In this study, we presents 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 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 α-casein and β-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 Shc1 complex, an immunopurified protein complex, which resulted in the identification of 15 pTyr sites. Bring together, this technique is best fitted to analyze the pTyr in minute amount of sample and will have broad application in fields where only limited amount of sample is available.

中文翻译：

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SH2超级结合修饰整体毛细管柱用于蛋白质酪氨酸磷酸化的灵敏分析。

在这项研究中，我们提出了一种从微量样品中特异性捕获磷酸酪氨酸（pTyr）肽的方法，用于蛋白质酪氨酸磷酸化的灵敏分析。我们将SH2超级粘合剂固定在整体毛细管柱上，以构建微反应器以富集pTyr肽。已经发现，通过微反应器可以从肽混合物中特异性富集合成的pTyr肽，该肽混合物是通过将合成的pTyr肽以1：1000：1000的摩尔比掺入α-酪蛋白和β-酪蛋白的胰蛋白酶消化物中而制得的。将该微反应器进一步应用于从过钒酸盐处理的HeLa细胞消化物中富集pTyr肽，以进行磷酸化蛋白质组学分析，从而鉴定了796个独特的pTyr位点。相比之下，传统的基于SH2超级结合剂的方法可为同一样品确定41个pTyr位点，微型反应器的数量仅为其的5.2％。最后，该微反应器还用于分析Shc1复合物（一种免疫纯化的蛋白质复合物）中的pTyr，从而鉴定了15个pTyr位点。综上所述，该技术最适合分析微量样品中的pTyr，并将在只有有限数量样品的领域中得到广泛应用。