Evaluation of enrichment techniques for mass spectrometry: identification of tyrosine phosphoproteins in cancer cells.

Phosphorylation of tyrosine residues by protein tyrosine kinases mediates numerous cellular processes. Deregulated tyrosine phosphorylation underlies constitutive activation of signaling pathways leading to oncogenesis. Analytical techniques for evaluation of the global phosphoproteome level are challenging and can be improved on to enhance yields. Here, we evaluated several approaches to enrich for tyrosine phosphoproteins in cancer cells for subsequent liquid chromatography-tandem mass spectrometry analysis using lysates from SU-DHL-1 cells, which express the nucleophosmin-anaplastic lymphoma kinase tyrosine kinase as a model system. Cells were grown in the presence or absence of the phosphatase inhibitor sodium orthovanadate, and tyrosine phosphoproteins were subsequently enriched by immunoprecipitation or immunoaffinity chromatography and protein identification performed by liquid chromatography-tandem mass spectrometry. Our results show that sodium orthovanadate improves enrichment and thus detection of tyrosine phosphoproteins. Immunoprecipitation of tyrosine phosphoproteins using two different antiphosphotyrosine antibodies increased the number of protein identifications. Finally, peptides from proteins enriched by immunoprecipitation were more abundant (n=338) than those enriched by immunoaffinity chromatography (n=138), and relatively few proteins were found in common (n=43). Our data demonstrate the utility of an enrichment strategy for the mass spectrometry-based identification of tyrosine phosphoproteins and show the advantage of complementary techniques for greater protein identification.