Verification of cerebrospinal fluid (CSF) biomarkers for multiple sclerosis and other neurological diseases is a major challenge due to a large number of candidates, limited sample material availability, disease and biological heterogeneity, and the lack of standardized assays. Furthermore, verification studies are often based on a low number of proteins from a single discovery experiment in medium-sized cohorts, where antibodies and surrogate peptides may differ, thus only providing an indication of proteins affected by the disease and not revealing the bigger picture or concluding on the validity of the markers. We here present a standard approach for locating promising biomarker candidates based on existing knowledge, resulting in high-quality assays covering the main biological processes affected by multiple sclerosis for comparable measurements over time. Biomarker candidates were located in CSF-PR (proteomics.uib.no/csf-pr), and further filtered based on estimated concentration in CSF and biological function. Peptide surrogates for internal standards were selected according to relevant criteria, parallel reaction monitoring (PRM) assays created, and extensive assay quality testing performed, i.e. intra- and inter-day variation, trypsin digestion status over time, and whether the peptides were able to separate multiple sclerosis patients and controls. Assays were developed for 25 proteins, represented by 72 peptides selected according to relevant guidelines and available literature and tested for assay peptide suitability. Stability testing revealed 64 peptides with low intra- and inter-day variations, with 44 also being stably digested after 16 h of trypsin digestion, and 37 furthermore showing a significant difference between multiple sclerosis and controls, thereby confirming literature findings. Calibration curves and the linear area of measurement have, so far, been determined for 17 of these peptides. We present 37 high-quality PRM assays across 21 CSF-proteins found to be affected by multiple sclerosis, along with a recommended workflow for future development of new assays. The assays can directly be used by others, thus enabling better comparison between studies. Finally, the assays can robustly and stably monitor biological processes in multiple sclerosis patients over time, thus potentially aiding in diagnosis and prognosis, and ultimately in treatment decisions.

中文翻译：

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开发用于多发性硬化症脑脊液生物标志物的稳健靶向蛋白质组学分析。

由于大量候选物、有限的样本材料可用性、疾病和生物异质性以及缺乏标准化测定，验证多发性硬化症和其他神经系统疾病的脑脊液 (CSF) 生物标志物是一项重大挑战。此外，验证研究通常基于中型队列中单个发现实验的少量蛋白质，其中抗体和替代肽可能不同，因此仅提供受疾病影响的蛋白质的指示，而不能揭示更大的图景或对标记的有效性作出结论。我们在这里提出了一种基于现有知识定位有希望的生物标志物候选者的标准方法，从而产生涵盖受多发性硬化症影响的主要生物过程的高质量测定，以便随着时间的推移进行可比较的测量。候选生物标志物位于 CSF-PR (proteomics.uib.no/csf-pr) 中，并根据 CSF 中的估计浓度和生物功能进一步过滤。根据相关标准选择用于内标的肽替代物，创建平行反应监测 (PRM) 测定，并进行广泛的测定质量测试，即日内和日间变化、胰蛋白酶消化状态随时间变化，以及肽是否能够将多发性硬化症患者和对照分开。针对 25 种蛋白质开发了检测方法，由根据相关指南和现有文献选择的 72 种肽代表，并测试了检测肽的适用性。稳定性测试揭示了 64 种肽段的日内和日间差异较小，其中 44 种肽在胰蛋白酶消化 16 小时后也被稳定消化，另外 37 种肽段显示多发性硬化症和对照之间存在显着差异，从而证实了文献发现。到目前为止，已经确定了其中 17 种肽的校准曲线和线性测量面积。我们针对 21 种被发现受多发性硬化症影响的 CSF 蛋白提供了 37 种高质量的 PRM 检测，以及未来开发新检测的推荐工作流程。这些化验可以直接被其他人使用，从而能够更好地比较研究。最后，随着时间的推移，这些检测方法可以稳健、稳定地监测多发性硬化症患者的生物过程，从而可能有助于诊断和预后，