期刊
JOURNAL OF PROTEOME RESEARCH
卷 17, 期 1, 页码 243-251出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.jproteome.7b00546
关键词
phosphoproteomics; SH2 superbinder; monolithic capillary column; protein tyrosine phosphorylation; peptides; phosphopeptides; enrichment; microreactor; LC-MS/MS; protein complex
资金
- China State Key Basic Research Program [2016YFA0501402]
- National Natural Science Foundation of China [21605140, 81600046, 21535008, 21235006]
- National Science Fund of China for Distinguished Young Scholars [21525524]
In this study, we present a method to specifically capture phosphotyrosine (pTyr) peptides from minute amount of sample for the sensitive analysis of protein tyrosine phosphorylation. We immobilized SH2 superbinder on a monolithic capillary column to construct a microreactor to enrich pTyr peptides. It was found that the synthetic pTyr peptide could be specifically enriched by the microreactor from the peptide mixture prepared by spiking of the synthetic pTyr peptide into the tryptic digests of alpha-casein and beta-casein with molar ratios of 1:1000:1000. The microreactor was further applied to enrich pTyr peptides from pervanadate-treated HeLa cell digests for phosphoproteomics analysis, which resulted in the identification of 796 unique pTyr sites. In contrast, the conventional SH2 superbinder-based method identified 41 pTyr sites for the same sample, only 5.2% of the number achieved by the microreactor. Finally, this microreactor was also applied to analyze the pTyr in Shcl complex, an immunopurified protein complex, which resulted in the identification of 15 pTyr sites. Together, this technique is best fitted to analyze the pTyr in minute amount of sample and will have broad application in fields where only a limited amount of sample is available.
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