Proteome and glycoproteome analysis of fibroblasts from patients with congenital disorders of glycosylation

In the biological systems, several genes are involved in protein glycosylation pathway. Congenital Disorders of Glycosylation (CDG) is known to be a multisystem disorder. Pathogenic variants in the glycosylation genes lead to abnormal N- or O-linked glycosylation that causes cellular stress. We used TMT-based N-glycoproteomics and proteomics on different patient-derived CDG and control fibroblasts to characterize the alteration in glycoproteome and proteome. 12 fractions of enriched glycopeptides after size exclusion chromatography (SEC) and 12 fractions after basic reverse phase liquid chromatography (bRPLC) were analyzed by LC-MS/MS for glycoproteomics and proteomics, respectively. A site-specific aberrant glycosylation was observed for several proteins such as mitochondrial, autophagy, extracellular matrix and cell adhesion proteins. By using quantitative proteomics, we observed alteration in abundances of several proteins separated the affected individuals and controls. The glycoproteomics and proteomics analysis in CDG patients provides the potential biomarkers and will increase our general understanding of its pathogenesis.

在生物系统中，多种基因参与蛋白质糖基化通路。先天性糖基化障碍（Congenital Disorders of Glycosylation，CDG）是一类多系统疾病。糖基化相关基因的致病变异会引发异常的N-连接或O-连接糖基化，进而导致细胞应激。本研究采用基于串联质量标签（TMT）的N-糖蛋白质组学与蛋白质组学技术，对不同患者来源的CDG成纤维细胞及对照成纤维细胞进行分析，以解析糖蛋白质组与蛋白质组的变化特征。我们先通过尺寸排阻色谱（Size Exclusion Chromatography，SEC）富集糖肽，得到12个组分；再通过碱性反相液相色谱（basic reverse phase liquid chromatography，bRPLC）获得12个组分，分别采用液相色谱-串联质谱（Liquid Chromatography-Tandem Mass Spectrometry，LC-MS/MS）进行糖蛋白质组学和蛋白质组学检测。研究在多种蛋白质中观察到了位点特异性的异常糖基化，这些蛋白质包括线粒体相关蛋白、自噬相关蛋白、细胞外基质蛋白及细胞黏附蛋白。通过定量蛋白质组学分析，我们发现受影响个体与对照样本的多种蛋白质丰度存在显著差异，可有效区分两组样本。对CDG患者的糖蛋白质组学与蛋白质组学分析，不仅可筛选出潜在的生物标志物，还能增进我们对该疾病发病机制的全面认知。