Medical Research Center, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
Department of Laboratory Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College Hospital, Beijing, China.
Front Immunol. 2022 May 23;13:855513. doi: 10.3389/fimmu.2022.855513. eCollection 2022.
We aimed to explore and identify candidate protein biomarkers of cryoglobulinemia (CGE) in disease control patients with negative cryoglobulin (DC) or healthy controls (HCs).
The tandem mass tag (TMT)-labeled serum quantitative proteomics approach was used to identify differentially expressed proteins between the CGE and DC groups. Ingenuity pathway analysis was used for functional annotation of differentially expressed proteins. Biomarker candidates were validated in another cohort using the parallel reaction monitoring (PRM) method. Apolipoprotein A1 (APOA1), apolipoprotein CIII (APOC3), adiponectin, and proprotein convertase subtilisin/kexin type-9 (PCSK9), which represent key proteins involved in the cholesterol metabolism pathway, were further verified in an increased number of samples by enzyme-linked immunosorbent assay (ELISA).
A total of 1004 proteins were identified, of which 109 proteins were differentially expressed between the CGE and DC groups. These differentially expressed proteins were primarily involved in hepatic fibrosis/hepatic stellate cell activation and immune/inflammation-related pathways. In the disease and biofunction analysis, these proteins were mainly associated with the adhesion of blood cells, leukocyte migration, cholesterol transport, and transport of lipids. Twelve candidate biomarkers were validated by PRM-based proteomics, and proteins involved in the cholesterol metabolism pathway were further verified. APOA1, APOC3, adiponectin and PCSK9 concentrations were increased in CGE patients compared with healthy controls (P=0.0123, 0.1136, 0.5760, and 0.0019, respectively).
This report describes the first application of a TMT-PRM-ELISA workflow to identify and validate CGE-specific biomarkers in serum. APOA1 and PCSK9 have been confirmed to be increased in CGE patients, demonstrating that proteins involved in cholesterol metabolism are also implicated in the development of CGE. These findings contribute to pathogenesis research and biomarker discovery in CGE.
本研究旨在探索和鉴定阴性冷球蛋白血症（DC）或健康对照（HC）患者疾病控制期的候选蛋白生物标志物。
采用串联质量标签（TMT）标记血清定量蛋白质组学方法鉴定 CGE 组与 DC 组之间差异表达的蛋白质。使用 IPA 对差异表达蛋白进行功能注释。采用平行反应监测（PRM）方法在另一队列中验证候选生物标志物。进一步通过酶联免疫吸附测定（ELISA）在更多样本中验证载脂蛋白 A1（APOA1）、载脂蛋白 CIII（APOC3）、脂联素和前蛋白转化酶枯草溶菌素/柯萨奇蛋白酶 9（PCSK9），这些蛋白代表了胆固醇代谢途径中的关键蛋白。
共鉴定到 1004 种蛋白质，其中 CGE 组与 DC 组之间有 109 种蛋白质差异表达。这些差异表达蛋白主要参与肝纤维化/肝星状细胞激活和免疫/炎症相关途径。在疾病和生物功能分析中，这些蛋白主要与血细胞黏附、白细胞迁移、胆固醇转运和脂质转运有关。通过 PRM 基于蛋白质组学验证了 12 种候选生物标志物，进一步验证了胆固醇代谢途径相关蛋白。与健康对照组相比，CGE 患者的 APOA1、APOC3、脂联素和 PCSK9 浓度升高（P=0.0123、0.1136、0.5760 和 0.0019）。
本研究首次应用 TMT-PRM-ELISA 工作流程在血清中鉴定和验证 CGE 特异性生物标志物。APOA1 和 PCSK9 在 CGE 患者中被证实增加，表明胆固醇代谢相关蛋白也参与 CGE 的发生。这些发现有助于 CGE 的发病机制研究和生物标志物发现。
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