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

• Published in: Talanta (Oxford) Vol. 199; pp. 254 - 261
• Main Authors: Jiang, Biyun, Huang, Jiangming, Yu, Zixiang, Wu, Mengxi, Liu, Mingqi, Yao, Jun, Zhao, Huanhuan, Yan, Guoquan, Ying, Wantao, Cao, Weiqian, Yang, Pengyuan
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.07.2019
• Subjects: Carcinoma, Hepatocellular - chemistry; Carcinoma, Hepatocellular - metabolism; Chromatography, Liquid; Enrichment; Glycopeptides - chemistry; Glycopeptides - metabolism; Glycosylation; Hepatocellular carcinoma; High throughput; High-Throughput Screening Assays; Humans; Liver Neoplasms - chemistry; Liver Neoplasms - metabolism; Mass Spectrometry; N-glycoproteome; Protein Interaction Mapping; Proteome - analysis; Proteome - metabolism; Tumor Cells, Cultured; High throughput; Enrichment; Hepatocellular carcinoma; N-glycoproteome; Mass spectrometry
• ISSN: 0039-9140; 1873-3573
• DOI: 10.1016/j.talanta.2019.02.010

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. [Display omitted] •Multi-parallel N-glycopeptide enrichment in a 96-well plate.•Achieving high specific and high throughput N-glycoproteome enrichment simultaneously.•Performance of complementary 2D fraction in large scale N-glycoprotein mapping.•In-depth mapping of N-glycoproteome in three HCC cell lines.