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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.