Lee Gil Ya Cancer and Diabetes Institute, Gachon University, 7-45 Songdo-dong, Yeonsu-ku, Incheon, 406-840, Republic of Korea.
Rapid Commun Mass Spectrom. 2013 Dec 30;27(24):2767-76. doi: 10.1002/rcm.6742.
Protein post-translational modifications (PTMs) are directly involved in protein function and cellular activities. Among them, glycosylation and phosphorylation are particularly important modifications on proteins located at extracellular and intracellular domains, respectively. However, the combined detection using phospho- and glycoproteomics is limited mainly due to protocol differences.
In this study, we developed a novel method for both phospho- and glycoproteome detection from a single sample batch, in which a titanium dioxide cartridge was used to capture the phosphoproteome, and the flow-through solution was processed for capturing N-linked glycopeptides using hydrazide resin.
By using 1 mg of protein from kidney tissue lysates from normal and diseased rats, we concurrently identified 437 glycosites/358 phosphosites and 468 glycosites/369 phosphosites in normal and disease kidneys, respectively, by liquid chromatography/tandem mass spectrometric analysis.
Compared with individual PTM analyses, the combined PTM analysis clearly provides more broad implications for PTMs related to the pathological status and discovery of biomarker candidates. Furthermore, the combined protocol thoroughly showed its advantages in enrichment efficiency and biological interpretation compared with current methods.
蛋白质翻译后修饰（PTMs）直接参与蛋白质功能和细胞活动。其中，糖基化和磷酸化分别是位于细胞外和细胞内结构域的蛋白质上特别重要的修饰。然而，由于方案差异，联合使用磷酸化和糖蛋白质组学的检测受到限制。
在这项研究中，我们开发了一种从单个样品批次同时检测磷酸化和糖蛋白质组的新方法，其中使用二氧化钛小柱捕获磷酸蛋白质组，而流穿溶液则使用酰肼树脂处理以捕获 N 连接的糖肽。
通过使用来自正常和患病大鼠肾脏组织裂解物的 1 毫克蛋白质，我们通过液相色谱/串联质谱分析分别在正常和患病肾脏中同时鉴定出 437 个糖基化位点/358 个磷酸化位点和 468 个糖基化位点/369 个磷酸化位点。
与单独的 PTM 分析相比，联合 PTM 分析显然为与病理状态相关的 PTM 及其生物标志物候选物的发现提供了更广泛的意义。此外，与当前方法相比，联合方案在富集效率和生物学解释方面具有明显优势。
