Chen Xiulan, Wei Shasha, Yang Fuquan
Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
University of Chinese Academy of Sciences, Beijing, China.
Methods Mol Biol. 2025;2884:259-278. doi: 10.1007/978-1-0716-4298-6_17.
Protein phosphorylation is an important post-translational modification that regulates almost all cellular processes, such as cellular metabolism, growth, differentiation, signal transduction, and gene regulation. Mass spectrometry, which acts as an automated and sensitive method, enables global analysis of protein phosphorylation. However, several technical challenges need to be addressed when analyzing protein phosphorylation in a global manner. Low-abundant phosphopeptides need to be enriched before analysis with LC-MS/MS, so specific enrichment of phosphopeptides is central for a successful analysis of the phosphoproteome. Due to the complexity of phosphoproteome, fractionation of phosphopeptides before LC-MS/MS is essential to increase it coverage. Here, we present a detailed protocol for in-depth analysis of tissue phosphoproteome, including collection of tissue samples, extraction of tissue proteins, proteolytic digestion of proteins into peptides, enrichment of phosphopeptides with TiO using lactic acid as non-phosphopeptide excluder, fractionation of phosphopeptides with TEA-based high-pH reversed-phase (HpH-RP) chromatography, and identification of phosphopeptides with LC-MS/MS. We also outline the essential steps for data processing.
蛋白质磷酸化是一种重要的翻译后修饰，几乎调节所有细胞过程，如细胞代谢、生长、分化、信号转导和基因调控。质谱作为一种自动化且灵敏的方法，能够对蛋白质磷酸化进行全局分析。然而，以全局方式分析蛋白质磷酸化时需要解决几个技术挑战。低丰度磷酸肽在通过液相色谱-串联质谱（LC-MS/MS）分析之前需要进行富集，因此磷酸肽的特异性富集是成功分析磷酸化蛋白质组的关键。由于磷酸化蛋白质组的复杂性，在LC-MS/MS之前对磷酸肽进行分级分离对于提高其覆盖率至关重要。在此，我们展示了一个用于深入分析组织磷酸化蛋白质组的详细方案，包括组织样本的收集、组织蛋白的提取、蛋白质酶解成肽、使用乳酸作为非磷酸肽排除剂通过二氧化钛（TiO）富集磷酸肽、基于三乙胺（TEA）的高pH值反相（HpH-RP）色谱对磷酸肽进行分级分离以及通过LC-MS/MS鉴定磷酸肽。我们还概述了数据处理的基本步骤。
