Development of protein biomarkers in cerebrospinal fluid for secondary progressive multiple sclerosis using selected reaction monitoring mass spectrometry (SRM-MS)

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.

Biomarker Report from the Phase II Lamotrigine Trial in Secondary Progressive MS – Neurofilament as a Surrogate of Disease Progression

“…Correction for multiple comparisons was not performed at this stage as the work is exploratory in nature and positive findings should be tested in future prospective studies. However, as a collaborative study with John Hopkins University the CSF samples were analysed using mass spectrometry based on serum drug adherence [22], which revealed a significant reduction in 11 protein biomarkers, including neurofilament.…”

A type 2 biomarker separates relapsing-remitting from secondary progressive multiple sclerosis

“…1 Identifying biomarkers that can distinguish between the different clinical phenotypes of MS is an important goal to ensure that the appropriate treatment regimens are adopted in a timely fashion. [2][3][4] Furthermore, such biomarkers may provide new insight into the pathologic basis for the progressive process and lead to the development of effective treatments for disability prevention.…”

The emergence of top-down proteomics in clinical research

“…Examples of MRM successes in clinical research include the following: the quantification of proteins in the cerebrospinal fluid to aid understanding of the later stages of multiple sclerosis [17]; the development of quantitative validation techniques for plasma biomarkers, with LOQs reaching picograms per milliliter [13]; and the demonstration of robust targeted assays for cancer-associated protein quantification in both plasma and urine samples from patients [18]. In the first example, Jia et al [17] used MRM to quantify 26 proteins from the cerebrospinal fluid of patients with secondary progressive multiple sclerosis.…”

“…In the first example, Jia et al [17] used MRM to quantify 26 proteins from the cerebrospinal fluid of patients with secondary progressive multiple sclerosis. They included patients with a non-inflammatory neurological disorder and healthy humans as controls.…”

“…They included patients with a non-inflammatory neurological disorder and healthy humans as controls. The many significant differences in the abundance of certain proteins between patient groups may hold true upon further sampling and could yield important insight and provide a new method for multiple sclerosis research [17]. In the second example, Keshishian et al [13] performed important empirical testing of serum-processing options and provided a method for achieving an LOQ appropriate for current serum biomarkers (low nanogram per milliliter), even while multiplexing the assay to monitor multiple analytes.…”