Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA.
Department of Biochemistry, Duke University, Durham, NC, USA.
Methods Mol Biol. 2021;2259:247-257. doi: 10.1007/978-1-0716-1178-4_16.
Protein phosphorylation is a critical posttranslational modification (PTM), with cell signaling networks being tightly regulated by protein phosphorylation. Despite recent technological advances in reversed-phase liquid chromatography (RPLC)-mass spectrometry (MS)-based proteomics, comprehensive phosphoproteomic coverage in complex biological systems remains challenging, especially for hydrophilic phosphopeptides that often have multiple phosphorylation sites. Herein, we describe an MS-based phosphoproteomics protocol for effective quantitative analysis of hydrophilic phosphopeptides. This protocol was built upon a simple tandem mass tag (TMT)-labeling method for significantly increasing peptide hydrophobicity, thus effectively enhancing RPLC-MS analysis of hydrophilic peptides. Through phosphoproteomic analyses of MCF7 cells, this method was demonstrated to greatly increase the number of identified hydrophilic phosphopeptides and improve MS signal detection. With the TMT labeling method, we were able to identify a previously unreported phosphopeptide from the G protein-coupled receptor (GPCR) CXCR3, QPpSSSR, which is thought to be important in regulating receptor signaling. This protocol is easy to adopt and implement and thus should have broad utility for effective RPLC-MS analysis of the hydrophilic phosphoproteome as well as other highly hydrophilic analytes.
蛋白质磷酸化是一种关键的翻译后修饰（PTM），细胞信号网络受到蛋白质磷酸化的严格调控。尽管反相液相色谱（RPLC）-质谱（MS）为基础的蛋白质组学技术最近取得了进展，但在复杂的生物系统中进行全面的磷酸蛋白质组学覆盖仍然具有挑战性，特别是对于具有多个磷酸化位点的亲水性磷酸肽。在此，我们描述了一种基于 MS 的磷酸蛋白质组学方法，用于有效定量分析亲水性磷酸肽。该方案建立在简单的串联质量标签（TMT）标记方法的基础上，该方法可显著增加肽的疏水性，从而有效增强亲水性肽的 RPLC-MS 分析。通过 MCF7 细胞的磷酸蛋白质组学分析，该方法极大地增加了鉴定的亲水性磷酸肽的数量，并提高了 MS 信号的检测。通过 TMT 标记方法，我们能够鉴定出 G 蛋白偶联受体（GPCR）CXCR3 中的一个以前未报道的磷酸肽 QPpSSSR，该磷酸肽被认为在调节受体信号中很重要。该方案易于采用和实施，因此应该在有效 RPLC-MS 分析亲水性磷酸蛋白质组以及其他高度亲水性分析物方面具有广泛的应用。
