Abstract

The lack of reliable diagnostic and/or prognostic biomarkers for multiple sclerosis (MS) is the major obstacle to timely and accurate patient diagnosis in MS patients. We performed a detailed proteomic analysis of CSF samples of patients newly diagnosed with relapsing-remitting MS (RRMS) and healthy controls to identify new proteins associated with MS. Reflecting significantly higher prevalence of MS in women we included only women patients and controls in the study. To eliminate a potential effect of therapy on the CSF composition, only the therapy-naïve patients were included. Pooled CSF samples were processed in a technical duplicate, and labeled with stable-isotope coded TMT tags. To maximize the proteome coverage peptide fractionation using 2D-LC preceded mass analysis using Orbitrap Fusion Tribrid Mass Spectrometer. Of the identified 900 CSF proteins we found 69 proteins to be differentially abundant between patients and controls. In addition to several proteins identified as differentially abundant in MS patients previously, we observed several linked to MS for the first time, namely eosinophil-derived neurotoxin and Nogo receptor. Differential concentration between patients and controls was verified for ceruloplasmin and lysozyme-C using specific antibodies. Our data confirm differential abundance of several previously proposed protein markers, and provide indirect support for involvement of copper-iron disbalance in MS. Most importantly, we identified two new differentially abundant CSF proteins that seem to be directly connected with myelin loss and axonal damage via TLR2 signaling and Nogo-receptor pathway in women newly diagnosed with RRMS.

摘要

缺乏可靠的多发性硬化症（MS）诊断和/或预后生物标记物是MS患者及时准确诊断的主要障碍。我们对新诊断为复发缓解型MS（RRMS）的患者和健康对照的CSF样本进行了详细的蛋白质组学分析，以鉴定与MS相关的新蛋白质。为了反映女性多发性硬化症的患病率，我们在研究中仅包括女性患者和对照组。为了消除治疗对脑脊液成分的潜在影响，仅纳入未接受过治疗的患者。合并的CSF样品进行技术重复处理，并用稳定同位素编码的TMT标签标记。为了使蛋白质组覆盖率最大化，在使用Orbitrap Fusion Tribrid质谱仪进行2D-LC质谱分析之前进行肽分析。在鉴定出的900种CSF蛋白中，我们发现69种蛋白在患者和对照组之间差异明显。除了先前在MS患者中鉴定出差异丰富的几种蛋白质外，我们还首次观察到了与MS相关的几种蛋白质，即嗜酸性粒细胞衍生的神经毒素和Nogo受体。使用特异性抗体验证了铜蓝蛋白和溶菌酶-C在患者和对照组之间的差异浓度。我们的数据证实了几种先前提出的蛋白质标记物的丰度差异，并为MS中铜铁失衡的参与提供了间接支持。最重要的是，我们鉴定了两个新发现的差异丰富的CSF蛋白，这些蛋白似乎与TLR2信号传导和Nogo受体途径的髓磷脂丢失和轴突损伤直接相关，而这些女性新诊断为RRMS。