Center for Proteomics and Metabolomics, Leiden University Medical Center, 2300 RC Leiden, The Netherlands.
Glycoscience Research Laboratory, Genos Ltd., Zagreb 10000, Croatia.
Anal Chem. 2020 Oct 6;92(19):13172-13181. doi: 10.1021/acs.analchem.0c02342. Epub 2020 Sep 22.
Fc gamma receptors (FcγRs) translate antigen recognition by immunoglobulin G (IgG) into various immune responses. A better understanding of this key element of immunity promises novel insights into mechanisms of (auto-/allo-)immune diseases and more rationally designed antibody-based drugs. Glycosylation on both IgG and FcγR impacts their interaction dramatically. Regarding FcγR glycosylation profiling, major analytical challenges are associated with the presence of multiple glycosylation sites in close proximity and large structural heterogeneity. To address these challenges, we developed a straightforward and comprehensive analytical methodology to map FcγRIIIb glycosylation in primary human cells. After neutrophil isolation and immunoprecipitation, glycopeptides containing a single site each were generated by a dual-protease in-gel digestion. The complex mixture was resolved by liquid chromatography-tandem mass spectrometry (LC-MS/MS) providing information on the level of individual donors. In contrast to recently published alternatives for FcγRIIIb, we assessed its site-specific glycosylation in a single LC-MS/MS run and simultaneously determined the donor allotype. Studying FcγRIIIb derived from healthy donor neutrophils, we observed profound differences as compared to the soluble variant and the homologous FcγRIIIa on natural killer cells. This method will allow assessment of differences in FcγRIII glycosylation between individuals, cell types, subcellular locations, and pathophysiological conditions.
Fc 受体（FcγRs）将免疫球蛋白 G（IgG）对抗原的识别转化为各种免疫反应。更好地了解这一免疫关键要素有望为（自身/同种）免疫疾病的机制提供新的见解，并能更合理地设计基于抗体的药物。IgG 和 FcγR 上的糖基化对它们的相互作用有很大的影响。关于 FcγR 糖基化分析，主要的分析挑战与多个糖基化位点的紧密接近和结构的巨大异质性有关。为了解决这些挑战，我们开发了一种简单而全面的分析方法来描绘原代人细胞中 FcγRIIIb 的糖基化。在分离中性粒细胞并进行免疫沉淀后，通过双蛋白酶胶内消化产生每个糖肽各含一个糖基化位点。通过液相色谱-串联质谱（LC-MS/MS）对复杂混合物进行分离，提供了关于单个供体水平的信息。与最近发表的用于 FcγRIIIb 的替代方法不同，我们在单个 LC-MS/MS 运行中评估了其特定位点的糖基化，并同时确定了供体同种异型。研究源自健康供体中性粒细胞的 FcγRIIIb，与可溶性变体和自然杀伤细胞中的同源 FcγRIIIa 相比，我们观察到了明显的差异。该方法将能够评估个体、细胞类型、亚细胞位置和病理生理条件下 FcγRIII 糖基化的差异。
