PXD005838-1

PXD005838 is an original dataset announced via ProteomeXchange.

Title	A sensitive and cost-effective approach for tyrosine phosphoproteome analysis based on SH2 superbinder
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.
HostingRepository	PRIDE
AnnounceDate	2017-08-29
AnnouncementXML	Submission_2017-08-29_06:14:35.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Mingming Dong
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090; scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	phosphorylated residue; monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2017-02-02 07:02:03	ID requested
⏵ 1	2017-08-29 06:14:36	announced
2	2024-10-22 04:36:10	announced	2024-10-22: Updated project metadata.

Dong M, Bian Y, Wang Y, Dong J, Yao Y, Deng Z, Qin H, Zou H, Ye M, Sensitive, Robust, and Cost-Effective Approach for Tyrosine Phosphoproteome Analysis. Anal Chem, 89(17):9307-9314(2017) [pubmed]

curator keyword: Technical

submitter keyword: SH2 superbinder, pTyr phosphoproteome, LC MS/MS

Mingliang Ye
contact affiliation	Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023, China
contact email	mingliang@dicp.ac.cn
lab head
Mingming Dong
contact affiliation	DICP,CAS
contact email	dongmm@dicp.ac.cn
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD005838
     1. Label: PRIDE project
     2. Name: A sensitive and cost-effective approach for tyrosine phosphoproteome analysis based on SH2 superbinder