title: Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry.
abstract: 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.
authors.affiliation: The Bindley Bioscience Center and Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907, USA.
authors.forename: W Andy
authors.initials: WA
authors.lastname: Tao
authors.forename: Bernd
authors.initials: B
authors.lastname: Wollscheid
authors.forename: Robert
authors.initials: R
authors.lastname: O'Brien
authors.forename: Jimmy K
authors.initials: JK
authors.lastname: Eng
authors.forename: Xiao-jun
authors.initials: XJ
authors.lastname: Li
authors.forename: Bernd
authors.initials: B
authors.lastname: Bodenmiller
authors.forename: Julian D
authors.initials: JD
authors.lastname: Watts
authors.forename: Leroy
authors.initials: L
authors.lastname: Hood
authors.forename: Ruedi
authors.initials: R
authors.lastname: Aebersold
mesh_terms: D002478:Cells, Cultured; D019281:Dimerization; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D015336:Molecular Probe Techniques; D010750:Phosphoproteins / Q000032:analysis* / Q000378:metabolism*; D020543:Proteome / Q000032:analysis* / Q000378:metabolism*; D013601:T-Lymphocytes / Q000378:metabolism*
chemical_list: D010750:Phosphoproteins; D020543:Proteome