Abstract
BackgroundMultiple 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.ResultsUsing 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.ConclusionsA 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.
Highlights
Multiple sclerosis (MS) is a chronic inflammatory disorder of the central nervous system (CNS)
Early MS associated symptoms will occur as a series of relapses and remissions whereby a patient will remain almost entirely asymptomatic for an extended period of time between temporary symptom “relapses.” Over a variable period of time, MS will typically evolve from relapsing-remitting multiple sclerosis (RRMS) into a slowly progressive form of neurological dysfunction termed secondary progressive multiple sclerosis (SPMS) [3]
cerebrospinal fluid (CSF) samples were obtained from patient cohorts consisting of 12 secondary progressive MS (SPMS) subjects from the National Institute of Neurological Disorders and Stroke (NINDS) at NIH, 10 SPMS subjects from the placebo group involved in clinical trial of lamotrigine, 12 non-inflammatory neurological disorder (NIND) controls from NINDS/NIH and 10 healthy controls from the University of Hawaii
Summary
Multiple sclerosis (MS) is a chronic inflammatory disorder of the central nervous system (CNS). Protein biomarkers differentially expressed in MS patients as compared to healthy individuals or patients suffering from other neurological disorders have great clinical potential - as early diagnostics, but as potential prognostic markers to be used for monitoring disease course and evaluating treatment efficacy [4,5,6]. Identification of such markers would further our current understanding of MS disease pathology, pinpointing novel protein targets or signalling pathways for therapeutic intervention
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