Nature Methods
2, 591 - 598 (2005)
Published online: 21 July 2005; | doi:10.1038/nmeth776
Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometryW Andy Tao1, 2, Bernd Wollscheid2, Robert O'Brien2, Jimmy K Eng2, Xiao-jun Li2, Bernd Bodenmiller3, Julian D Watts2, Leroy Hood2
& Ruedi Aebersold2, 3, 41
The Bindley Bioscience Center and Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907, USA. 2
Institute for Systems Biology, 1441 North 34th Street, Seattle, Washington 98103, USA. 3
Institute for Molecular Systems Biology, Federal Institute of Technology, Campus Hoenggerberg, HPT E78, Wolfgang Pauli Strasse 16, CH-8093, Zurich, Switzerland. 4
Faculty of Natural Sciences, University of Zurich, Winterthurerstr. 190, 8057, Zurich, Switzerland.
Correspondence should be addressed to Ruedi Aebersold aebersold@imsb.biol.ethz.ch We present a robust and general method for the identification and relative quantification of phosphorylation sites in complex protein mixtures. It is based on a new chemical derivatization strategy using a dendrimer as a soluble polymer support and tandem mass spectrometry (MS/MS). In a single step, phosphorylated peptides are covalently conjugated to a dendrimer in a reaction catalyzed by carbodiimide and imidazole. Modified phosphopeptides are released from the dendrimer via acid hydrolysis and analyzed by MS/MS. When coupled with an initial antiphosphotyrosine protein immunoprecipitation step and stable-isotope labeling, in a single experiment, we identified all known tyrosine phosphorylation sites within the immunoreceptor tyrosine-based activation motifs (ITAM) of the T-cell receptor (TCR) CD3 chains, and previously unknown phosphorylation sites on total 97 tyrosine phosphoproteins and their interacting partners in human T cells. The dynamic changes in phosphorylation were quantified in these proteins.
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