Dataset Information

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 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 ?1,3- and ?1,6-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.

SUBMITTER: Reiding KR

PROVIDER: S-EPMC6317482 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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

Reiding Karli R KR Bondt Albert A Hennig René R Gardner Richard A RA O'Flaherty Roisin R Trbojević-Akmačić Irena I Shubhakar Archana A Hazes Johanna M W JMW Reichl Udo U Fernandes Daryl L DL Pučić-Baković Maja M Rapp Erdmann E Spencer Daniel I R DIR Dolhain Radboud J E M RJEM Rudd Pauline M PM Lauc Gordan G Wuhrer Manfred M

Molecular & cellular proteomics : MCP 20180921 1

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 ...[more]

PMID: 30242110

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Project description:BackgroundLittle is known about gene expression changes induced by pregnancy in women with rheumatoid arthritis (RA) and healthy women because the few studies previously conducted did not have pre-pregnancy samples available as baseline. We have established a cohort of women with RA and healthy women followed prospectively from a pre-pregnancy baseline. In this study, we tested the hypothesis that pregnancy-induced changes in gene expression among women with RA who improve during pregnancy (pregDASimproved) overlap substantially with changes observed among healthy women and differ from changes observed among women with RA who worsen during pregnancy (pregDASworse).MethodsGlobal gene expression profiles were generated by RNA sequencing (RNA-seq) from 11 women with RA and 5 healthy women before pregnancy (T0) and at the third trimester (T3). Among the women with RA, eight showed an improvement in disease activity by T3, whereas three worsened. Differential expression analysis was used to identify genes demonstrating significant changes in expression within each of the RA and healthy groups (T3 vs T0), as well as between the groups at each time point. Gene set enrichment was assessed in terms of Gene Ontology processes and protein networks.ResultsA total of 1296 genes were differentially expressed between T3 and T0 among the 8 pregDASimproved women, with 161 genes showing at least two-fold change (FC) in expression by T3. The majority (108 of 161 genes) were also differentially expressed among healthy women (q<0.05, FC?2). Additionally, a small cluster of genes demonstrated contrasting changes in expression between the pregDASimproved and pregDASworse groups, all of which were inducible by type I interferon (IFN). These IFN-inducible genes were over-expressed at T3 compared to the T0 baseline among the pregDASimproved women.ConclusionsIn our pilot RNA-seq dataset, increased pregnancy-induced expression of type I IFN-inducible genes was observed among women with RA who improved during pregnancy, but not among women who worsened. These findings warrant further investigation into expression of these genes in RA pregnancy and their potential role in modulation of disease activity. These results are nevertheless preliminary and should be interpreted with caution until replicated in a larger sample.

Dataset Information

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

Publications

High-throughput Serum <i>N</i>-Glycomics: Method Comparison and Application to Study Rheumatoid Arthritis and Pregnancy-associated Changes.

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