Glyco-CPLL: An Integrated Method for In-Depth and Comprehensive N-Glycoproteome Profiling of Human Plasma
Glyco-CPLL: An Integrated Method for In-Depth and Comprehensive N-Glycoproteome Profiling of Human Plasma
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Glyco-CPLL：一种对人血浆进行深入、全面的 N-糖蛋白质组分析的集成方法
DOI：
10.1021/acs.jproteome.9b00557
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发表时间：
2020-02-01
影响因子：
4.4
通讯作者：
Yang, Hao
中科院分区：
文献类型：
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作者：
Zhang, Yong;Mao, Yonghong;Yang, Hao
N-糖蛋白参与多种生物过程。特定糖蛋白上的某些独特的糖形式提高了癌症诊断的特异性和/或敏感性。因此，血浆N-糖蛋白组学特性的研究对于发现新的生物标志物是至关重要的。缺乏合适的分析方法对低丰度血浆糖蛋白进行深入和大规模的分析，这使得研究糖基化的作用具有挑战性。在本研究中，我们开发了一种名为Glyco-CPLL的集成方法，该方法集成了组合多肽配基文库、高pH反相预分离、亲水相互作用层析、胰酶和PNGase F酶切、鸟枪式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质组分析。然后，我们利用该方法进行了对比研究，并以健康对照组为参照，探索了甲状腺乳头状癌相关蛋白和糖基化。最终建立了一个完整的人血浆N-糖蛋白数据库，该数据库包含786个蛋白质、369个N-糖蛋白、862个糖位、171个糖组分和1644个独特的完整N-糖肽。此外，还鉴定出几种低丰度的血浆糖蛋白，包括SVEP1(~0.54 ng/mL)、F8(~0.83 ng/mL)、ADAMTS13(~1.2 ng/mL)。这些结果表明，该方法将在今后的研究中用于血浆完整糖肽的分析。此外，Glyco-CPLL方法具有很大的临床应用潜力。数据可通过标识符为PXD016428的ProteomeXchange获得。
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 new biomarker discovery. Absence of suitable analytical methods for in-depth and large-scale analyses of low-abundance plasma glycoproteins make 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 pre-fractionation, 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), 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.