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.
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Acknowledgements
This project has been funded in part with Federal funds from the National Heart, Lung and Blood Institute, National Institutes of Health, under contract No. N01-HV-28179. W.A.T. is a Damon Runyon Fellow supported by the Damon Runyon Cancer Research Foundation (DRG 1740-02).
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Supplementary Table 1
Phosphorylated peptides from immuno-purified Jurkat lysates. (PDF 64 kb)
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Tao, W., Wollscheid, B., O'Brien, R. et al. Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry. Nat Methods 2, 591–598 (2005). https://doi.org/10.1038/nmeth776
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DOI: https://doi.org/10.1038/nmeth776
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