All samples were transferred to their designated positions on a 96-well plate according to predetermined experimental design, that was blocked on case–control status, sex and age. Standard and blank samples were included in every batch (96-well plate) for quality control and batch correction.

The sensitivity of the method for IgG N-glycome profiling was previously determined [29] based on the minimal starting amount of IgG (µg) as well as on the proportion of its starting amount which is finally analysed chromatographically (i.e. applied to the column). Namely, the minimal starting amount of IgG is 10 µg, i.e. the minimal amount of IgG required for the reliable quantification of its released N-glycans using fluorescence detection is 0.42 µg. The precision of the method is reported with coefficients of variation (CV, %) that are calculated from the relative abundance of each glycan peak (%) of standard samples. Herein, five standard samples per plate were analysed, giving the average CV value for directly measured IgG glycan peaks of 4.28% (range 0.44–15.65%), whereas calculated derived glycan traits gave the average CV value of 1.63% (range 0.17–4.39%).

IgG was isolated from plasma samples by affinity chromatography as described previously [30]. In brief, IgG was isolated in a high-throughput manner, using 96-well protein G monolithic plates (BIA Separations, Slovenia), starting from 100 μl of plasma. Plasma was diluted 7× with phosphate buffered saline (PBS; Merck, Germany) and applied to the protein G plate. IgG was eluted with 1 ml of 0.1 M formic acid (Merck, Germany) and immediately neutralised with 1 M ammonium bicarbonate (Acros Organics, USA).

Isolated IgG samples were dried in a vacuum centrifuge. After drying, IgG was denatured with the addition of 30 μl of 1.33% SDS (w/v) (Invitrogen, USA) and by incubation at 65 °C for 10 min. Plasma samples (10 μl) were denatured with the addition of 20 μl of 2% SDS (w/v) (Invitrogen, USA) and by incubation at 65 °C for 10 min. From this point on, the procedure was identical for both IgG and plasma samples. After denaturation, 10 μl of 4% Igepal-CA630 (v/v) (Sigma Aldrich, USA) was added to the samples, and the mixture was shaken 15 min on a plate shaker (GFL, Germany). N-glycans were released with the addition of 1.2 U of PNGase F (Promega, USA) and overnight incubation at 37 °C.

The released N-glycans were labelled with 2-aminobenzamide (2-AB). The labelling mixture consisted of 2-AB (19.2 mg/ml; Sigma Aldrich, USA) and 2-picoline borane (44.8 mg/ml; Sigma Aldrich, USA) in dimethyl sulfoxide (Sigma Aldrich, USA) and glacial acetic acid (Merck, Germany) mixture (70:30 v/v). To each sample, 25 μl of labelling mixture was added, followed by 2 h incubation at 65 °C. Free label and reducing agent were removed from the samples using hydrophilic interaction liquid chromatography solid-phase extraction (HILIC-SPE). After incubation samples were brought to 96% of acetonitrile (ACN) by adding 700 μl of ACN (J.T. Baker, USA) and applied to each well of a 0.2 μm GHP filter plate (Pall Corporation, USA). Solvent was removed by application of vacuum using a vacuum manifold (Millipore Corporation, USA). All wells were prewashed with 70% ethanol (Sigma-Aldrich, St. Louis, MO, USA) and water, followed by equilibration with 96% ACN. Loaded samples were subsequently washed 5× with 96% ACN. N-glycans were eluted with water and stored at −20 °C until usage.

Fluorescently labelled N-glycans were separated by hydrophilic interaction liquid chromatography (HILIC) on Acquity UPLC H-Class instrument (Waters, USA) consisting of a quaternary solvent manager, sample manager, and a fluorescence detector, set with excitation and emission wavelengths of 250 and 428 nm, respectively. The instrument was under the control of Empower 3 software, build 3471 (Waters, Milford, USA). Labelled N-glycans were separated on a Waters BEH Glycan chromatography column, with 100 mM ammonium formate, pH 4.4, as solvent A and ACN as solvent B. In the case of IgG N-glycans, separation method used linear gradient of 75–62% acetonitrile at flow rate of 0.4 ml/min in a 27-min analytical run. For plasma protein N-glycans separation method used linear gradient of 70–53% acetonitrile at flow rate of 0.561 ml/min in a 25-min analytical run. The system was calibrated using an external standard of hydrolysed and 2-AB labelled glucose oligomers from which the retention times for the individual glycans were converted to glucose units (GU). Data processing was performed using an automatic processing method with a traditional integration algorithm after which each chromatogram was manually corrected to maintain the same intervals of integration for all the samples. The chromatograms were all separated in the same manner into 24 peaks (GP1–GP24) for IgG N-glycans and 39 peaks (GP1–GP39) for plasma protein N-glycans and are depicted in Supplementary Fig. 2 and Supplementary Fig. 3, respectively. Detailed description of glycan structures corresponding to each glycan peak is presented in Supplementary Table 1. Glycan peaks were analysed based on their elution positions and measured in glucose units, then compared to the reference values in the “GlycoStore” database (available at: https://glycostore.org/) for structure assignment. The amount of glycans in each peak was expressed as a percentage of the total integrated area. For IgG N-glycans, in addition to 24 directly measured glycan traits, eight derived traits were calculated (Supplementary Table 2). In the case of TwinsUK cohort, IgG N-glycan derived traits were calculated from plasma protein glycan profiles, based on known elution positions of predominat IgG N-glycan structures (Supplementary Table 3). In general, derived glycan traits average particular glycosylation features, such as galactosylation, fucosylation, bisecting GlcNAc and sialylation.

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