Mass spectrometric analysis of core fucosylation and sequence variation in a human–camelid monoclonal antibody

Donald, Lynda J.; Spearman, Maureen; Mishra, Neha; Komatsu, Emy; Butler, Michael; Perreault, Hélène

doi:10.1039/c9mo00168a

Cited by 4 publications

(2 citation statements)

References 64 publications

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“…PNGase F treatment of intact glycopeptides was once used; however, the difference between CF glycoforms and non‐CF glycoforms would be lost (Segu et al., 2010; Zhang et al., 2015). Treatment by use of one or a combination of exo‐ or endoglycosidases, which left the simplified glycopeptides containing the disaccharide structure (fucosylated N‐acetyl glucosamine, FucHexNac), were attempted to study the CF glycoproteome (Donald et al., 2020; Lang et al., 2018; Ma et al., 2018; Segu et al., 2010). MS parameters were also evaluated from different aspects to increase the identification of CF glycoproteins, for example, combination of high collisional dissociation (HCD) and electron transfer dissociation (Ma et al., 2016), use of stepped collision energy (Cao et al., 2014; Liu et al., 2017), and the reference of Y1F/Y1 ratios at 20% and lower HCD energies (Chen et al., 2022).…”

Section: Commentarymentioning

confidence: 99%

Identification of Core‐Fucosylated Glycoproteins by Single‐Step Truncation of N‐Glycans

Min,

Zhao,

Ying

2024

Current Protocols

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Alpha‐1,6 core fucosylation (CF) is a unique glycoform of N‐glycans, and studies showed that CF modifications are involved in the occurrence and progression of various diseases and may provide potential disease biomarkers. Current strategies for the CF glycoproteome are often based on multistep enrichment of glycoproteins or glycopeptides and sequential cleavage with different glycosidases to truncate the N‐glycans. Although the detection ability of low‐abundance glycoproteins is improved, sample loss, high cost, and the time‐consuming multistep operation also affect the reproducibility of results and the practicality of the method. Here we developed a single‐step truncation (SST) strategy and evaluated its potential for the CF glycoproteome of human serum. The SST strategy has the advantages of fewer operational steps, lower cost, higher number of identifications, and better quantitative stability compared with previous approaches and provides an efficient solution for large‐scale quantitative analysis of the CF glycoproteome. © 2024 Wiley Periodicals LLC.Basic Protocol 1: Single‐step truncation strategy for core fucosylation glycoproteome analysis in human serumBasic Protocol 2: Liquid chromatography–tandem mass spectrometry quantification of site‐specific core fucosylation glycopeptidesAlternate Protocol: Pretreatment of cellular samples of core fucosylation glycoproteome with single‐step truncation strategy

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Section: Commentarymentioning

confidence: 99%

Identification of Core‐Fucosylated Glycoproteins by Single‐Step Truncation of N‐Glycans

Min,

Zhao,

Ying

2024

Current Protocols

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“…Over the past decade, lectin- and mass spectrometry-based approaches have been widely used to recognize and identify core-fucosylated glycans. − During mass spectrometry analysis of intact glycopeptides, core fucosylation of N- glycans can theoretically be determined based on the feature Y1F [peptide+HexNAc1+Fuc1] to Y4α/4βF [peptide+HexNAc2+Hex3+Fuc1] ions in MS/MS spectra . However, some feature peaks like the Y1F ion will also appear in the spectra of antenna-fucosylated glycopeptides, which is mainly due to fucose migration. , The migrated fucoses from glycan branches can bind to the normal Y peaks to form Yn+Fucose ions during MS/MS analysis, which would lead to the misidentification of these antenna-fucosylated glycopeptides as core-fucosylated glycopeptides.…”

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confidence: 99%

Recognition of Core-Fucosylated Glycopeptides Based on the Y1+Fuc/Y1 Ratio in Low-Energy HCD Spectra

et al. 2022

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Accurate identification of core fucosylation on Nglycopeptides remains challenging due to fucose migration during mass spectrometry analysis. Here, we introduce a simple and straightforward method for core-fucosylated glycopeptide recognition based on the relative intensities of Y1+Fuc ions compared with their corresponding Y1 ions (labeled as Y1+Fuc/Y1 or simply Y1F/Y1 ratio > 0.1) in low-energy HCD-based spectra. The method was first developed by systematically evaluating the influence of fucose migration on the Y1F ion from antenna fucoses based on the distribution of the Y1F/Y1 ratios in the MS/MS spectra of antenna-fucosylated glycopeptides from Fut8 −/− mouse brain. The feasibility of the method was then confirmed by using two standard glycoproteins, comparison with glycopeptides in Fut8 +/+ mouse brain with/without in silico core-fucosylation removal, and Y1F/Y1 ratio alterations under a lower HCD energy. This method will be applicable to the manual interpretation and software-based high-throughput analysis of core-fucosylated glycopeptides.

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Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2019–2020

Harvey

2023

Mass Spectrometry Reviews

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This review is the tenth update of the original article published in 1999 on the application of matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings coverage of the literature to the end of 2020. Also included are papers that describe methods appropriate to analysis by MALDI, such as sample preparation techniques, even though the ionization method is not MALDI. The review is basically divided into three sections: (1) general aspects such as theory of the MALDI process, matrices, derivatization, MALDI imaging, fragmentation, quantification and the use of arrays. (2) Applications to various structural types such as oligo‐ and polysaccharides, glycoproteins, glycolipids, glycosides and biopharmaceuticals, and (3) other areas such as medicine, industrial processes and glycan synthesis where MALDI is extensively used. Much of the material relating to applications is presented in tabular form. The reported work shows increasing use of incorporation of new techniques such as ion mobility and the enormous impact that MALDI imaging is having. MALDI, although invented nearly 40 years ago is still an ideal technique for carbohydrate analysis and advancements in the technique and range of applications show little sign of diminishing.

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Mass spectrometric analysis of core fucosylation and sequence variation in a human–camelid monoclonal antibody

Abstract: ESI-MS fucosylation studies on an intact EG2-hFc monoclonal antibody reveal the presence of fucose on both Fc N-glycans.

Cited by 4 publications

References 64 publications

Identification of Core‐Fucosylated Glycoproteins by Single‐Step Truncation of N‐Glycans

Identification of Core‐Fucosylated Glycoproteins by Single‐Step Truncation of N‐Glycans

Recognition of Core-Fucosylated Glycopeptides Based on the Y1+Fuc/Y1 Ratio in Low-Energy HCD Spectra

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2019–2020

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