Dataset Information

Identification of phosphotyrosine (pTyr)-containing T cell proteins

ABSTRACT: This project aimed at identifying phosphotyrosine (pTyr)-containing T cell proteins that bind directly to the pTyr-binding pocket in the N-terminal C2 domain of protein kinase C-theta. We used an immobilized recombinant GST-C2 fusion protein to pull-down proteins present in lysates of stimulated human leukemic Jurkat T cell line. After eluting and separating C2-bound proteins by SDS-PAGE, protein bands in the ~60-70 kDa range detected by Coomassie blue staining or anti-pTr immunoblotting were excised from the gel, destained, and subjected to trypsinization. The resulting peptides were extracted, dried, dissolved in 0.1% formic acid, and analyzed by online nano flow LC-MS/MS.

INSTRUMENT(S): LTQ Orbitrap, 1200 series LC/MSD SL

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

SUBMITTER: Amnon Altman

PROVIDER: MSV000083562 | MassIVE | Wed Mar 13 11:19:00 GMT 2019

REPOSITORIES: MassIVE

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

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.

Dataset Information

Identification of phosphotyrosine (pTyr)-containing T cell proteins

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets