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 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.

Keywords: Chromatography; Electrophoresis; Glycomics; Glycosylation; High Throughput Screening; Mass Spectrometry; Plasma or Serum Analysis; Pregnancy; Rheumatoid Arthritis.

Fig. 1.

The respective profiles of the same total plasma protein N-glycan standard as recorded by HILIC-UHPLC-FLD, xCGE-LIF and MALDI-TOF-MS. (A) Chromatogram as obtained by HILIC-UHPLC-FLD after 2-aminobenzamide labeling. (B) Electropherogram as obtained by xCGE-LIF after APTS labeling. (C) Mass spectrum as obtained by MALDI-TOF-MS after ethyl esterification, with species assigned as [M+Na]⁺. Signals of all recordings have been annotated to the best of knowledge, making use of the detections across the methods as well as established literature on biochemical pathways and plasma/serum N-glycosylation. The display of linkage has been restricted to the N-acetylneuraminic acids (sialic acids), which was principally acquired by MALDI-TOF-MS. Branching differences (galactose arm, bisection, fucose position) were only distinguishable by HILIC-UHPLC-FLD and xCGE-LIF. For full assignments of the signals, see supplemental Tables S1–S3, as well as supplemental Fig. S1.

Fig. 2.

Heat map visualizing the Pearson correlation between signals from MALDI-TOF-MS, HILIC-UHPLC-FLD (participant 2) and xCGE-LIF. (A) HILIC-UHPLC-FLD (horizontal) with MALDI-TOF-MS (vertical). (B) xCGE-LIF (horizontal) with MALDI-TOF-MS (vertical). (C) HILIC-UHPLC-FLD (horizontal) with xCGE-LIF (vertical). Signal correlations were calculated on clinical data, and represent similarity of behavior with biological phenotypes such as pregnancy and RA as well as technical variation. Crosses (X) indicate correlations significant below a p value of 1·10⁻⁵, whereas dots (.) indicate correlation below a p value of 0.05. For similar heat maps between non-MS methods and derived traits see supplemental Figs. S4–S6. H = hexose, N = N-acetylhexosamine, F = deoxyhexose (fucose), L = (lactonized) α2,3-linked N-acetylneuraminic acid, and E = (ethyl esterified) α2,6-linked N-acetylneuraminic acid.

Fig. 3.

Comparability of HILIC-UHPLC-FLD (participant 2) (left), xCGE-LIF (middle), and MALDI-TOF-MS (right) for the detection of clinical characteristics of pregnancy, RA and RA disease activity. (A) Comparability of single glycan signals (% area) with pregnancy (preconception = pc, third trimester of pregnancy = tm3, 26+ weeks postpartum = pp3), and RA (healthy = white, RA = gray). (B) Comparability of derived glycosylation traits with pregnancy and RA. (C) Derived trait differences detected uniquely by a single method with pregnancy and RA. (D) Association of derived glycosylation traits with RA disease activity (DAS28(3)-CRP). For a legend of the derived traits see supplemental Table S6.