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A multi-parallel N-glycopeptide enrichment strategy for high-throughput and in-depth mapping of the N-glycoproteome in metastatic human hepatocellular carcinoma cell lines

Jiang, Biyun, Huang, Jiangming, Yu, Zixiang, Wu, Mengxi, Liu, Mingqi, Yao, Jun, Zhao, Huanhuan, Yan, Guoquan, Ying, Wantao, Cao, Weiqian, Yang, Pengyuan
Talanta 2019 v.199 pp. 254-261
acetonitrile, biomarkers, fractionation, glycoproteins, glycoproteomics, glycosylation, hepatoma, human cell lines, humans, hydrophilic interaction chromatography, metastasis, neoplasm cells, washing, zwitterions
N-glycosylation is deeply involved in many biological processes, and approximately 50% of mammalian proteins are predicted to be glycosylated. Many large-scale studies have been carried out to reveal the glycosylation status involved in different physiological pathologies across species. However, the lack of a highly specific and high-throughput N-glycosylated enrichment method not only results in extended time requirements but also limits the depth of mapping when handling a large number of samples. In this study, we firstly optimized traditional zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) enrichment and found that using of 70% acetonitrile (ACN), 0.1% trifluoroacetic acid (TFA) as the enrichment buffer, 2800 g as the washing speed and 600 μL as the washing volume achieved the best specificity, which is higher than 75%. On this basis, we developed a multi-parallel enrichment strategy assisted by a filter-coated 96-well plate, which achieved high specificity and high throughput simultaneously. This strategy allowed us to enrich large numbers of fractionated samples from hepatocellular carcinoma (HCC) cell lines in less than 2 h. Its good specificity helped us achieve in-depth mapping of the N-glycoproteome in metastatic HCC cell lines. A total of 5466 N-glycosites from 2383 glycoproteins were identified, among which 1900 N-glycosites were unannotated in UniProt. The in-depth glycoproteome mapping provides insight into the N-glycosylation status in HCC cell lines with differences in metastatic potential and contributes to biomarker discovery.