Immunoaffinity profiling of tyrosine phosphorylation in cancer cells John Rush 1 , Albrecht Moritz 1 , Kimberly A Lee 1 , Ailan Guo 1 , Valerie L Goss 1 , Erik J Spek 1 , Hui Zhang 1,2 , Xiang-Ming Zha 1,2 , Roberto D Polakiewicz 1 & Michael J Comb 1 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. Unregulated tyrosine kinase activity can drive malignancy and tumor formation by generating inappropriate proliferation and survival signals 1 . The ability of researchers to recognize unregulated tyrosine kinases and the pathways driving tumor growth and survival could play a pivotal role in the development of effective drug therapies 2 and in the development of assays to identify drug-responsive patients. Progress in these areas is limited in part by a need for experimental approaches that can isolate and identify tyrosine-phosphorylated peptides in large numbers, without preconceived biases about where tyrosine phosphorylation sites will be found. Current phosphopro- teomic approaches generally reveal only small numbers of tyrosine phosphorylation sites, in keeping with the low level of phosphotyr- osine relative to phosphoserine and phosphothreonine residues 3 . For example, one current phosphoproteomic approach 4,5 uses immobilized metal affinity chromatography (IMAC) to purify phos- phopeptides in bulk from protease-digested cellular protein extracts, which have been methylated to reduce nonspecific binding of acidic unphosphorylated peptides to IMAC resin. The isolated peptides are then identified by reversed- phase liquid chromatography–tandem mass spectrometry (LC-MS/MS). In a yeast protein extract, this approach identified 216 phosphopeptides and 324 phosphorylation sites but only 3 tyrosine phosphorylation sites 4 . Similarly this approach identified only 3 tyrosine phosphorylation sites among 17 phosphopeptides purified from capacitated human sperm cells 5 . A targeted phosphoprotein identification approach uses phos- photyrosine-specific antibodies to immunoprecipitate tyrosine- phosphorylated proteins from cell extracts. Immunoprecipitated proteins are separated by gel electrophoresis, digested in gel bands with a protease and analyzed by MS/MS. Typically, immunoprecipi- tated proteins are identified without identifying their tyrosine phos- phorylation sites. For example, vav-2 6 , STAM2 7 and Odin 8 were isolated from epidermal growth factor–treated HeLa cells by immu- noprecipitation with a phosphotyrosine-specific antibody. After diges- tion these three proteins were identified by MS/MS, but no tyrosine phosphorylation sites were found even though all three proteins were known to be tyrosine-phosphorylated. A third approach combines immunoprecipitation of phosphotyr- osine proteins and purification of phosphopeptides by the methyl- esterification/IMAC method to increase the number of identified tyrosine phosphorylation sites 5,9,10 . This resulted in the identification of 19 tyrosine phosphorylation sites from Jurkat cells stimulated by T- cell receptor ligation 9 , a total of 38 phosphotyrosine sites from two cell lines expressing the Bcr-Abl fusion tyrosine kinase 9 , and after method optimization 10 , as many as 66 phosphotyrosine sites from 10 9 Jurkat cells treated with the tyrosine phosphatase inhibitor pervanadate. Here we describe a simple strategy that uses a phosphotyrosine- specific antibody to immunoprecipitate phosphotyrosine peptides directly from digested cellular protein extracts and standard LC-MS/MS methods to identify large numbers of tyrosine phosphor- ylation sites. Our results contradict the prevailing view that phos- phospecific antibodies are not generally suitable for purifying phosphopeptides 3,11,12 . Using this strategy we have identified 688 nonredundant phosphotyrosine-containing peptides and 628 phos- photyrosine sites, the majority of which are novel, from three distinct cell types. We show that this immunoaffinity profiling strategy can be used to identify protein kinases that are abnormally activated in cancer cells as well as their substrates and other downstream elements. To evaluate the feasibility of our approach for purifying tyrosine- phosphorylated peptides from complex peptide mixtures, we used © 2004 Nature Publishing Group http://www.nature.com/naturebiotechnology Published online 12 December 2004; doi:10.1038/nbt1046 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.). Correspondence should be addressed to M.J.C. (mcomb@cellsignal.com). NATURE BIOTECHNOLOGY ADVANCE ONLINE PUBLICATION 1 LETTERS