A precise approach in large scale core-fucosylated glycoprotein identification with low- and high-normalized collision energy.

UNLABELLED

The core fucosylation (CF) of N-glycoproteins plays important roles in regulating protein functions during biological development, and it has also been shown to be up-regulated in several high metastasis cancer cell lines. Therefore, global profiling and quantitative characterization of CF-glycoproteins may reveal potent biomarkers for clinical applications. However, due to the complex fragmentation pattern of CF-glycopeptides, accurately identifying CF-glycosylation sites via mass spectrometry with high throughput remains a formidable challenge. In this study, we established a precise CF-glycosylation site identification strategy with UHPLC LTQ-Orbitrap Elite under low- and high-normalized collision energy (LHNCE) conditions. To demonstrate the feasibility of LHNCE, the CF-glycopeptides of target proteins in clinical plasma samples were applied and compared as a preliminary demonstration and resulted in the assignment of 357 unique CF-glycosylation sites from 209 CF-glycoproteins. In this study, the largest human plasma CF-glycosylation site database was constructed, and at least three-fold more CF-sites were identified compared to previously published studies. The results further demonstrated that LHNCE provides an important approach for CF-glycoprotein function studies and biomarker screening in cancer research.

BIOLOGICAL SIGNIFICANCE

Core-fucosylation (CF) is a kind of N-linked glycosylation in which an α1,6-linked fucose is added to the innermost N-acetylglucosamine (GlcNAc) residue. It has been proved that core-fucosylation is involved in regulating biological processes in mammals. Abnormal core-fucosylation has been demonstrated in human pathological processes, such as metastasis. For example, the CF-glycosylation of an α-fetoprotein isoform (AFP-L3) was approved as a biomarker of hepatocellular carcinoma (HCC). In addition, GP73 is also a well-known biomarker and its CF-glycosylation level will increase in liver cancer patients. Therefore, it is crucial to develop a strategy for mapping human CF-glycosylation.