD
Comb
Michael J
MJ
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D019169:Jurkat Cells; D013058:Mass Spectrometry; D051379:Mice; D041681:NIH 3T3 Cells; D009369:Neoplasms / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction; D014443:Tyrosine / Q000737:chemistry*
D010455:Peptides; D019000:Phosphotyrosine; D014443:Tyrosine