Development of novel affinity reagents for detecting protein tyrosine phosphorylation based on superbinder SH2 domain in tumor cells
Development of novel affinity reagents for detecting protein tyrosine phosphorylation based on superbinder SH2 domain in tumor cells
复制标题
开发新型亲和试剂，用于检测肿瘤细胞中基于 superbinder SH2 结构域的蛋白质酪氨酸磷酸化。
DOI：
10.1016/j.aca.2018.05.045
复制
发表时间：
2018-11-22
影响因子：
6.2
通讯作者：
Cao, Xuan
中科院分区：
文献类型：
--
作者：
Ke, Ai-Qing;Liu, An-Dong;Cao, Xuan
酪氨酸磷酸化作为细胞信号转导的标志，对细胞增殖、代谢、运动和生存等多种细胞过程都很重要。酪氨酸异常磷酸化在许多疾病，特别是癌症中起着重要的作用。检测癌细胞或组织中的蛋白磷酸化状态对于评估病理阶段、发现癌症生物标志物和确定药物靶点至关重要。然而，常见的生化检测方法仍然是通过抗ptyr抗体，这种方法灵敏度有限，重现性差，成本高。近年来的研究证明，在磷酸化蛋白质组学分析中，超结合物SH2结构域是抗pTyr抗体的良好替代品，可用于特异性富集pTyr肽。在这项工作中，我们开发了一系列基于Src蛋白超结合剂SH2的亲和试剂，用于检测含有ptyrr的蛋白，以取代免疫印迹和免疫荧光技术中的抗py抗体。HRP-sSH2和EGFP-sSH2的优异性能通过对几种不同肿瘤细胞样本的分析得到验证，并与最常用的商业抗体进行了比较。EGFP-sSH2-(Arg)(9)可作为探针，有效穿透细胞膜，特异结合pTyr蛋白，在活细胞中进行直接荧光成像。综上所述，我们开发了三种新颖、方便、灵敏、经济的亲和试剂，在蛋白质酪氨酸磷酸化分析中具有广泛的应用前景，可用于肿瘤研究和临床诊断。(C) 2018 Elsevier B.V.版权所有
Tyrosine phosphorylation, as a hallmark in cellular signal transduction, is important for a diverse array of cellular processes, such as proliferation, metabolism, motility, and survival. Aberrant tyrosine phosphorylation plays a causal role in many diseases, especially the cancer. Detecting protein phosphorylation status in the cancer cells or tissues is vital for assessing the pathological phase, discovering the cancer biomarkers, and identifying the drug targets. However, the common biochemical detection methods remain through anti-pTyr antibodies, which are known to have limited sensitivity, poor reproducibility and high cost. Recent studies have proved that superbinder SH2 domain is a good replacement of anti-pTyr antibodies for the specific enrichment of pTyr peptides in phosphoproteomics analysis. In this work, we exploited a series of affinity reagents based on superbinder SH2 derived from Src protein for detecting the pTyr-containing proteins to replace anti-pY antibodies in immunoblotting and immunofluorescence techniques. The excellent performance of HRP-sSH2 and EGFP-sSH2 was verified by the analysis of several different tumor cell samples and was compared with most commonly used commercial antibodies. EGFP-sSH2-(Arg)(9) might be applied as the probe for direct fluorescence imaging in live cells via efficiently penetrating cell membranes and specifically binding with pTyr proteins. In summary, we have developed three novel, convenient, sensitive, and cost-effective affinity reagents that would have wide applications in protein tyrosine phosphorylation analysis for the tumor research and clinical diagnosis. (C) 2018 Elsevier B.V. All rights reserved.