Dataset Information

Evaluation of phospho-tyrosine antibodies for label-free phosphoproteomics

ABSTRACT: 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.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Dr Connie Ramona Jimenez

PROVIDER: MSV000080270 | MassIVE | Fri Oct 21 16:54:00 BST 2016

SECONDARY ACCESSION(S): PXD001565

REPOSITORIES: MassIVE

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Evaluation of different phospho-tyrosine antibodies for label-free phosphoproteomics.

van der Mijn Johannes C JC Labots Mariette M Piersma Sander R SR Pham Thang V TV Knol Jaco C JC Broxterman Henk J HJ Verheul Henk M HM Jiménez Connie R CR

Journal of proteomics 20150416 Pt B

<h4>Background</h4>Mass spectrometry based phosphoproteomics emerged as advantageous approach for the analysis of tyrosine phosphorylation on proteins and tyrosine kinase signaling. Immunoaffinity purification is required for comprehensive analysis. Here we compared the performance of two antibodies for label-free phosphotyrosine-based phosphoproteomics.<h4>Methods</h4>Phosphopeptide immunoprecipitation of six technical replicates corresponding to 10mg protein from HCT116 cells was performed usi ...[more]

PMID: 25890253

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