High-throughput Serum N-Glycomics: Method Comparison and Application to Study Rheumatoid Arthritis and Pregnancy-associated Changes

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 highthroughput manner. Here we evaluate and compare the performance of three high-throughput released N-glycome analysis methods. Included were hydrophilic-interaction ultra-highperformance 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-offlight mass spectrometry (MALDI-TOF-MS) with linkagespecific 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-gly-From the ‡Center for Proteomics and Metabolomics,

N‐glycome signatures in human plasma: associations with physiology and major diseases

“…Recently, these limitations have been addressed: Methods have been developed and validated allowing the analysis of larger sample sets in a robust and reproducible manner, facilitating the comparison of results between laboratories and disease‐specific cohorts . Notably, next to fluorescence detection of glycans, also methods with mass spectrometric detection have been proven suitable.…”

Antibody Panel Based N‐Glycan Imaging for N‐Glycoprotein Biomarker Discovery

“…The discovery of N ‐glycan biomarkers in serum could lead to clinical implementation for disease detection. The majority of serum N ‐glycan methods focus on the analysis of a pool of N ‐glycans released from all proteins in the serum (Gornik et al., ; Kirmiz et al., ; Novokmet et al., ; Reiding et al., ; Ruhaak et al., ; Ruhaak, Xu, Li, Goonatilleke, & Lebrilla, ), or the analysis of one target protein's N ‐glycan profile (Comunale et al., ; Pompach et al., ; Ruhaak et al., , ; Shubhakar et al., ; Simunovic et al., ; Šimurina et al., ; Theodoratou et al., ; Zhang et al., ). Pooled serum analyses have shown trends in overall N ‐glycan changes in the presence of cancer, such as increased fucosylation, branching, and bisects (Gebrehiwot et al., ; Hecht et al., ; Snyder et al., ; Vučković et al., ).…”

“…Current Protocols in Protein Science 2014; Reiding et al, 2019;Ruhaak et al, 2008;Ruhaak, Xu, Li, Goonatilleke, & Lebrilla, 2018), or the analysis of one target protein's N-glycan profile (Comunale et al, 2010;Pompach et al, 2016;Ruhaak et al, 2013Ruhaak et al, , 2018Shubhakar et al, 2016;Simunovic et al, 2019;Šimurina et al, 2018;Theodoratou et al, 2016;Zhang et al, 2016). Pooled serum analyses have shown trends in overall N-glycan changes in the presence of cancer, such as increased fucosylation, branching, and bisects (Gebrehiwot et al, 2019;Hecht et al, 2015;Snyder et al, 2016;Vučković et al, 2016).…”

Mass Spectrometry‐Based Analysis of Time‐Resolved Proteome Quantification

“…Depending on the approach used for enrichment and quantification, different numbers of phosphorylations have been possible to monitor. There are examples of extensive approaches that use both fractionation and enrichment methods that allowed the time resolved quantitative comparison of >10 000 sites . In one example of an advanced study, SCX fractionation and titanium dioxide enrichment were combined, and 6600 phosphorylation sites were monitored in HeLa cells upon EGF stimulation .…”