Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama, USA.
Department of Molecular and Cellular Biology, Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, Texas, USA.
Mol Cell Proteomics. 2021;20:100094. doi: 10.1016/j.mcpro.2021.100094. Epub 2021 May 13.
Identifying biomarkers is important for assessment of disease progression, prediction of symptom development, and determination of treatment effectiveness. While unbiased analyses of differential gene expression using next-generation sequencing methods are now routinely conducted, proteomics studies are more challenging because of traditional methods predominantly being low throughput and offering a limited dynamic range for simultaneous detection of hundreds of proteins that drastically differ in their intracellular abundance. We utilized a sensitive and high-throughput proteomic technique, reverse phase protein array (RPPA), to attain protein expression profiles of primary fibroblasts obtained from patients with Friedreich's ataxia (FRDA) and unaffected controls (CTRLs). The RPPA was designed to detect 217 proteins or phosphorylated proteins by individual antibody, and the specificity of each antibody was validated prior to the experiment. Among 62 fibroblast samples (44 FRDA and 18 CTRLs) analyzed, 30 proteins/phosphoproteins were significantly changed in FRDA fibroblasts compared with CTRL cells (p < 0.05), mostly representing signaling molecules and metabolic enzymes. As expected, frataxin was significantly downregulated in FRDA samples, thus serving as an internal CTRL for assay integrity. Extensive bioinformatics analyses were conducted to correlate differentially expressed proteins with critical disease parameters (e.g., selected symptoms, age of onset, guanine-adenine-adenine sizes, frataxin levels, and Functional Assessment Rating Scale scores). Members of the integrin family of proteins specifically associated with hearing loss in FRDA. Also, RPPA data, combined with results of transcriptome profiling, uncovered defects in the retinoic acid metabolism pathway in FRDA samples. Moreover, expression of aldehyde dehydrogenase family 1 member A3 differed significantly between cardiomyopathy-positive and cardiomyopathy-negative FRDA cohorts, demonstrating that metabolites such as retinol, retinal, or retinoic acid could become potential predictive biomarkers of cardiac presentation in FRDA.
鉴定生物标志物对于评估疾病进展、预测症状发展和确定治疗效果非常重要。虽然使用下一代测序方法进行无偏差异基因表达分析现在已经很常规，但蛋白质组学研究更具挑战性，因为传统方法主要是低通量的，并且对于同时检测数百种在细胞内丰度上差异很大的蛋白质提供的动态范围有限。我们利用一种敏感的高通量蛋白质组学技术，即反相蛋白质阵列（RPPA），获得了来自弗里德里希共济失调（FRDA）患者和未受影响对照（CTRL）的原代成纤维细胞的蛋白质表达谱。RPPA 设计用于通过单个抗体检测 217 种蛋白质或磷酸化蛋白质，并且在实验之前验证了每个抗体的特异性。在分析的 62 个成纤维细胞样本（44 个 FRDA 和 18 个 CTRL）中，与 CTRL 细胞相比，FRDA 成纤维细胞中有 30 种蛋白质/磷酸化蛋白质发生显著变化（p < 0.05），主要代表信号分子和代谢酶。如预期的那样，FRDA 样本中的 frataxin 显著下调，因此作为测定完整性的内部对照。进行了广泛的生物信息学分析，以将差异表达的蛋白质与关键疾病参数（例如，选定的症状、发病年龄、鸟嘌呤-腺嘌呤-腺嘌呤大小、frataxin 水平和功能评估评分）相关联。整联蛋白家族的成员与 FRDA 中的听力损失特别相关。此外，RPPA 数据与转录组谱分析结果相结合，揭示了 FRDA 样本中视黄酸代谢途径的缺陷。此外，醛脱氢酶家族 1 成员 A3 的表达在心肌病阳性和心肌病阴性 FRDA 队列之间存在显著差异，表明视黄醇、视网膜或视黄酸等代谢物可能成为 FRDA 心脏表现的潜在预测生物标志物。
