Abstract
Multiple sclerosis is a chronic demyelinating disorder characterized by the infiltration of auto-reactive immune cells from the periphery into the central nervous system resulting in axonal injury and neuronal cell death. Experimental autoimmune encephalomyelitis represents the best characterized animal model as common clinical, histological, and immunological features are recapitulated. A label-free mass spectrometric proteomics approach was used to detect differences in protein abundance within specific fractions of disease-affected tissues including the soluble lysate derived from the spinal cord and membrane protein-enriched peripheral blood mononuclear cells. Tissues were harvested from actively induced experimental autoimmune encephalomyelitis mice and sham-induced ("vehicle" control) counterparts at the disease peak followed by subsequent analysis by nanoflow liquid chromatography tandem mass spectrometry. Relative protein quantitation was performed using both intensity- and fragmentation-based approaches. After statistical evaluation of the data, over 500 and 250 differentially abundant proteins were identified in the spinal cord and peripheral blood mononuclear cell data sets, respectively. More than half of these observations have not previously been linked to the disease. The biological significance of all candidate disease markers has been elucidated through rigorous literature searches, pathway analysis, and validation studies. Results from comprehensive targeted mass spectrometry analyses have confirmed the differential abundance of ∼ 200 candidate markers (≥ twofold dysregulated expression) at a 70% success rate. This study is, to our knowledge, the first to examine the cell-surface proteome of peripheral blood mononuclear cells in experimental autoimmune encephalomyelitis. These data provide a unique mechanistic insight into the dynamics of peripheral immune cell infiltration into CNS-privileged sites at a molecular level and has identified several candidate markers, which represent promising targets for future multiple sclerosis therapies. The mass spectrometry proteomics data associated with this manuscript have been deposited to the ProteomeXchange Consortium with the data set identifier PXD000255.
Highlights
From the ‡Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria, 3010, Australia; §Banting and Best Department of Medical Research, Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada M5S 3E1; ¶Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, 3800, Australia; ʈDepartment of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 3E1
There are two fundamentally different approaches for performing label-free quantitation: [1] measuring the area under the chromatographic elution peak (AUC) based on each peptide precursor ion or the peptide signal intensity produced from the MS1 spectrum that correlates with peptide abundance in a complex mixture and [33, 34] [2] spectral counting (SC), which calculates the number of acquired fragment spectra (MS2) used to identify peptides from a given protein and is proportional to its abundance [35]
This is the first comparative proteomics analysis of this subcellular fraction derived from an animal model of Multiple sclerosis (MScl) with which a novel mass spectrometry-compatible detergent, MNG, has been used to solubilize the membrane protein-enriched fraction of PBMCs
Summary
Differential gene and protein expression profiles have been generated based on comparative analyses of healthy control and disease-affected tissues derived from clinical samples [7,8,9,10,11,12,13,14,15,16,17,18] and animal models (19 –29) These biomarker discovery platforms include gel-based approaches such as two-dimensional gel electrophoresis (2D-GE) [10, 17, 30], 2D-difference image gel electrophoresis (2D-DIGE) [9, 14], as well as shotgun proteomics techniques [11, 13, 16, 31, 32] incorporating the use of label-free or stable isotope labeling LC-MS-based strategies for quantitative proteomic studies. PBMCs are comprised of various lymphocyte populations including T and B cells, the major cellular components of the adaptive immune response in EAE and MScl and are known to infiltrate sites of CNS damage
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