Sensitive, Robust, and Cost-Effective Approach for Tyrosine Phosphoproteome Analysis
Sensitive, Robust, and Cost-Effective Approach for Tyrosine Phosphoproteome Analysis
复制标题
灵敏、稳健且经济高效的酪氨酸磷酸化蛋白质组分析方法
DOI：
10.1021/acs.analchem.7b02078
复制
发表时间：
2017-09-05
影响因子：
7.4
通讯作者：
Ye, Mingliang
中科院分区：
文献类型：
--
作者：
Dong, Mingming;Bian, Yangyang;Ye, Mingliang
尽管Tyr磷酸化（pTyr）的丰度远低于Ser/Thr磷酸化（pSer/pThr），但它被认为是细胞信号转导中的标志。然而，其在蛋白质组水平的分析仍然具有挑战性。已知使用对pTyr位点具有特异性的抗体的常规免疫纯化（IP）方法具有低灵敏度、差再现性和高成本。我们最近的研究表明，SH 2结构域衍生的pTyr-superbinder是一个很好的替代pTyr抗体的特异性富集pTyr肽磷酸蛋白质组学分析。在这项研究中，我们提出了一个有效的SH 2 superbinder为基础的工作流程，酪氨酸磷酸化蛋白质组的灵敏度分析。该方法比原方法多识别41%的pTyr肽。通过对各种不同样品的分析，证明了其优异的性能。对于高度酪氨酸磷酸化的样品，例如，过钒酸盐处理的Jurkat T细胞，其仅从2 mg蛋白质中鉴定出超过1800个高置信度的pTyr位点。对于pTyr事件很少发生的未刺激的Jurkat细胞，它从5 mg蛋白质中鉴定出343个高置信度的pTyr位点，这比通过基于抗体的方法获得的多31%。对于组织的异质样品，其从小鼠骨骼肌的5 mg蛋白质消化物中鉴定出197个高置信度pTyr位点。总的来说，它是一种灵敏、稳健和具有成本效益的方法，在研究酪氨酸磷酸化在各种生理和病理过程中的调节作用方面具有广泛的应用。
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.