Glycans, Glycosite, and Intact Glycopeptide Analysis of N‑Linked Glycoproteins Using Liquid Handling Systems

Aberrant glycosylation has been shown to associate with disease progression, and with glycoproteins representing the major protein component of biological fluids this makes them attractive targets for disease monitoring. Leveraging glycoproteomic analysis via mass spectrometry (MS) could provide the insight into the altered glycosylation patterns that relate to disease progression. However, investigation of large sample cohorts requires rapid, efficient, and highly reproducible sample preparation. To address the limitation, we developed a high-throughput method for characterizing glycans, glycosites, and intact glycopeptides (IGPs) derived from N-linked glycoproteins. We combined disparate peptide enrichment strategies (i.e., hydrophilic and hydrophobic) and a liquid handling platform allowing for a high throughput and rapid enrichment of IGP in a 96-well plate format. The C18/MAX-Tip workflow reduced sample processing time and facilitated the selective enrichment of IGPs from complex samples. Furthermore, our approach enabled the analysis of deglycosylated peptides and glycans from enriched IGPs following PNGase F digest. Following development and optimization of the C18/MAX-Tip methodology using the standard glycoprotein, fetuin, we investigated normal urine samples to obtain N-linked glycoprotein information. Together, our method enables a high-throughput enrichment of glycan, glycosites, and IGPs from biological samples.

异常糖基化（aberrant glycosylation）已被证实与疾病进展密切相关；鉴于糖蛋白（glycoprotein）是生物体液中的主要蛋白质组分，其成为疾病监测的理想靶点。借助质谱（mass spectrometry, MS）开展糖蛋白质组学分析，可深入解析与疾病进展相关的异常糖基化模式。然而，针对大规模样本队列的研究需要快速、高效且重复性极佳的样本前处理方案。为解决这一局限，本研究开发了一种可表征N-连接糖蛋白（N-linked glycoprotein）来源的聚糖（glycan）、糖基化位点（glycosite）以及完整糖肽（intact glycopeptide, IGP）的高通量分析方法。我们将不同的肽段富集策略（即亲水与疏水策略）与适配96孔板形式的液体处理平台相结合，实现完整糖肽的高通量快速富集。C18/MAX-Tip 流程缩短了样本处理时长，并可从复杂样本中选择性富集完整糖肽。此外，该方法可在经PNGase F（肽N-糖苷酶F）酶解后，对富集得到的完整糖肽的去糖基化肽段与聚糖进行分析。本研究以标准糖蛋白胎球蛋白（fetuin）为对象完成C18/MAX-Tip方法的开发与优化后，对正常尿液样本进行分析以获取N-连接糖蛋白相关信息。综上，本方法可实现从生物样本中高通量富集聚糖、糖基化位点及完整糖肽。