Sensitive, Robust, and Cost-Effective Approach for Tyrosine Phosphoproteome Analysis

被引:29
作者
Dong, Mingming [1 ,2 ]
Bian, Yangyang [1 ,3 ]
Wang, Yan [1 ,2 ]
Dong, Jing [1 ]
Yao, Yating [1 ,2 ]
Deng, Zhenzhen [1 ,2 ]
Qin, Hongqiang [1 ]
Zou, Hanfa [1 ]
Ye, Mingliang [1 ]
机构
[1] Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog R&A Ctr, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China
[2] Univ Chinese Acad Sci, Beijing 100049, Peoples R China
[3] Zhengzhou Univ, Affiliated Hosp 1, Med Res Ctr, Zhengzhou 450052, Henan, Peoples R China
基金
中国国家自然科学基金;
关键词
PHOSPHORYLATION SITES; SIGNALING PATHWAYS; ENRICHMENT; MEDIATORS; EGF;
D O I
10.1021/acs.analchem.7b02078
中图分类号
O65 [分析化学];
学科分类号
070302 ; 081704 ;
摘要
Albeit much less abundant than Ser/Thr phosphorylation (pSer/pThr), Tyr phosphorylation (pTyr) is considered as a hallmark in cellular signal transduction. However, its analysis at the proteome level remains challenging. The conventional immunopurification (IP) approach using antibodies specific to pTyr sites is known to have low sensitivity, poor reproducibility and high cost. Our recent study indicated that 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 presented an efficient SH2 superbinder based workflow for the sensitive analysis of tyrosine phosphoproteome. This new method can identify 41% more pTyr peptides than the previous'method. Its excellent performance was demonstrated by the analysis of a variety of different samples. For the highly tyrosine phosphorylated sample, for example, pervanadate-treated Jurkat T cells, it identified over 1800 high confident pTyr sites from only 2 mg of proteins. For the unstimulated Jurkat cells, where the pTyr events rarely occurred, it identified 343 high confident pTyr sites from 5 mg of proteins, which was 31% more than that obtained by the antibody-based method. For the heterogeneous sample of tissue, it identified 197 high confident pTyr sites from 5 mg protein digest of mouse skeletal muscle. In general, it is a sensitive, robust and cost-effective approach and would have wide applications in the study of the regulatory role of tyrosine phosphorylation in diverse physiological and pathological processes.
引用
收藏
页码:9307 / 9314
页数:8
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