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Characterization of core fucosylation via sequential enzymatic treatments of intact glycopeptides and mass spectrometry analysis.

ABSTRACT: Core fucosylation of N-linked glycoproteins has been linked to the functions of glycoproteins in physiological and pathological processes. However, quantitative characterization of core fucosylation remains challenging due to the complexity and heterogeneity of N-linked glycosylation. Here we report a mass spectrometry-based method that employs sequential treatment of intact glycopeptides with enzymes (STAGE) to analyze site-specific core fucosylation of glycoproteins. The STAGE method utilizes Endo F3 followed by PNGase F treatment to generate mass signatures for glycosites that are formerly modified by core fucosylated N-linked glycans. We benchmark the STAGE method and use it to characterize site specific core fucosylation of glycoproteins from human hepatocellular carcinoma and pancreatic ductal adenocarcinoma, resulting in the identification of 1130 and 782 core fucosylated glycosites, respectively. These results indicate that our STAGE method enables quantitative characterization of core fucosylation events from complex protein mixtures, which may benefit our understanding of core fucosylation functions in various diseases.

SUBMITTER: Cao L

PROVIDER: S-EPMC9262967 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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Characterization of core fucosylation via sequential enzymatic treatments of intact glycopeptides and mass spectrometry analysis.

Cao Liwei L Lih T Mamie TM Hu Yingwei Y Schnaubelt Michael M Chen Shao-Yung SY Zhou Yangying Y Guo Chuanyu C Dong Mingming M Yang Weiming W Eguez Rodrigo Vargas RV Chen Lijun L Clark David J DJ Sodhi Akrit A Li Qing Kay QK Zhang Hui H

Nature communications 20220707 1

Core fucosylation of N-linked glycoproteins has been linked to the functions of glycoproteins in physiological and pathological processes. However, quantitative characterization of core fucosylation remains challenging due to the complexity and heterogeneity of N-linked glycosylation. Here we report a mass spectrometry-based method that employs sequential treatment of intact glycopeptides with enzymes (STAGE) to analyze site-specific core fucosylation of glycoproteins. The STAGE method utilizes ...[more]

PMID: 35798744

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Project description:Fucosylation (Fuc) of glycoproteins plays an important role in regulating protein function and has been associated with the development of several cancer types including prostate cancer (Pca). Therefore, the research of Fuc glycoproteins has attracted increasing attention recently in the analytical field. Herein, a strategy based on lectin affinity enrichments of intact glycopeptides followed by mass spectrometry has been established to evaluate the specificities of various Fuc-binding lectins for glycosite-specific Fuc analysis of nonaggressive (NAG) and aggressive (AG) Pca cell lines. The enrichment specificities of Fuc glycopeptides using lectins (LCA, PSA, AAL, LTL, UEA I, and AOL) and MAX extraction cartridges alone, or in tandem, were evaluated. Our results showed that the use of lectin enrichment significantly increased the ratio of fucosylated glycopeptides to total glycopeptides compared to MAX enrichment. Furthermore, tandem use of lectin followed by MAX increased the number of identifications of Fuc glycopeptides compared to using lectin enrichment alone. LCA, PSA, and AOL showed stronger binding capacity than AAL, LTL, and UEA I. Also, LCA and PSA bound specifically to core Fuc, whereas AOL, AAL, and UEA I showed binding to both core Fuc and branch Fuc. The optimized enrichment method with tandem enrichment of LCA followed by MAX (LCA-MAX) was then applied to examine the Fuc glycoproteomes in two NAG and two AG Pca cell lines. In total, 973 intact Fuc glycopeptides were identified and quantified from 252 Fuc proteins by using the tandem-mass-tags (TMT) labeling and nanoliquid chromatography-mass spectrometry (nanoLC-MS/MS) analysis. Further data analysis revealed that 51 Fuc glycopeptides were overexpressed more than 2-fold in AG cell lines compared to NAG cells. The analysis of protein core fucosylation has great potential for aiding our understanding of invasive activity of AG Pca and may lead to the development of diagnostic approaches for AG Pca.

Dataset Information

Characterization of core fucosylation via sequential enzymatic treatments of intact glycopeptides and mass spectrometry analysis.

Publications

Characterization of core fucosylation via sequential enzymatic treatments of intact glycopeptides and mass spectrometry analysis.

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