N-glycoproteins are involved in various biological processes. Certain distinctive glycoforms on specific glycoproteins enhance the specificity and/or sensitivity of cancer diagnosis. Therefore, the characterization of plasma N-glycoproteome is essential for a new biomarker discovery. The absence of suitable analytical methods for in-depth and large-scale analyses of low-abundance plasma glycoproteins makes it challenging to investigate the role of glycosylation. In this study, we developed an integrated method termed Glyco-CPLL, which integrates combinatorial peptide ligand libraries, high-pH reversed-phase prefractionation, hydrophilic interaction chromatography, trypsin and PNGase F digestion, shotgun proteomics, and various analysis software (MaxQuant and pGlyco2.0) for the low-abundance plasma glycoproteomic profiling. Then, we utilized the method to perform a comparative study and to explore papillary thyroid carcinoma-related proteins and glycosylations with reference to healthy controls. Finally, a large and comprehensive human plasma N-glycoproteomic database was established, containing 786 proteins, 369 N-glycoproteins, 862 glycosites, 171 glycan compositions, and 1644 unique intact N-glycopeptides. Additionally, several low-abundance plasma glycoproteins were identified, including SVEP1 (∼0.54 ng/mL), F8 (∼0.83 ng/mL), and ADAMTS13 (∼1.2 ng/mL). These results suggest that this method will be useful for analyzing plasma intact glycopeptides in future studies. Besides, the Glyco-CPLL method has a great potential to be translated to clinical applications. Data are available via ProteomeXchange with identifier PXD016428.

中文翻译：

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Glyco-CPLL：一种用于人类血浆的深度和全面N-糖蛋白组分析的综合方法。

N-糖蛋白参与各种生物学过程。特定糖蛋白上的某些独特糖型可增强癌症诊断的特异性和/或敏感性。因此，血浆N-糖蛋白组的表征对于发现新的生物标志物至关重要。缺乏用于低丰度血浆糖蛋白的深入和大规模分析的合适分析方法，使得研究糖基化的作用具有挑战性。在这项研究中，我们开发了一种称为Glyco-CPLL的集成方法，该方法集成了组合肽配体库，高pH反相预分离，亲水相互作用色谱，胰蛋白酶和PNGase F消化，shot弹枪蛋白质组学以及各种分析软件（MaxQuant和pGlyco2 .0）用于低丰度血浆糖蛋白组学分析。然后，我们利用该方法进行了比较研究，并参考了健康对照来探索甲状腺乳头状癌相关蛋白和糖基化。最后，建立了一个庞大而全面的人类血浆N-糖蛋白数据库，包含786个蛋白质，369个N-糖蛋白，862个糖位，171个聚糖成分和1644个完整的N-糖肽。此外，还鉴定了几种低丰度血浆糖蛋白，包括SVEP1（〜0.54 ng / mL），F8（〜0.83 ng / mL）和ADAMTS13（〜1.2 ng / mL）。这些结果表明，该方法将在将来的研究中用于分析血浆完整糖肽。此外，Glyco-CPLL方法具有很大的潜力可转化为临床应用。数据可通过ProteomeXchange获得，其标识符为PXD016428。