In the Early Edition of the
The approach has several advantages over conventional 2D-gel electrophoresis techniques. Whole cell extracts are first enriched for phosphotyrosine-containing polypeptides by immunoprecipitation using a specific anti-phosphotyrosine antibody. After tryptic digestion, samples were further enriched by methyl esterification and immobilized metal affinity chromatography. Reversed phase HPLC and tandem mass spectrometry methods allowed unambiguous assignment of many sites of tyrosine phosphorylation.
Salomon et al. tested the technique by looking at tyrosine phosphorylation events following T cell receptor ligation in Jurkat cells or BCR-ABL signaling in leukemia cells. A large number of the phosphotyrosine sites that they identified have been previously found using more traditional methods.