Autoantibodies are a hallmark of autoimmune diseases. Autoantibody screening by indirect immunofluorescence staining of HEp-2 cells with patient sera is a current standard in clinical practice. Differential diagnosis of autoimmune disorders is based on commonly recognizable nuclear and cytoplasmic staining patterns. In this study, we attempted to identify as many autoantigens as possible from HEp-2 cells using a unique proteomic DS-affinity enrichment strategy. HEp-2 cells were cultured and lysed. Total proteins were extracted from cell lysate and fractionated with DS-Sepharose resins. Proteins were eluted with salt gradients, and fractions with low to high affinity were collected and sequenced by mass spectrometry. Literature text mining was conducted to verify the autoantigenicity of each protein. Protein interaction network and pathway analyses were performed on all identified proteins. This study identified 107 proteins from fractions with low to high DS-affinity. Of these, 78 are verified autoantigens with previous reports as targets of autoantibodies, whereas 29 might be potential autoantigens yet to be verified. Among the 107 proteins, 82 can be located to nucleus and 15 to the mitotic cell cycle, which may correspond to the dominance of nuclear and mitotic staining patterns in HEp-2 test. There are 55 vesicle-associated proteins and 12 ribonucleoprotein granule proteins, which may contribute to the diverse speckled patterns in HEp-2 stains. There are also 32 proteins related to the cytoskeleton. Protein network analysis indicates that these proteins have significantly more interactions among themselves than would be expected of a random set, with the top 3 networks being mRNA metabolic process regulation, apoptosis, and DNA conformation change. This study provides a proteomic repertoire of confirmed and potential autoantigens for future studies, and the findings are consistent with a mechanism for autoantigenicity: how self-molecules may form molecular complexes with DS to elicit autoimmunity. Our data contribute to the molecular etiology of autoimmunity and may deepen our understanding of autoimmune diseases.

中文翻译：

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从经典自身抗体临床测试底物 HEp-2 细胞中鉴定出的自身抗原的蛋白质组库


自身抗体是自身免疫性疾病的标志。通过用患者血清对 HEp-2 细胞进行间接免疫荧光染色来筛选自身抗体是临床实践中的现行标准。自身免疫性疾病的鉴别诊断基于通常可识别的细胞核和细胞质染色模式。在本研究中，我们尝试使用独特的蛋白质组 DS 亲和力富集策略从 HEp-2 细胞中鉴定尽可能多的自身抗原。培养并裂解 HEp-2 细胞。从细胞裂解液中提取总蛋白并用 DS-Sepharose 树脂分级。用盐梯度洗脱蛋白质，收集具有低到高亲和力的级分并通过质谱法进行测序。进行文献文本挖掘以验证每种蛋白质的自身抗原性。对所有已识别的蛋白质进行蛋白质相互作用网络和途径分析。这项研究从 DS 亲和力从低到高的级分中鉴定出了 107 种蛋白质。其中，78 种是已验证的自身抗原，之前有报道作为自身抗体的目标，而 29 种可能是尚未验证的潜在自身抗原。在107个蛋白中，82个可以定位到细胞核，15个可以定位到有丝分裂细胞周期，这可能对应于HEp-2测试中核和有丝分裂染色模式的优势。有 55 种囊泡相关蛋白和 12 种核糖核蛋白颗粒蛋白，这可能导致 HEp-2 染色中出现不同的斑点图案。还有32种与细胞骨架相关的蛋白质。蛋白质网络分析表明，这些蛋白质之间的相互作用明显多于随机组的预期，其中前 3 个网络是 mRNA 代谢过程调节、细胞凋亡和 DNA 构象变化。 这项研究为未来的研究提供了已证实和潜在的自身抗原的蛋白质组库，并且研究结果与自身抗原性机制一致：自身分子如何与 DS 形成分子复合物以引发自身免疫。我们的数据有助于自身免疫的分子病因学研究，并可能加深我们对自身免疫疾病的理解。