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

Cao, Liwei; Lih, Tung-Shing Mamie; Hu, Yingwei; Schnaubelt, Michael; Chen, Shao-Yung; Zhou, Yangying; Guo, Chuanyu; Dong, Mingming; Yang, Weiming; Eguez, Rodrigo Vargas; Chen, Lijun; Clark, David; Sodhi, Akrit; Li, Qing Kay; Zhang, Hui

doi:10.1038/s41467-022-31472-4

Cited by 22 publications

(25 citation statements)

References 67 publications

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“…These PPI network analyses also accord well with the previously reported results. 36,54,55 The site information about where glycosylation occurred is the prerequisite to understanding the biological functions of protein glycosylation. Therefore, there are many works on the global analysis of the core-fucosylated glycoproteome and O-GlcNAcylated glycoproteome from cancer cell lines.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“Two Birds One Stone” Strategy for the Site-Specific Analysis of Core Fucosylation and O-GlcNAcylation

et al. 2023

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Core fucosylation and O-GlcNAcylation are the two most famous protein glycosylation modifications that regulate diverse physiological and pathological processes in living organisms. Here, a "two birds one stone" strategy has been described for the site-specific analysis of core fucosylation and O-GlcNAcylation. Taking advantage of two mutant endoglycosidases (EndoF3-D165A and EndoCC-N180H), which efficiently and specifically recognize core fucose and O-GlcNAc, glycopeptides can be labeled using a biantennary N-glycan probe bearing azido and oxazoline groups. Then, a temperature-sensitive poly(N-isopropylacrylamide) polymer functionalized with dibenzocyclooctyne was introduced to facilitate the enrichment of the labeled glycopeptides from the complex mixture. The captured glycopeptides can be further released enzymatically by wild-type endoglycosidases (EndoF3 and EndoCC) in a traceless manner for mass spectrometry (MS) analysis. The described strategy allows simultaneous profiling of corefucosylated glycoproteome and O-GlcNAcylated glycoproteome from one complex sample by MS technology and searching the database using different variable modifications.

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Section: ■ Results and Discussionmentioning

confidence: 99%

“Two Birds One Stone” Strategy for the Site-Specific Analysis of Core Fucosylation and O-GlcNAcylation

et al. 2023

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“… 31 , 109 , 129 Permutations of this standard workflow have identified many aberrant glycoforms and glycopeptides in various forms of cancer, for example, increased high-mannose N-glycans and sialylated O-glycans in aggressive ovarian cancers 130 and specific core fucosylated glycopeptides associated with liver tumors. 131 …”

Section: Glycopeptide Mass Spectrometrymentioning

confidence: 99%

Mass Spectrometry-Based Glycoproteomic Workflows for Cancer Biomarker Discovery

Doud

Yeh

2023

Technol Cancer Res Treat

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Glycosylation has a clear role in cancer initiation and progression, with numerous studies identifying distinct glycan features or specific glycoproteoforms associated with cancer. Common findings include that aggressive cancers tend to have higher expression levels of enzymes that regulate glycosylation as well as glycoproteins with greater levels of complexity, increased branching, and enhanced chain length1. Research in cancer glycoproteomics over the last 50-plus years has mainly focused on technology development used to observe global changes in glycosylation. Efforts have also been made to connect glycans to their protein carriers as well as to delineate the role of these modifications in intracellular signaling and subsequent cell function. This review discusses currently available techniques utilizing mass spectrometry-based technologies used to study glycosylation and highlights areas for future advancement.

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“… 16 A recent report 17 from our research team combined HILIC enrichment and high-pH reversed-phase peptide fractionation to enhance signal intensity of intact glycopeptides in patient sera. Other groups have used one-step lectin enrichment 12 , 13 , 18 or MAX solid-phase extraction, 19 followed by Endo F3 digestion. In quantitative proteomics, isobaric tags for relative and absolute quantitation (iTRAQ) labeling has often been used to determine the differences in the expression of CF peptides and was believed to improve identification of targeted peptides by facilitating MS/MS fragmentation.…”

Section: Introductionmentioning

confidence: 99%

Discovery of Core-Fucosylated Glycopeptides as Diagnostic Biomarkers for Early HCC in Patients with NASH Cirrhosis Using LC-HCD-PRM-MS/MS

et al. 2023

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Aberrant changes in site-specific core fucosylation (CF) of serum proteins contribute to cancer development and progression, which enables them as potential diagnostic markers of tumors. An optimized data-dependent acquisition (DDA) workflow involving isobaric tags for relative and absolute quantitationlabeling and enrichment of CF peptides by lens culinaris lectin was applied to identify CF of serum proteins in a test set of patients with nonalcoholic steatohepatitis (NASH)-related cirrhosis (N = 16) and hepatocellular carcinoma (HCC, N = 17), respectively. A total of 624 CF peptides from 343 proteins, with 683 CF sites, were identified in our DDA−mass spectrometry (MS) analysis. Subsequently, 19 candidate CF peptide markers were evaluated by a target parallel reaction-monitoring−MS workflow in a validation set of 58 patients, including NASH-related cirrhosis (N = 29), early-stage HCC (N = 21), and late-stage HCC (N = 8). Significant changes (p < 0.01) were observed in four CF peptides between cirrhosis and HCC, where peptide LGSFEGLVn 160 LTFIHLQHNR from LUM in combination with AFP showed the best diagnostic performance in discriminating HCC from cirrhosis, with an area under curve (AUC) of 0.855 compared to AFP only (AUC = 0.717). This peptide in combination with AFP also significantly improved diagnostic performance in distinguishing early HCC from cirrhosis, with an AUC of 0.839 compared to AFP only (AUC = 0.689). Validation of this novel promising biomarker panel in larger cohorts should be performed.

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

Cited by 22 publications

References 67 publications

“Two Birds One Stone” Strategy for the Site-Specific Analysis of Core Fucosylation and O-GlcNAcylation

“Two Birds One Stone” Strategy for the Site-Specific Analysis of Core Fucosylation and O-GlcNAcylation

Mass Spectrometry-Based Glycoproteomic Workflows for Cancer Biomarker Discovery

Discovery of Core-Fucosylated Glycopeptides as Diagnostic Biomarkers for Early HCC in Patients with NASH Cirrhosis Using LC-HCD-PRM-MS/MS

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