SH2 Superbinder Modified Monolithic Capillary Column for the Sensitive Analysis of Protein Tyrosine Phosphorylation
SH2 Superbinder Modified Monolithic Capillary Column for the Sensitive Analysis of Protein Tyrosine Phosphorylation
复制标题
SH2 Superbinder 改性整体毛细管柱用于蛋白质酪氨酸磷酸化的灵敏分析
DOI：
10.1021/acs.jproteome.7b00546
复制
发表时间：
2018
影响因子：
4.4
通讯作者：
Ye Mingliang
中科院分区：
文献类型：
--
作者：
Yao Yating;Bian Yangyang;Dong Mingming;Wang Yan;Lv Jiawen;Chen Lianfang;Wang Hongwei;Mao Jiawei;Dong Jing;Ye Mingliang
在这项研究中，我们提出了一种从微量样品中特异捕获磷酸酪氨酸(PTyr)多肽的方法，用于蛋白质酪氨酸磷酸化的灵敏分析。我们将SH2超结合剂固定在整体毛细管柱上，构建了一个浓缩pTyr多肽的微反应器。结果表明，在α-酪蛋白和β-酪蛋白(摩尔比为1：1000：1000)的胰酶消化液中加入合成的pTyr多肽，可使合成的pTyr多肽被微反相特异地浓缩。将该微反应器进一步用于从过钒酸盐处理的HeLa细胞消化液中富集pTyr多肽进行磷酸蛋白质组学分析，结果鉴定出796个独特的pTyr位点。相比之下，传统的基于SH2超结合剂的方法为同一样本确定了41个pTyr位点，仅为微反应器所获得数量的5.2%。最后，利用该微反应器对免疫纯化的蛋白复合体Shc1中的pTyr进行了分析，鉴定出15个pTyr位点。总而言之，这项技术最适合于微量样品的分析，在样品数量有限的领域将有广泛的应用。
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 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. 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.