In the past decade multiple tyrosine kinase inhibitors (TKIs) have been implemented in standard treatment regimens for patients with cancer. Unfortunately the majority of patients develops resistance to these drugs. Reliable tools for analysis of pharmacodynamic effects and drug resistance mechanisms are therefore warranted. Phosphoproteomics has meanwhile emerged as tool for the analysis of tyrosine protein phosphorylation. These studies rely on antibodies for enrichment of tyrosine-phosphorylated peptides. Here we compared two commercially available phosphotyrosine antibodies and show that P-Tyr-1000 yields 64% more phosphopeptides than the 4G10 antibody. To investigate performance of P-Tyr-1000 in a label-free comparative phosphoproteomics analysis, a commonly used cell culture model was employed. U87 glioma cells with or without EGFRvIII mutation, lacking the extracellular ligand binding domains (exon 2 - 7) and displaying constitutive signaling activity, were analyzed with the workflow described above. U87 cells and the isogenic version with EGFRvIII were both treated with or without the TKI Erlotinib. In total 1330 phosphopeptides were detected derived from 683 phosphorylated proteins.
[dataset license: CC0 1.0 Universal (CC0 1.0)]
Keywords: Human ; label-free ; single-shot ; phosphoproteomics ; phosphotyrosine ; pTyr IP ; glioblastoma ; U87
Principal Investigators: (in alphabetical order) |
Dr Connie Ramona Jimenez |
Submitting User: | ccms |
van der Mijn JC, Labots M, Piersma SR, Pham TV, Knol JC, Broxterman HJ, Verheul HM, Jiménez CR.
Evaluation of different phospho-tyrosine antibodies for label-free phosphoproteomics.
J Proteomics. 2015 Sep 8;127(Pt B):259-63. Epub 2015 Apr 16.
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