Reiding, Karli R. and Bondt, Albert and Hennig, René and Gardner, Richard A. and O'Flaherty, Roisin and Trbojevic-Akmacic, Irena and Shubhakar, Archana and Hazes, Johanna M. W. and Reichi, Udo and Fernandes, Daryl L. and Pucic-Bakovic, Maja and Rapp, Erdmann and Spencer, Daniel I. R. and Dolhain, Radboud J. E. M. and Rudd, Pauline M and Lauc, Gordan and Wuhrer, Manfred (2019) High-throughput Serum N-Glycomics: Method Comparison and Application to Study Rheumatoid Arthritis and Pregnancy-associated Changes. Molecular and Cellular Proteomics (MCP), 18 (1). pp. 3-15. ISSN 1535-9476

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Abstract

N-Glycosylation is a fundamentally important protein modification with a major impact on glycoprotein characteristics such as serum half-life and receptor interaction. More than half of the proteins in human serum are glycosylated, and the relative abundances of protein glycoforms often reflect alterations in health and disease. Several analytical methods are currently capable of analyzing the total serum N-glycosylation in a high-throughput manner. Here we evaluate and compare the performance of three high-throughput released N-glycome analysis methods. Included were hydrophilic-interaction ultra-high-performance liquid chromatography with fluorescence detection (HILIC-UHPLC-FLD) with 2-aminobenzamide labeling of the glycans, multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF) with 8-aminopyrene-1,3,6-trisulfonic acid labeling, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) with linkage-specific sialic acid esterification. All methods assessed the same panel of serum samples, which were obtained at multiple time points during the pregnancies and postpartum periods of healthy women and patients with rheumatoid arthritis (RA). We compared the analytical methods on their technical performance as well as on their ability to describe serum protein N-glycosylation changes throughout pregnancy, with RA, and with RA disease activity. Overall, the methods proved to be similar in their detection and relative quantification of serum protein N-glycosylation. However, the non-MS methods showed superior repeatability over MALDI-TOF-MS and allowed the best structural separation of low-complexity N-glycans. MALDI-TOF-MS achieved the highest throughput and provided compositional information on higher-complexity N-glycans. Consequentially, MALDI-TOF-MS could establish the linkage-specific sialylation differences within pregnancy and RA, whereas HILIC-UHPLC-FLD and xCGE-LIF demonstrated differences in α1,3- and α1,6-branch galactosylation. While the combination of methods proved to be the most beneficial for the analysis of total serum protein N-glycosylation, informed method choices can be made for the glycosylation analysis of single proteins or samples of varying complexity.

Item Type:	Article
Keywords:	Glycomics; Glycosylation; High Throughput Screening; Plasma or Serum Analysis; Electrophoresis; Chromatography; Mass Spectrometry; Pregnancy; Rheumatoid Arthritis;
Academic Unit:	Faculty of Science and Engineering > Chemistry
Item ID:	15022
Depositing User:	Roisin O'Flaherty
Date Deposited:	16 Nov 2021 12:08
Journal or Publication Title:	Molecular and Cellular Proteomics (MCP)
Publisher:	Elsevier
Refereed:	Yes
URI:	Publisher
Use Licence:	This item is available under a Creative Commons Attribution Non Commercial Share Alike Licence (CC BY-NC-SA). Details of this licence are available here

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