CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
Anal Chem. 2021 May 25;93(20):7473-7480. doi: 10.1021/acs.analchem.1c00645. Epub 2021 May 11.
Bottom-up proteomics has been increasingly applied in clinical research to study the disease pathophysiology and to discover disease biomarkers. However, glycoproteomic analysis always requires tedious experimental steps for intact glycopeptide enrichment, which has been the technique bottleneck for large-scale analysis of clinical samples. Herein, we developed an automated glycopeptide enrichment method for the analysis of serum site-specific N-glycoproteome. This automated method allowed for processing one sample within 20 min. It showed higher enrichment specificity, more intact glycopeptide identifications, and better quantitative reproducibility than the traditional manual method using microtip enrichment devices. We further applied this method to investigate the serum site-specific N-glycosylation changes between four patients with pancreatic cancer and seven healthy controls. The principal component analysis of intact N-glycopeptides showed good clustering across cancer and normal groups. Furthermore, we found that the site-specific glycoforms, monofucosylated and nonsialylated oligosaccharides, on IgG1 site 180 expressed a significant decrease in pancreatic cancer patients compared to healthy controls. Together, the automated method is a powerful tool for site-specific N-glycoproteomic analysis of complex biological samples, and it has great potential for clinical utilities.
自下而上的蛋白质组学在临床研究中被越来越多地应用于研究疾病病理生理学和发现疾病生物标志物。然而，糖蛋白质组学分析通常需要繁琐的实验步骤来富集完整的糖肽，这一直是临床样本大规模分析的技术瓶颈。在这里，我们开发了一种自动化的糖肽富集方法，用于分析血清中特定位置的 N-糖蛋白质组。这种自动化方法可以在 20 分钟内处理一个样本。与传统的使用微吸嘴富集装置的手动方法相比，它显示出更高的富集特异性、更多完整的糖肽鉴定和更好的定量重现性。我们进一步将该方法应用于研究 4 名胰腺癌患者和 7 名健康对照者之间血清中特定位置 N-糖基化的变化。完整的 N-糖肽的主成分分析显示癌症组和正常组之间有很好的聚类。此外，我们发现与健康对照组相比，在 IgG1 位点 180 上，单岩藻糖基化和非唾液酸化的寡糖的特定位置糖型表达显著降低。总之，自动化方法是一种用于复杂生物样品特定位置 N-糖蛋白质组分析的强大工具，具有很大的临床应用潜力。
