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Method development of immunoglobulin G purification from micro-volumes of human serum for untargeted and targeted proteomics-based antibody repertoire studies

机译:从微量人血清中纯化免疫球蛋白G的方法开发,用于基于蛋白质组学的非靶向和靶向抗体库研究

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Immunoglobulins (Igs) are major serum proteins which play important roles in immunity. Both untargeted and targeted proteomic workflows can be applied to investigate antigen-binding sites and the glycosylation profiles of Igs. For a more-comprehensive picture of IgG from human serum, we developed an IgG purification process and coupled the standardized method to untargeted and targeted proteomic workflows for IgG investigations. Parameters such as the type of purification beads, volume of the bead slurry, incubation conditions, and binding capacities were evaluated in this study. Only 2?μL of human serum was required for each sample. The performance of coupling the purification process to untargeted proteomics in the IgG analysis was evaluated by comparing normalized abundances of IgG subclass-specific peptides with quantification results from an ELISA. Pearson's correlation values were all 0.82. Targeted proteomic workflow was applied to serum samples from patients with autoimmune pancreatitis and from healthy controls, and the results corresponded to clinical findings that IgG4-related peptides/glycopeptides showed higher abundances in the diseased group. The developed IgG purification process is simple and requires small sample volume, and it can be coupled to targeted and untargeted proteomic workflows for clinical investigations in the future.
机译:免疫球蛋白(Igs)是主要的血清蛋白,在免疫中起重要作用。非靶向和靶向蛋白质组学工作流程均可用于研究Igs的抗原结合位点和糖基化谱。为了更全面地了解人血清中的IgG,我们开发了IgG纯化工艺,并将标准化方法与用于IgG研究的非靶向和靶向蛋白质组学工作流程相结合。在这项研究中评估了诸如纯化珠的类型,珠浆的体积,孵育条件和结合能力等参数。每个样品仅需要2?L人体血清。通过将IgG亚类特异性肽的标准化丰度与ELISA定量结果进行比较,评估了IgG分析中将纯化过程与非靶向蛋白质组学偶联的性能。皮尔逊的相关值均> 0.82。靶向蛋白质组学工作流程应用于自体免疫性胰腺炎患者和健康对照组的血清样品,其结果与临床发现相一致,即在患病组中IgG4相关肽/糖肽显示出更高的丰度。所开发的IgG纯化过程非常简单,需要的样品量很小,并且可以与靶向和非靶向的蛋白质组学工作流程相结合,以用于将来的临床研究。

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