Combined phospho- and glycoproteome enrichment in nephrocalcinosis tissues of phytate-fed rats.
Combined phospho- and glycoproteome enrichment in nephrocalcinosis tissues of phytate-fed rats.
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DOI：
10.1002/rcm.6742
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发表时间：
2013-12
期刊：
影响因子：
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通讯作者：
T. Tran;Jong-Moon Park;O. Kim;Bora Kim;D. Choi;Jeonghwa Lee;K. Kim;Byung-Chul Oh;Hookeun Lee
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文献类型：
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作者：
T. Tran;Jong-Moon Park;O. Kim;Bora Kim;D. Choi;Jeonghwa Lee;K. Kim;Byung-Chul Oh;Hookeun Lee
蛋白质翻译后修饰(PTM)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胞外区和胞内区的蛋白质的重要修饰。然而，使用磷酸和糖蛋白组学的联合检测受到限制，主要是由于方案的差异。方法在本研究中，我们建立了一种从单个样品批次中同时检测磷酸和糖蛋白质组的新方法，该方法使用二氧化钛小柱捕获磷酸蛋白质组，并用肼树脂处理流过的溶液以捕获N-连接的糖肽。结果从正常大鼠和疾病大鼠肾组织裂解液中提取1 mg蛋白质，用液相色谱/串联质谱仪同时检测出正常大鼠和疾病大鼠肾脏中437个糖基/358个亚磷基和468个糖基/369个亚磷基。结论与单独的PTM分析相比，联合PTM分析清楚地为PTM提供了与病理状态和发现候选生物标记物相关的更广泛的含义。此外，与现有方法相比，该组合方法在浓缩效率和生物学解释方面的优势得到了充分的体现。
RATIONALE 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.