Simultaneous 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 obtained nanoLC-MS data were subjected to semiautomated, targeted feature integration and quality control. The combined and simplified protocol displayed high overall method repeatability, as assessed using pooled plasma and serum standards. Taking all samples together, 143 individual N- and O-glycopeptides were reliably quantified. These glycopeptides were attributable to 11 different peptide backbones, derived from IgG1, IgG2/3, IgG4, IgA1, IgA2, and the joining chain from dimeric IgA. Using this method, novel associations were found between IgA N- and O-glycosylation and age. Furthermore, previously reported associations of IgG Fc glycosylation with age in healthy individuals were confirmed. In conclusion, the new method paves the way for high-throughput multiprotein plasma glycoproteomics.免疫球蛋白（Immunoglobulin, Ig）糖基化因其对免疫球蛋白周转及效应功能的调控作用而受到广泛认可。然而，在生物医学场景中开展免疫球蛋白糖基化的大规模谱分析仍面临诸多挑战：不同免疫球蛋白同种型与亚类的存在、各异的血清浓度，以及单条免疫球蛋白上存在多个糖基化位点。本研究开发了一种基于高通量纳升液相色谱（LC）-质谱（MS）的方法，可同时分析免疫球蛋白G（IgG）与免疫球蛋白A（IgA）的糖肽，并将其应用于185名健康供体的血清样本集。该方法采用96孔板形式，仅需微量血清即可完成样品前处理：在胰蛋白酶消化前，同时富集IgG与IgA，随后进行一步法变性、还原与烷基化处理。所获得的纳LC-MS数据经过半自动化靶向特征整合与质量控制流程。使用混合血浆及血清标准品评估后发现，该整合简化的实验方案具有优异的整体方法重复性。对所有样本进行分析后，共可靠定量得到143种独立的N-糖肽与O-糖肽，这些糖肽源自11种不同的肽骨架，分别对应IgG1、IgG2/3、IgG4、IgA1、IgA2以及二聚体IgA的连接链。利用该方法，本研究发现了IgA的N-糖基化与O-糖基化与年龄之间的全新关联；同时验证了此前已报道的健康个体中IgG Fc段糖基化与年龄的关联。综上，本研究开发的新方法为高通量多蛋白血浆糖蛋白质组学研究铺平了道路。