Wang Yuqiu, Gao Jing, Xie Wenfan, Tang Minchu, Chen Xin, Chen Lv, Chen Hongxu, Yang Zhicheng, Gao Qiang, Liu Yansheng, Zhou Hu
Department of Otolaryngology, Eye & ENT Hospital, Fudan University, Shanghai 200031, China.
Department of Analytical Chemistry, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
J Proteome Res. 2025 May 2;24(5):2443-2453. doi: 10.1021/acs.jproteome.4c01091. Epub 2025 Apr 10.
Phosphorylation (-linked and -linked) plays an important role in biological functions and cell signaling. Here, we employed a one-dimensional online alkaline-pH reversed-phase nanoelectrospray-tandem mass spectrometry (alkaline-pH-MS/MS) for the investigation of global phosphorylation. In this method, phosphopeptides were separated on a nanoflow C18 column with an alkaline-pH gradient and directly introduced to the mass spectrometer through nanoelectrospray ionization. Although the phosphosites and phosphopeptides identified by alkaline-pH-MS/MS were slightly lower than those of traditional online low-pH reversed-phase tandem MS (low-pH-MS/MS), these two methods were highly complementary to each other. This alkaline-pH-MS/MS may affect the actual polarity and CSD of phosphopeptides, consequently improving the identification of multiply phosphorylated peptides. Moreover, alkaline-pH-MS/MS was compatible with other peptide fractionation and phosphopeptide enrichment techniques, such as offline high-pH or low-pH reversed-phase liquid chromatography fractionation. The complementarity of alkaline-pH-MS/MS and low-pH-MS/MS was further demonstrated by the tandem mass tag (TMT)-based quantitative phosphoproteomic analysis of five pairs of hepatocellular carcinoma (HCC) tumors and normal adjacent tissues (NATs). Furthermore, unique information on significantly changed phosphosites was observed by alkaline-pH-MS/MS. This study provided an alternative and complementary tool for global analysis of both - and -phosphoproteome, which may be beneficial for the discovery of phosphoproteins with significant biological functions.
磷酸化（-连接和-连接）在生物功能和细胞信号传导中起着重要作用。在此，我们采用一维在线碱性pH反相纳米电喷雾串联质谱（碱性pH-MS/MS）来研究全局磷酸化。在该方法中，磷酸肽在具有碱性pH梯度的纳流C18柱上分离，并通过纳米电喷雾电离直接引入质谱仪。尽管碱性pH-MS/MS鉴定出的磷酸位点和磷酸肽略低于传统的在线低pH反相串联质谱（低pH-MS/MS），但这两种方法相互高度互补。这种碱性pH-MS/MS可能会影响磷酸肽的实际极性和CSD，从而提高对多重磷酸化肽的鉴定。此外，碱性pH-MS/MS与其他肽段分级分离和磷酸肽富集技术兼容，如离线高pH或低pH反相液相色谱分级分离。通过对五对肝细胞癌（HCC）肿瘤组织和正常相邻组织（NAT）进行基于串联质量标签（TMT）的定量磷酸化蛋白质组学分析，进一步证明了碱性pH-MS/MS和低pH-MS/MS的互补性。此外，通过碱性pH-MS/MS观察到了显著变化的磷酸位点的独特信息。本研究为全局分析-和-磷酸化蛋白质组提供了一种替代和互补的工具，这可能有利于发现具有重要生物学功能的磷酸化蛋白质。
