biostudies-literatureMomcilovic ADutch Research Council (NWO)European Commission4518-4526https://www.ebi.ac.uk/biostudies/studies/S-EPMC7252899biostudies-literatureUnknown92(6)Immunoglobulin (Ig) glycosylation is recognized for its influence on Ig turnover and effector functions. However, the large-scale profiling of Ig glycosylation in a biomedical setting is challenged by the existence of different Ig isotypes and subclasses, their varying serum concentrations, and the presence of multiple glycosylation sites per Ig. Here, a high-throughput nanoliquid chromatography (LC)- mass spectrometry (MS)-based method for simultaneous analysis of IgG and IgA glycopeptides was developed and applied on a serum sample set from 185 healthy donors. Sample preparation from minute amounts of serum was performed in 96-well plate format. Prior to trypsin digestion, IgG and IgA were enriched simultaneously, followed by a one-step denaturation, reduction, and alkylation. The obtainAnalytical chemistrySimultaneous Immunoglobulin A and G Glycopeptide Profiling for High-Throughput Applications.PMC725289967642191116004722.016.008Koeleman CAMHipgrave Ederveen ALMesker WEFalck Dde Haan Nde Neef LATollenaar RWuhrer MBondt Ade Ru AMomcilovic Avan Veelen PDotz VfalseSimultaneous Immunoglobulin A and G Glycopeptide Profiling for High-Throughput Applications.Immunoglobulin (Ig) glycosylation is recognized for its influence on Ig turnover and effector functions. However, the large-scale profiling of Ig glycosylation in a biomedical setting is challenged by the existence of different Ig isotypes and subclasses, their varying serum concentrations, and the presence of multiple glycosylation sites per Ig. Here, a high-throughput nanoliquid chromatography (LC)- mass spectrometry (MS)-based method for simultaneous analysis of IgG and IgA glycopeptides was developed and applied on a serum sample set from 185 healthy donors. Sample preparation from minute amounts of serum was performed in 96-well plate format. Prior to trypsin digestion, IgG and IgA were enriched simultaneously, followed by a one-step denaturation, reduction, and alkylation. The obtain2020-01-01T00:00:00Z2020 Mar2025-04-04T09:04:50.269Z2020-06-02T07:10:37ZS-EPMC72528993209188910.1021/acs.analchem.9b05722