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- Title
Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry.
- Authors
Tao, W Andy; Wollscheid, Bernd; O'Brien, Robert; Eng, Jimmy K; Li, Xiao-jun; Bodenmiller, Bernd; Watts, Julian D; Hood, Leroy; Aebersold, Ruedi
- 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.
- Publication
Nature methods, 2005, Vol 2, Issue 8, p591
- ISSN
1548-7091
- Publication type
Journal Article
- DOI
10.1038/nmeth776