Wuhrer Manfred, Stam Jord C, van de Geijn Fleur E, Koeleman Carolien A M, Verrips C Theo, Dolhain Radboud J E M, Hokke Cornelis H, Deelder André M
Biomolecular Mass Spectrometry Unit, Department of Parasitology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.
Proteomics. 2007 Nov;7(22):4070-81. doi: 10.1002/pmic.200700289.
All four subclasses of human serum IgG contain a single N-glycosylation site in the constant region of their heavy chain, which is occupied by biantennary, largely core-fucosylated and partially truncated oligosaccharides, that may carry a bisecting N-acetylglucosamine and sialic acid residues. IgG glycosylation has been shown to be altered under various physiological and pathological circumstances. IgG N-glycan profiles vary with age, and galactosylation for example is enhanced during pregnancy. Several diseases including rheumatoid arthritis are associated with a reduction in galactosylation of the IgG N-glycans. Here, we describe a robust method for the isolation of IgG subclasses using protein A (binds IgG1, IgG2, and IgG4) and protein G (binds additionally IgG3) at the 96-well plate level, which is suitable for automation. Isolated IgGs were digested with trypsin, and obtained glycopeptides were analyzed by nano-LC-MS. Glycopeptides were characterized by CID as well as electron transfer dissociation (ETD). The method provided glycosylation profiles for IgG1, IgG2, IgG3, and IgG4 and revealed distinct differences in N-glycosylation between the four IgG subclasses. The changes in galactosylation associated with rheumatoid arthritis could readily be monitored. This method is suitable for the subclass-specific analysis of IgG glycosylation from clinical samples.
人血清IgG的所有四个亚类在其重链恒定区均含有一个N-糖基化位点，该位点被双触角型、大部分为核心岩藻糖基化且部分截短的寡糖占据，这些寡糖可能带有一个平分型N-乙酰葡糖胺和唾液酸残基。已证明IgG糖基化在各种生理和病理情况下会发生改变。IgG N-聚糖谱随年龄变化，例如在怀孕期间半乳糖基化会增强。包括类风湿性关节炎在内的几种疾病与IgG N-聚糖的半乳糖基化减少有关。在此，我们描述了一种在96孔板水平上使用蛋白A（结合IgG1、IgG2和IgG4）和蛋白G（还结合IgG3）分离IgG亚类的稳健方法，该方法适用于自动化操作。将分离的IgG用胰蛋白酶消化，所得糖肽通过纳升液相色谱-质谱联用仪进行分析。通过碰撞诱导解离（CID）以及电子转移解离（ETD）对糖肽进行表征。该方法提供了IgG1、IgG2、IgG3和IgG4的糖基化谱，并揭示了四个IgG亚类之间N-糖基化的明显差异。与类风湿性关节炎相关的半乳糖基化变化能够很容易地被监测到。该方法适用于对临床样本中IgG糖基化进行亚类特异性分析。
