Comment in:
Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry.
Tao WA, Wollscheid B, O'Brien R, Eng JK, Li XJ, Bodenmiller B, Watts JD, Hood L, Aebersold R.
The Bindley Bioscience Center and Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907, USA.
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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PMID: 16094384 [PubMed - indexed for MEDLINE]