Department of Pharmacology and Molecular Sciences, School of Medicine, Johns Hopkins University, Baltimore, MD, 21205, USA.
Clin Proteomics. 2012 Jul 30;9(1):9. doi: 10.1186/1559-0275-9-9.
Multiple sclerosis (MS) is a chronic inflammatory disorder of the central nervous system (CNS). It involves damage to the myelin sheath surrounding axons and to the axons themselves. MS most often presents with a series of relapses and remissions but then evolves over a variable period of time into a slowly progressive form of neurological dysfunction termed secondary progressive MS (SPMS). The reasons for this change in clinical presentation are unclear. The absence of a diagnostic marker means that there is a lag time of several years before the diagnosis of SPMS can be established. At the same time, understanding the mechanisms that underlie SPMS is critical to the development of rational therapies for this untreatable stage of the disease.
Using high performance liquid chromatography-coupled mass spectrometry (HPLC); we have established a highly specific and sensitive selected reaction monitoring (SRM) assay. Our multiplexed SRM assay has facilitated the simultaneous detection of surrogate peptides originating from 26 proteins present in cerebrospinal fluid (CSF). Protein levels in CSF were generally ~200-fold lower than that in human sera. A limit of detection (LOD) was determined to be as low as one femtomol. We processed and analysed CSF samples from a total of 22 patients with SPMS, 7 patients with SPMS treated with lamotrigine, 12 patients with non-inflammatory neurological disorders (NIND) and 10 healthy controls (HC) for the levels of these 26 selected potential protein biomarkers. Our SRM data found one protein showing significant difference between SPMS and HC, three proteins differing between SPMS and NIND, two proteins between NIND and HC, and 11 protein biomarkers showing significant difference between a lamotrigine-treated and untreated SPMS group. Principal component analysis (PCA) revealed that these 26 proteins were correlated, and could be represented by four principal components. Overall, we established an efficient platform to develop and verify protein biomarkers in CSF, which can be easily adapted to other proteins of interest related to neurodegenerative diseases.
A highly specific and sensitive multiplex SRM-MS assay was established for development and verification of CSF protein biomarkers in SPMS. Five proteins were found to be expressed significantly differently between the three cohorts, SPMS, NIND and HC and 11 proteins associated with lamotrigine treatment, which we expect will further our current understanding of SPMS disease pathology and/or therapeutic intervention.
多发性硬化症（MS）是一种中枢神经系统（CNS）的慢性炎症性疾病。它涉及到轴突周围的髓鞘和轴突本身的损伤。MS 最常见的表现是一系列复发和缓解，但随后在一段时间内演变为一种称为继发进展性多发性硬化症（SPMS）的缓慢进展性神经功能障碍。这种临床表现变化的原因尚不清楚。由于缺乏诊断标志物，因此在能够确立 SPMS 的诊断之前，会有几年的时间延迟。同时，了解 SPMS 背后的机制对于开发针对这种无法治愈疾病阶段的合理疗法至关重要。
我们使用高效液相色谱-串联质谱（HPLC）建立了一种高度特异和敏感的选择反应监测（SRM）测定法。我们的多重 SRM 测定法促进了来自 26 种存在于脑脊液（CSF）中的蛋白质的替代肽的同时检测。CSF 中的蛋白质水平通常比人类血清中的低 200 倍。检测限（LOD）低至 1 飞摩尔。我们处理和分析了总共 22 例 SPMS 患者、7 例接受拉莫三嗪治疗的 SPMS 患者、12 例非炎症性神经疾病（NIND）患者和 10 名健康对照（HC）的 CSF 样本，以检测这些 26 种选定的潜在蛋白质生物标志物的水平。我们的 SRM 数据发现，1 种蛋白质在 SPMS 和 HC 之间存在显著差异，3 种蛋白质在 SPMS 和 NIND 之间存在差异，2 种蛋白质在 NIND 和 HC 之间存在差异，11 种蛋白质生物标志物在拉莫三嗪治疗和未治疗的 SPMS 组之间存在显著差异。主成分分析（PCA）表明，这 26 种蛋白质相互关联，可由四个主成分表示。总的来说，我们建立了一个高效的平台，用于开发和验证 SPMS 中的 CSF 蛋白质生物标志物，该平台可以很容易地适用于与神经退行性疾病相关的其他感兴趣的蛋白质。
我们建立了一种高度特异和敏感的多重 SRM-MS 测定法，用于开发和验证 SPMS 中的 CSF 蛋白质生物标志物。在三个队列（SPMS、NIND 和 HC）之间，有 5 种蛋白质的表达差异显著，11 种蛋白质与拉莫三嗪治疗相关，我们预计这将进一步加深我们对 SPMS 疾病病理和/或治疗干预的理解。
