title: N-glycoproteome analysis of the secretome of human metastatic hepatocellular carcinoma cell lines combining hydrazide chemistry, HILIC enrichment and mass spectrometry.
abstract: Cancer cell metastasis is a major cause of cancer death. Unfortunately, the underlying molecular mechanisms remain unknown, which results in the lack of efficient diagnosis, therapy and prevention approaches. Nevertheless, the dysregulation of the cancer cell secretome is known to play key roles in tumor transformation and progression. The majority of proteins in the secretome are secretory proteins and membrane-released proteins, and, mostly, the glycosylated proteins. Until recently, few studies have explored protein N-glycosylation changes in the secretome, although protein glycosylation has received increasing attention in the study of tumor development processes. Here, the N-glycoproteins in the secretome of two human hepatocellular carcinoma (HCC) cell lines with low (MHCC97L) or high (HCCLM3) metastatic potential were investigated with a in-depth characterization of the N-glycosites by combining two general glycopeptide enrichment approaches, hydrazide chemistry and zwitterionic hydrophilic interaction chromatography (zic-HILIC), with mass spectrometry analysis. A total of 1,213 unique N-glycosites from 611 N-glycoproteins were confidently identified. These N-glycoproteins were primarily localized to the extracellular space and plasma membrane, supporting the important role of N-glycosylation in the secretory pathway. Coupling label-free quantification with a hierarchical clustering strategy, we determined the differential regulation of several N-glycoproteins that are related to metastasis, among which AFP, DKK1, FN1, CD151 and TGFβ2 were up-regulated in HCCLM3 cells. The inclusion of the well-known metastasis-related proteins AFP and DKK1 in this list provides solid supports for our study. Further western blotting experiments detecting FN1 and FAT1 confirmed our discovery. The glycoproteome strategy in this study provides an effective means to explore potential cancer biomarkers.
authors: The College of Life Science and Bio-engineering, Beijing University of Technology, Beijing, China ; State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing, China.
authors: Xianyu
authors: X
authors: Li
authors: Jing
authors: J
authors: Jiang
authors: Xinyuan
authors: X
authors: Zhao
authors: Jifeng
authors: J
authors: Wang
authors: Huanhuan
authors: H
authors: Han
authors: Yan
authors: Y
authors: Zhao
authors: Bo
authors: B
authors: Peng
authors: Rugang
authors: R
authors: Zhong
authors: Wantao
authors: W
authors: Ying
authors: Xiaohong
authors: X
authors: Qian
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000473:pathology*; D045744:Cell Line, Tumor; D002845:Chromatography; D016000:Cluster Analysis; D019295:Computational Biology; D006020:Glycopeptides / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D006834:Hydrazines / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D008113:Liver Neoplasms / Q000378:metabolism* / Q000473:pathology; D013058:Mass Spectrometry / Q000379:methods*; D009362:Neoplasm Metastasis; D060066:Protein Interaction Maps; D040901:Proteomics; D015203:Reproducibility of Results; D013194:Staining and Labeling; D015854:Up-Regulation
chemical_list: D006020:Glycopeptides; D006023:Glycoproteins; D006834:Hydrazines