© BioMed Central Ltd 2003
Published: 9 January 2003
In the Early Edition of the Proceedings of the National Academy of Sciences Salomon et al. describe a sensitive method, exploiting multidimensional liquid chromatography/mass spectrometry, for measuring dynamic phosphotyrosine modifications (Proc Natl Acad Sci USA 2002, 10.1073 pnas.2436191100). The approach has several advantages over conventional two-dimensional gel electrophoresis. Whole cell extracts are first enriched for phosphotyrosine-containing polypeptides by immunoprecipitation using a specific anti-phosphotyrosine antibody. After tryptic digestion samples, are further enriched by methyl esterification and immobilized metal affinity chromatography. Reversed phase HPLC and tandem mass spectrometry allowed Salomon et al.unambiguously to assign many sites of tyrosine phosphorylation. They tested the technique by looking at tyrosine phosphorylation events following T-cell-receptor ligation in Jurkat cells and following BCR-Abl signaling in leukemia cells. A large number of the phosphotyrosine sites that they identified have previously been found using more traditional methods.
- Proceedings of the National Academy of Sciences , [http://www.pnas.org]
- Phosphoamino acid analysis.