title: Identification of N-glycosylation sites on secreted proteins of human hepatocellular carcinoma cells with a complementary proteomics approach.
abstract: N-linked glycosylation is prevalent in proteins destined for extracellular environments; nearly all secreted proteins are glycosylated. However, with respect to their glycosylation sites, little attention has been paid. Here, we report the analysis of N-glycosylation sites on secreted proteins of human hepatocellular carcinoma cells. For the enrichment of glycopeptides, capture methods with hydrophilic affinity (HA) and hydrazide chemistry (HC) were used complementarily. With the use of both methods in combination with nano-LC-ESI-MS/MS analysis, 300 different glycosylation sites within 194 unique glycoproteins were identified, and 172 glycosites have not been determined experimentally previously. A direct comparison between HA and HC methods was also investigated for the first time. In brief, in terms of selectivity for glycopeptides, HC is superior to HA (92.9% vs 51.3%); however, based on the number of glycosites identified, HA outweighs HC (265 vs 159). Furthermore, unavoidable contaminants such as actin and bovine serum albumin which are not N-glycosylated could be easily depleted by using this glycoproteomic strategy. As a consequence, more low-abundance and genuinely secreted proteins were identified. Among the glycoproteins identified, alpha-fetoprotein, CD44 and laminin have been reported to be implicated in HCC and its metastasis.
authors.affiliation: Department of Chemistry, Fudan University, Shanghai, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Cao
authors.forename: Chengping
authors.initials: C
authors.lastname: Shen
authors.forename: Hong
authors.initials: H
authors.lastname: Wang
authors.forename: Huali
authors.initials: H
authors.lastname: Shen
authors.forename: Yaohan
authors.initials: Y
authors.lastname: Chen
authors.forename: Aiying
authors.initials: A
authors.lastname: Nie
authors.forename: Guoquan
authors.initials: G
authors.lastname: Yan
authors.forename: Haojie
authors.initials: H
authors.lastname: Lu
authors.forename: Yinkun
authors.initials: Y
authors.lastname: Liu
authors.forename: Pengyuan
authors.initials: P
authors.lastname: Yang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D045744:Cell Line, Tumor; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008113:Liver Neoplasms / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*
chemical_list: D006023:Glycoproteins; D009363:Neoplasm Proteins