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 (human)

TISSUE(S): Brain, Epithelial Cell, Colon

DISEASE(S): Brain Glioblastoma Multiforme

SUBMITTER: Sander Piersma

LAB HEAD: Connie Ramona Jimenez

PROVIDER: PXD001565 | Pride | 2015-12-09

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	MaxQuantP-Tyr-1000vs4G10.zip	Other
	MaxQuant_U87.zip	Other
	QE2_130305_OPL1013_U87pTyrIP_KM_mut_05.raw	Raw
	QE2_130305_OPL1013_U87pTyrIP_KM_mut_Erl_06.raw	Raw
	QE2_130305_OPL1013_U87pTyrIP_KM_wt_03.raw	Raw

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Publications

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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Project description:Albeit much less abundant than Ser/Thr phosphorylation (pSer/pThr), Tyr phosphorylation (pTyr) is considered a hallmark in cellular signal transduction. However, its analysis remains a challenge. The conventional immunopurification (IP) approach using antibodies pan-specific to pTyr sites is known to have low sensitivity, poor reproducibility and high cost. SH2 domain-derived pTyr-superbinder is a good replacement of pTyr antibody for the specific enrichment of pTyr peptides for phosphoproteomics analysis. In this study, we aim to optimize the approach using SH2 superbinder for tyrosine phosphoproteome analysis. The present work covalently immobilized SH2 superbinders to agrose beads, designed and evaluated four different SH2 superbinder based experimental workflows for phosphotyrosine analysis. After made a head to head comparison of them, we observed that the combined strategy employing Ti4+-IMAC and covalently immobilized SH2 superbinder enrichment in sequence generated the largest pTyr peptide dataset (3519) with the best selectivity (90%). Next the optimal method was applied for pTyr profiling from normal mouse tissues, and resulted in the identification of 197 pTyr sites (of which 73 were novel) from 5 mg protein digests, which validated its high sensitivity. An comparison with antibody 4G10 by isolating pTyr peptides from 5 mg unstimulated Jurkat cell digests were made, our optimal strategy identified 343 while the antibody 4G10 based method identified 242 pTyr sites, respectively, further confirmed the robustness of the method. Finally, the optimal strategy was applied for quantitative pTyr phosphorylation analysis of EGF stimulated/unstimulated HeLa cells, 261 pTyr sites were successfully quantified from 5 mg stable-isotope dimethyl labling protein digests. In general, the combination of Ti4+-IMAC and SH2 superbinder enrichment in sequence generated a facial and robust strategy for qualitative and quantitative analysis of pTyr proteome.