Dataset Information

Quantative pTyr protein complex

ABSTRACT: Our current research focuses on the complex-centric quantification of pTyr protein complexes. We initially constructed a tri-functional probe based on pTyr superbinder (SH2-S) for the enrichment of pTyr protein complexes. The pTyr protein complexes were separated by ion exchange chromatography (IEC). To further quantify the complexes, we developed an algorithm for co-fractionation/mass spectrometry (CF/MS) protein complex screening and quantification.

ORGANISM(S): Homo Sapiens

SUBMITTER: Mingxia Gao

PROVIDER: PXD052742 | iProX | Fri May 31 00:00:00 BST 2024

REPOSITORIES: iProX

ACCESS DATA

Publications

Integrated Platform for Large-Scale Quantitative Profiling of Phosphotyrosine Signaling Complexes Based on Cofractionation/Mass Spectrometry and Complex-Centric Algorithm.

Liu Wei W Wang Xuantang X Yu Hailong H Yan Guoquan G Shen Shun S Gao Mingxia M Zhang Xiangmin X

Analytical chemistry 20240605 24

The scarcity and dynamic nature of phosphotyrosine (pTyr)-modified proteins pose a challenge for researching protein complexes with pTyr modification, which are assembled through multiple protein-protein interactions. We developed an integrated complex-centric platform for large-scale quantitative profiling of pTyr signaling complexes based on cofractionation/mass spectrometry (CoFrac-MS) and a complex-centric algorithm. We initially constructed a trifunctional probe based on pTyr superbinder (S ...[more]

PMID: 38836774

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

Quantative pTyr protein complex

Publications

Integrated Platform for Large-Scale Quantitative Profiling of Phosphotyrosine Signaling Complexes Based on Cofractionation/Mass Spectrometry and Complex-Centric Algorithm.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets