Large-Scale Identification of Core-Fucosylated Glycopeptide Sites in Pancreatic Cancer Serum Using Mass Spectrometry

Abstract

Glycosylation has significant effects on protein function and cell metastasis, which are important in cancer progression. It is of great interest to identify site-specific glycosylation in search of potential cancer biomarkers. However, the abundance of glycopeptides is low compared to that of nonglycopeptides after trypsin digestion of serum samples, and the mass spectrometric signals of glycopeptides are often masked by coeluting nonglycopeptides due to low ionization efficiency. Selective enrichment of glycopeptides from complex serum samples is essential for mass spectrometry (MS)-based analysis. Herein, a strategy has been optimized using LCA enrichment to improve the identification of core-fucosylation (CF) sites in serum of pancreatic cancer patients. The optimized strategy was then applied to analyze CF glycopeptide sites in 13 sets of serum samples from pancreatic cancer, chronic pancreatitis, healthy controls, and a standard reference. In total, 630 core-fucosylation sites were identified from 322 CF proteins in pancreatic cancer patient serum using an Orbitrap Elite mass spectrometer. Further data analysis revealed that 8 CF peptides exhibited a significant difference between pancreatic cancer and other controls, which may be potential diagnostic biomarkers for pancreatic cancer.

Mass-Selected Site-Specific Core-Fucosylation of Serum Proteins in Hepatocellular Carcinoma

“…For CF level quantification, there are two methods: one is label-free relative quantification 11,18 and the other one is absolute quantification with iTRAQ labeling. 10 iTRAQ labeling was used to quantify the CF peptides in this work.…”

Site-Specific Fucosylation Analysis Identifying Glycoproteins Associated with Aggressive Prostate Cancer Cell Lines Using Tandem Affinity Enrichments of Intact Glycopeptides Followed by Mass Spectrometry

“…There are two major strategies for Fuc glycoproteins analyses: (1) targeted Fuc proteins measurement by ELISA, western/lectin blot, or mass spectrometry (MS) 6,13,14,16,19,20 and (2) large-scale Fuc glycoproteomes profiling by MS analysis. 8,10,18,21–24 The second approach may be more comprehensive as it facilitates the understanding of underlying mechanisms associated with tumorigenesis and metastasis as well as the discovery of novel potential biomarkers for early clinical diagnosis. Currently, large-scale Fuc glyco-proteomics analyses often employ a divide-and-conquer approach that profiles glycans or deglycosylated peptides separately, utilizing enzymatic or chemical methods to selectively cleave either the glycan or glycopeptide.…”

“…Currently, large-scale Fuc glyco-proteomics analyses often employ a divide-and-conquer approach that profiles glycans or deglycosylated peptides separately, utilizing enzymatic or chemical methods to selectively cleave either the glycan or glycopeptide. 8,10,18,20–25 However, a key drawback of this strategy is that information linking the site of glycosylation to its respective glycan structure is lost, and aspects such as glycan microheterogeneity at specific glycosites cannot be examined. 26 Therefore, strategies to profile the large-scale glycoproteomes, including Fuc, by analyzing intact glycopeptides directly are needed.…”

Glycans and glycoproteins as specific biomarkers for cancer

“…A method for screening and quantifying core fucosylated (CF) peptides in the depleted serum was recently developed [130]. The method uses lens culinaris agglutinin (LCA) to enrich for the core fucosylated peptides.…”