Abstract:
Protein glycosylation is an essential post-translational modification in all domains of life. Its impairment in humans can result in severe diseases named congenital disorders of glycosylation (CDGs). Most of the glycosyltransferases (GTs) responsible for proper glycosylation are polytopic membrane proteins that represent challenging targets in proteomics. We established a multiple reaction monitoring (MRM) assay to comprehensively quantify GTs involved in the processes of N-glycosylation and O- and C-mannosylation in the endoplasmic reticulum. High robustness was achieved by using an enriched membrane protein fraction of isotopically labeled HEK 293T cells as an internal protein standard. The analysis of primary skin fibroblasts from eight CDG type I patients with impaired ALG1, ALG2, and ALG11 genes, respectively, revealed a substantial reduction in the corresponding protein levels. The abundance of the other GTs, however, remained unchanged at the transcript and protein levels, indicating that there is no fail-safe mechanism for the early steps of glycosylation in the endoplasmic reticulum. The established MRM assay was shared with the scientific community via the commonly used open source Skyline software environment, including Skyline Batch for automated data analysis. We demonstrate that another research group could easily reproduce all analysis steps, even while using different LC-MS hardware.
摘要:
蛋白质糖基化是生命所有域中必不可少的翻译后修饰。它在人类中的损害可导致称为先天性糖基化障碍(CDG)的严重疾病。负责适当糖基化的大多数糖基转移酶(GT)是代表蛋白质组学中具有挑战性的靶标的多能膜蛋白。我们建立了多反应监测(MRM)测定法,以全面定量内质网中N-糖基化以及O-和C-甘露糖基化过程中涉及的GTs。通过使用同位素标记的HEK293T细胞的富集膜蛋白级分作为内部蛋白标准品实现了高稳健性。对8个ALG1、ALG2和ALG11基因受损的CDGⅠ型患者的原代皮肤成纤维细胞进行分析,分别,显示相应的蛋白质水平大幅下降。丰富的其他GT,然而,在转录本和蛋白质水平上保持不变,表明内质网糖基化的早期步骤没有失效安全机制。已建立的MRM测定通过常用的开源Skyline软件环境与科学界共享,包括用于自动数据分析的Skyline批处理。我们证明了另一个研究小组可以轻松地复制所有分析步骤,即使使用不同的LC-MS硬件。