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

Nat Methods. 2005 Aug;2(8):591-8. doi: 10.1038/nmeth776.


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 types

  • Evaluation Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Cells, Cultured
  • Dimerization
  • Gene Expression Profiling / methods*
  • Humans
  • Mass Spectrometry / methods*
  • Molecular Probe Techniques*
  • Phosphoproteins / analysis*
  • Phosphoproteins / metabolism*
  • Proteome / analysis*
  • Proteome / metabolism*
  • T-Lymphocytes / metabolism*


  • Phosphoproteins
  • Proteome