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