Molecular components in blood, such as proteins, are used as biomarkers to detect or predict disease states, guide clinical interventions and aid in the development of therapies. While multiplexing proteomics methods promote discovery of such biomarkers, their translation to clinical use is difficult due to the lack of substantial evidence regarding their reliability as quantifiable indicators of disease state or outcome. To overcome this challenge, a novel orthogonal strategy was developed and used to assess the reliability of biomarkers and analytically corroborate already identified serum biomarkers for Duchenne muscular dystrophy (DMD). DMD is a monogenic incurable disease characterized by progressive muscle damage that currently lacks reliable and specific disease monitoring tools. Two technological platforms are used to detect and quantify the biomarkers in 72 longitudinally collected serum samples from DMD patients at 3 to 5 timepoints. Quantification of the biomarkers is achieved by detection of the same biomarker fragment either through interaction with validated antibodies in immuno-assays or through quantification of peptides by Parallel Reaction Monitoring Mass Spectrometry assay (PRM-MS). Five, out of ten biomarkers previously identified by affinity-based proteomics methods, were confirmed to be associated with DMD using the mass spectrometry-based method. Two biomarkers, carbonic anhydrase III and lactate dehydrogenase B, were quantified with two independent methods, sandwich immunoassays and PRM-MS, with Pearson correlations of 0.92 and 0.946 respectively. The median concentrations of CA3 and LDHB in DMD patients was elevated in comparison to those in healthy individuals by 35- and 3-fold, respectively. Levels of CA3 vary between 10.26 and 0.36 ng/ml in DMD patients whereas those of LDHB vary between 15.1 and 0.8 ng/ml. These results demonstrate that orthogonal assays can be used to assess the analytical reliability of biomarker quantification assays, providing a means to facilitate the translation of biomarkers to clinical practice. This strategy also warrants the development of the most relevant biomarkers, markers that can be reliably quantified with different proteomics methods.

中文翻译：

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正交蛋白质组学方法保证了杜氏肌营养不良症生物标志物的发展

血液中的分子成分，例如蛋白质，被用作生物标志物来检测或预测疾病状态、指导临床干预并帮助开发疗法。虽然多重蛋白质组学方法促进了此类生物标志物的发现，但由于缺乏大量证据证明它们作为疾病状态或结果的可量化指标的可靠性，因此很难将其转化为临床应用。为了克服这一挑战，开发了一种新的正交策略，用于评估生物标志物的可靠性，并通过分析证实已经确定的杜氏肌营养不良症 (DMD) 血清生物标志物。DMD 是一种单基因无法治愈的疾病，其特征是进行性肌肉损伤，目前缺乏可靠和特定的疾病监测工具。两个技术平台被用来检测和量化 72 个纵向收集的 DMD 患者血清样本在 3 到 5 个时间点的生物标志物。通过在免疫测定中与经过验证的抗体相互作用或通过平行反应监测质谱测定 (PRM-MS) 对肽进行定量来检测相同的生物标志物片段，从而实现生物标志物的量化。先前通过基于亲和力的蛋白质组学方法鉴定的十个生物标志物中有五个被证实与使用基于质谱的方法的 DMD 相关。两种生物标志物，碳酸酐酶 III 和乳酸脱氢酶 B，用两种独立的方法（夹心免疫测定和 PRM-MS）进行量化，皮尔逊相关性分别为 0.92 和 0.946。与健康个体相比，DMD 患者的 CA3 和 LDHB 浓度中值分别升高了 35 倍和 3 倍。DMD 患者的 CA3 水平在 10.26 和 0.36 ng/ml 之间变化，而 LDHB 的水平在 15.1 和 0.8 ng/ml 之间变化。这些结果表明，正交分析可用于评估生物标志物定量分析的分析可靠性，提供一种促进生物标志物转化为临床实践的方法。该策略还保证了最相关的生物标志物的开发，这些标志物可以用不同的蛋白质组学方法可靠地量化。这些结果表明，正交分析可用于评估生物标志物定量分析的分析可靠性，提供一种促进生物标志物转化为临床实践的方法。该策略还保证了最相关的生物标志物的开发，这些标志物可以用不同的蛋白质组学方法可靠地量化。这些结果表明，正交分析可用于评估生物标志物定量分析的分析可靠性，提供一种促进生物标志物转化为临床实践的方法。该策略还保证了最相关的生物标志物的开发，这些标志物可以用不同的蛋白质组学方法可靠地量化。