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Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry

Nature Methods volume 2, pages 591–598 (2005)Cite this article

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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Figure 1: Isolation and tagging of phosphopeptides, and validation with β-casein digests.
Figure 2: Quantitation of phosphopeptides in standard protein mixtures.
Figure 3: Quantitative phosphopeptide analysis.
Figure 4: Abundance ratios of tyrosine phosphopeptides isolated from T cells treated with pervanadate over 10 min or 2 min.

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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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Authors and Affiliations

  1. The Bindley Bioscience Center and Department of Biochemistry, Purdue University, West Lafayette, 47907, Indiana, USA

    W Andy Tao

  2. Institute for Systems Biology, 1441 North 34th Street, Seattle, 98103, Washington, USA

    W Andy Tao, Bernd Wollscheid, Robert O'Brien, Jimmy K Eng, Xiao-jun Li, Julian D Watts, Leroy Hood & Ruedi Aebersold

  3. Institute for Molecular Systems Biology, Federal Institute of Technology, Campus Hoenggerberg, HPT E78, Wolfgang Pauli Strasse 16, Zurich, CH-8093, Switzerland

    Bernd Bodenmiller & Ruedi Aebersold

  4. Faculty of Natural Sciences, University of Zurich, Winterthurerstr. 190, Zurich, 8057, Switzerland

    Ruedi Aebersold

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Supplementary information

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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