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. 2022:2370:267-280.
doi: 10.1007/978-1-0716-1685-7_13.

N-glycan Characterization by Liquid Chromatography Coupled with Fluorimetry and Mass Spectrometry

Richard A Gardner  1 , Paulina A Urbanowicz  2 , Daniel I R Spencer  3
Affiliations

N-glycan Characterization by Liquid Chromatography Coupled with Fluorimetry and Mass Spectrometry

Richard A Gardner et al. Methods Mol Biol. 2022.

Abstract

Human blood plasma and serum have been a source of biomarkers for the indication and progression of many diseases for a few decades now. Human blood plasma is also an excellent source material to enable patients to monitor their health, with a multitude of biomarkers detectable for the assessment of health status. Blood sampling kits are increasingly available for use in the home with no specialist clinical skills required to obtain good quality samples for pathology lab analysis. Many of the proteins that constitute plasma are glycosylated with both N- and O-type glycans. There is increasing interest in the scientific community to identify potential glycan biomarkers or glycan features that are indicative of disease, and in particular disease at an early stage. The quality and reproducibility of glycan analysis data is key in order to identify and utilise glycan-based blood biomarkers with sufficient specificity and sensitivity; hence, the required analytical tools need to be robust. In this chapter, we describe an analytical method for the UHPLC separation of plasma N-glycans which utilizes both glycan reducing terminus fluorophore labeling, to ensure stoichiometric analysis of relative glycan abundance, and online mass spectrometry for glycan identification. Exoglycosidase digestions were employed as example technique to aid and enable structure identification.

Keywords: Biomarker; Fluorophore; Glycoprofiling; Glycosylation; LC-MS; N-glycan; Plasma; Reductive amination.

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References

    1. Singh SS, Naber A, Dotz V, Schoep E, Memarian E, Slieker RC, Elders PJM, Vreeker G, Nicolardi S, Wuhrer M, Sijbrands EJG, Lieverse AG, ‘t Hart LM, van Hoek M (2020) Metformin and statin use associate with plasma protein N-glycosylation in people with type 2 diabetes. BMJ Open Diabetes Res Care 8(1):e001230. https://doi.org/10.1136/bmjdrc-2020-001230 - DOI - PubMed - PMC
    1. Matsumoto T, Hatakeyama S, Yoneyama T, Tobisawa Y, Ishibashi Y, Yamamoto H, Yoneyama T, Hashimoto Y, Ito H, Nishimura SI, Ohyama C (2019) Serum N-glycan profiling is a potential biomarker for castration-resistant prostate cancer. Sci Rep 9(1):16761. https://doi.org/10.1038/s41598-019-53384-y - DOI - PubMed - PMC
    1. Reiding KR, Bondt A, Hennig R, Gardner RA, O’Flaherty R, Trbojević-Akmačić I, Shubhakar A, Hazes JMW, Reichl U, Fernandes DL, Pučić-Baković M, Rapp E, Spencer DIR, Dolhain RJEM, Rudd PM, Lauc G, Wuhrer M (2019) High-throughput serum N-glycomics: method comparison and application to study rheumatoid arthritis and pregnancy-associated changes. Mol Cell Proteomics 18(1):3–15. https://doi.org/10.1074/mcp.RA117.000454 - DOI - PubMed
    1. Thanabalasingham G, Huffman JE, Kattla JJ, Novokmet M, Rudan I, Gloyn AL, Hayward C, Adamczyk B, Reynolds RM, Muzinic A, Hassanali N, Pucic M, Bennett AJ, Essafi A, Polasek O, Mughal SA, Redzic I, Primorac D, Zgaga L, Kolcic I, Hansen T, Gasperikova D, Tjora E, Strachan MW, Nielsen T, Stanik J, Klimes I, Pedersen OB, Njølstad PR, Wild SH, Gyllensten U, Gornik O, Wilson JF, Hastie ND, Campbell H, McCarthy MI, Rudd PM, Owen KR, Lauc G, Wright AF (2013) Mutations in HNF1A result in marked alterations of plasma glycan profile. Diabetes 62(4):1329–1337. https://doi.org/10.2337/db12-0880 - DOI - PubMed - PMC
    1. Scott DA, Drake RR (2019) Glycosylation and its implications in breast cancer. Expert Rev Proteomics 16(8):665–680. https://doi.org/10.1080/14789450.2019.1645604 - DOI - PubMed - PMC

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