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Integrated and High-Throughput Approach for Sensitive Analysis of Tyrosine Phosphoproteome
Analytical Chemistry ( IF 6.7 ) Pub Date : 2022-09-30 , DOI: 10.1021/acs.analchem.2c01807 Qian Kong 1, 2, 3 , Yicheng Weng 1, 2 , Zhendong Zheng 1, 2 , Wendong Chen 1 , Pengfei Li 1, 2, 4 , Zongwei Cai 3 , Ruijun Tian 1, 2, 4
Analytical Chemistry ( IF 6.7 ) Pub Date : 2022-09-30 , DOI: 10.1021/acs.analchem.2c01807 Qian Kong 1, 2, 3 , Yicheng Weng 1, 2 , Zhendong Zheng 1, 2 , Wendong Chen 1 , Pengfei Li 1, 2, 4 , Zongwei Cai 3 , Ruijun Tian 1, 2, 4
Affiliation
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Tyrosine phosphorylation (pTyr) regulates various signaling pathways under normal and cancerous states. Due to their low abundance and transient and dynamic natures, systematic profiling of pTyr sites is challenging. Antibody and engineered binding domain-based approaches have been well applied to pTyr peptide enrichment. However, traditional methods have the disadvantage of a long sample preparation process, which makes them unsuitable for processing limited amount of samples, especially in a high-throughput manner. In this study we developed a 96-well microplate-based approach to integrate all the sample preparation steps starting from cell culture to MS-compatible pTyr peptide enrichment in three consecutive 96-well microplates. By assembling an engineered SH2 domain onto a microplate, nonspecific adsorption of phosphopeptides is greatly reduced, which allows us to remove the Ti-IMAC purification and three C18 desalting steps (after digestion, pTyr enrichment, and Ti-IMAC purification) and, therefore, greatly simplifies the entire pTyr peptide enrichment workflow, especially when processing a large number of samples. Starting with 96-well microplate-cultured, pervanadate-stimulated cells, our approach could enrich 21% more pTyr sites than the traditional serial pTyr enrichment approach and showed good sensitivity and reproducibility in the range of 200 ng to 200 μg peptides. Importantly, we applied this approach to profile tyrosine kinase inhibitor-mediated EGFR signaling pathway and could well differentiate the distinct response of different pTyr sites. Collectively, the integrated 96-well microplate-based approach is valuable for profiling pTyr sites from limited biological samples and in a high-throughput manner.
中文翻译：
用于酪氨酸磷酸蛋白质组敏感分析的集成和高通量方法
酪氨酸磷酸化 (pTyr) 在正常和癌变状态下调节各种信号通路。由于它们的低丰度和瞬态和动态性质，pTyr 位点的系统分析具有挑战性。基于抗体和工程结合域的方法已很好地应用于 pTyr 肽富集。然而，传统方法的缺点是样品制备过程长，这使得它们不适合处理有限量的样品，尤其是在高通量的情况下。在这项研究中，我们开发了一种基于 96 孔微孔板的方法，将所有样品制备步骤从细胞培养到与 MS 兼容的 pTyr 肽富集整合到三个连续的 96 孔微孔板中。通过将工程化 SH2 结构域组装到微孔板上，磷酸肽的非特异性吸附大大减少，这使我们能够去除 Ti-IMAC 纯化和三个 C18 脱盐步骤（消化后、pTyr 富集和 Ti-IMAC 纯化后），因此大大简化了整个 pTyr 肽富集工作流程，尤其是在处理大量样本时。从 96 孔微孔板培养的过钒酸盐刺激细胞开始，我们的方法可以比传统的连续 pTyr 富集方法多富集 21% 的 pTyr 位点，并在 200 ng 至 200 μg 肽的范围内显示出良好的灵敏度和重现性。重要的是，我们应用这种方法来分析酪氨酸激酶抑制剂介导的 EGFR 信号通路，并且可以很好地区分不同 pTyr 位点的不同反应。集体，
更新日期：2022-09-30
中文翻译：
用于酪氨酸磷酸蛋白质组敏感分析的集成和高通量方法
酪氨酸磷酸化 (pTyr) 在正常和癌变状态下调节各种信号通路。由于它们的低丰度和瞬态和动态性质，pTyr 位点的系统分析具有挑战性。基于抗体和工程结合域的方法已很好地应用于 pTyr 肽富集。然而，传统方法的缺点是样品制备过程长，这使得它们不适合处理有限量的样品，尤其是在高通量的情况下。在这项研究中，我们开发了一种基于 96 孔微孔板的方法，将所有样品制备步骤从细胞培养到与 MS 兼容的 pTyr 肽富集整合到三个连续的 96 孔微孔板中。通过将工程化 SH2 结构域组装到微孔板上，磷酸肽的非特异性吸附大大减少，这使我们能够去除 Ti-IMAC 纯化和三个 C18 脱盐步骤（消化后、pTyr 富集和 Ti-IMAC 纯化后），因此大大简化了整个 pTyr 肽富集工作流程，尤其是在处理大量样本时。从 96 孔微孔板培养的过钒酸盐刺激细胞开始，我们的方法可以比传统的连续 pTyr 富集方法多富集 21% 的 pTyr 位点，并在 200 ng 至 200 μg 肽的范围内显示出良好的灵敏度和重现性。重要的是，我们应用这种方法来分析酪氨酸激酶抑制剂介导的 EGFR 信号通路，并且可以很好地区分不同 pTyr 位点的不同反应。集体，
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