Background Autoantibodies are produced when tolerance to self-antigens is broken and they can be mediators of tissue injury and systemic inflammation. They are excellent biomarkers because they are minimally invasive to screen and are highly abundant in serum due to limited proteolysis and slow clearance. Conventionally used methods of identifying autoantibodies in patient sera include indirect immunofluorescence, enzyme-linked immunoabsorbent assays (ELISAs) and protein microarrays. Here we present a novel proteome-wide immuno-mass spectrometric method to identify serum autoantibody targets. Methods Serum samples from patients with inflammatory bowel disease (IBD) were analyzed by ELISA for the presence of autoantibodies to CUB and zona pellucida-like domain-containing protein 1 (CUZD1). Protein was extracted from the human pancreas as well as 16 other human tissues to make a complex tissue lysate protein mixture. Antibodies in patient sera were immobilized and purified on protein G magnetic beads and subsequently incubated with pancreatic lysate containing CUZD1 or the aforementioned complex tissue lysate. After extensive washing, antibody-bound protein antigens were trypsin-digested and identified using shotgun mass spectrometry. Results The protocol was optimized for the immunoaffinity purification of autoantibody targets from tissue lysate, using CUZD1 from pancreatic lysate and anti-CUZD1 autoantibodies present in IBD patient serum as a proof-of-concept. Pancreatic secretory granule membrane major glycoprotein 2, whose autoantibodies are a known biomarker of Crohn's disease, was also immunoprecipitated from IBD patient serum, as an additional internal positive control. Conclusions This study demonstrates the effectiveness of a proteomic approach to identify serum autoantibody targets, using immunoaffinity purification followed by tandem mass spectrometry. Our methodology is applicable for proteome-wide analysis of autoantibody targets in a wide variety of clinical settings.

中文翻译：

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血清自身抗体的全蛋白质组免疫质谱鉴定。

背景 当对自身抗原的耐受性被破坏时会产生自身抗体，它们可能是组织损伤和全身炎症的介质。它们是优秀的生物标志物，因为它们对筛选的侵入性微乎其微，并且由于有限的蛋白水解和缓慢的清除而在血清中含量非常丰富。鉴定患者血清中自身抗体的常规使用方法包括间接免疫荧光法、酶联免疫吸附测定法 (ELISA) 和蛋白质微阵列。在这里，我们提出了一种新的全蛋白质组免疫质谱方法来识别血清自身抗体靶标。方法 通过 ELISA 分析炎症性肠病 (IBD) 患者的血清样本中是否存在针对 CUB 和透明带样结构域蛋白 1 (CUZD1) 的自身抗体。从人胰腺以及其他 16 个人体组织中提取蛋白质，制成复杂的组织裂解物蛋白质混合物。将患者血清中的抗体固定并纯化在蛋白 G 磁珠上，然后与含有 CUZD1 的胰腺裂解物或上述复杂组织裂解物一起孵育。大量洗涤后，抗体结合蛋白抗原被胰蛋白酶消化，并使用鸟枪质谱法鉴定。结果 该方案针对组织裂解物中自身抗体靶标的免疫亲和纯化进行了优化，使用来自胰腺裂解物的 CUZD1 和 IBD 患者血清中存在的抗 CUZD1 自身抗体作为概念验证。胰腺分泌颗粒膜主要糖蛋白 2，其自身抗体是克罗恩病的已知生物标志物，也从 IBD 患者血清中免疫沉淀，作为额外的内部阳性对照。结论 本研究证明了使用免疫亲和纯化和串联质谱法鉴定血清自身抗体靶点的蛋白质组学方法的有效性。我们的方法适用于各种临床环境中自身抗体靶点的全蛋白质组分析。