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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.
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