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Central nervous system-specific proteins (CSPs), transported across the damaged blood-brain-barrier (BBB) to cerebrospinal fluid (CSF) and blood (serum), might be promising diagnostic, prognostic and predictive protein biomarkers of disease in individual multiple sclerosis (MS) patients because they are not expected to be present at appreciable levels in the circulation of healthy subjects. We hypothesized that microwave & magnetic (M2) proteomics of CSPs in brain tissue might be an effective means to prioritize putative CSP biomarkers for future immunoassays in serum. To test this hypothesis, we used M2 proteomics to longitudinally assess CSP expression in brain tissue from mice during experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Confirmation of central nervous system (CNS)-infiltrating inflammatory cell response and CSP expression in serum was achieved with cytokine ELISPOT and ELISA immunoassays, respectively, for selected CSPs. M2 proteomics (and ELISA) revealed characteristic CSP expression waves, including synapsin-1 and -II-spectrin, which peaked at day 7 in brain tissue (and serum) and preceded clinical EAE symptoms that began at day 10 and peaked at day 20. Moreover, M2 proteomics supports the concept that relatively few CNS-infiltrating inflammatory cells can have a disproportionally large impact on CSP expression prior to clinical manifestation of EAE. Introduction Multiple sclerosis (MS) is a debilitating neurological disease that affects approximately 2.5 million people globally1. MS patients experience episodes of inflammation and demyelination that are believed to be mediated by an autoimmune attack directed against central nervous system (CNS)-specific proteins (CSPs) such as components of the myelin sheath on axons, including: myelin basic protein, proteolipid protein and myelin oligodendrocyte glycoprotein. The autoimmune attack promotes an inflammatory cascade in the CNS highlighted by recruitment of innate- and adaptive-immune cells and release of inflammatory mediators that act in concert to damage the myelin sheath and neuronal axons. Autoreactive T cells have been shown to be key mediators of this attack against the myelin sheath. Once activated, these T cells migrate and infiltrate into the CNS, crossing the blood-brain-barrier (BBB) in a multistep process2,3. Infiltrating autoreactive T cells release inflammatory cytokines that modulate the activation of microglia, infiltrating macrophages and dendritic cells to release neurotoxic mediators, including nitric oxide and reactive-oxygen species (ROS)4,5. Macrophages, microglia, and dendritic cells are also actively involved in the inflammatory response6,7. While MS research has historically focused on inflammatory events in the CNS, such as the pathological role of cytokines8,9, a more detailed molecular understanding of the biology of other proteins, particularly CSPs transported across the BBB into cerebrospinal fluid (CSF) or serum, is critical to further our understanding of MS and to develop new biomarkers and treatments. Clinical diagnosis of MS, versus other similar neurological diseases, and classification into the consensus definitions of the four major subtypes of MS is based on a limited diagnostic repertoire, including: clinical appearance, disease history and laboratory imaging and/or diagnostics2,10,11,12,13,14. Currently, MS is classified into relapsing-remitting (RRMS), secondary-progressive (SPMS), primary-progressive (PPMS) and progressive-relapsing (PRMS). Approximately 80% of the patients initially develop the RRMS form of the disease, characterized by clinical attacks (relapses) with diverse neurological dysfunctions, followed by functional recovery (remission). More than half of these patients will eventually develop SPMS, characterized by progressive residual neurological deficiencies with or without attacks during the progressive phase13. Current immunomodulatory treatments ameliorate, but do not cure, MS, including: beta-interferons, therapeutic antibodies, glucocorticoids and glatiramer acetate. Responses to treatments are highly variable between patients and no accurate means exist to predict efficacy of a particular drug. Individual responses to treatment are typically evaluated by clinical measures of disease progression such as the expanded disability status scale (EDSS)15 and magnetic resonance imaging (MRI) of brain lesion volume16,17,18,19. However, these clinical measures lack sensitivity and specificity for a large population of MS patients, and they fail to show a strong correlation between specific treatments and their efficacy in slowing disease progression in individual MS patients. Thus, there is an urgent need for molecular biochemical markers with improved diagnostic, prognostic and predictive power. However, poorly understood variations of genetic, environmental, and socioeconomic factors in the MS patient population present profound challenges for biomarker research. A diagnostic matrix with a particular combination of biomarkers might enable more precise molecular stratification of individual patients into treatment groups. Moreover, a particular combination of biomarkers might be necessary because not all of these molecules are expected to be exclusive to MS and might also be found in other diseases and neurological disorders. Studying the relation of protein expression trajectories to disease progression within individual MS patients is expected to mitigate population variability to a certain degree and account for potential patient-specific factors. More than 24,000 genes are translated and post-translationally modified into an estimated 2 million protein isoforms in humans, encoding far more molecular diversity than the relatively static genome or transcriptome. Paradoxically, less than 100 proteins are routinely quantified in serum today20,21. Thus, the most sensitive (most true-positive) and specific (least false-positive) biomarkers are expected to be at the protein level. Notably, proteins must be measured directly due to the poor correlation between the transcriptome and proteome due to alternative splicing, post-translational modifications, single nucleotide polymorphisms, limiting ribosomes available for translation, mRNA and protein stability, and other factors (e.g., microRNA). CSPs, transported across the damaged BBB to CSF and blood (serum), might be promising diagnostic, prognostic and predictive (therapeutic) protein biomarkers of disease in individual MS patients because they are not expected to be present at appreciable levels in the circulation of healthy subjects. Compared to the highly variable clinical spectrum of individual MS patients, disease in groups of mice with experimental autoimmune encephalomyelitis (EAE), the major animal model for MS, is less heterogeneous and more synchronous, providing a strong rationale for preclinical biomarker studies. Our laboratory has pioneered microwave and magnetic (M2) proteomics for quantitative liquid chromatography-tandem mass spectrometry (LC/MS/MS) of relatively large numbers of CSPs and brain tissue specimens in murine EAE22,23. M2 proteomics synergizes off-line microwave-assisted chemical modification of CSPs bound to magnetic C8 microparticles, multiplexed isobaric encoding, and automated sample preparation with 96-well plates. M2 proteomics is also amino acid sequence- and post-translational modification-specific24,25,26,27. Despite its advantages, LC/MS/MS-based proteomics of low abundance CSPs can be confounded by masking effects due to high abundance proteins, particularly in CSF and serum where protein abundance can span up to 12 orders of magnitude28,29. In this study, we hypothesized that M2 proteomics of CSPs in brain tissue might be an effective means to prioritize putative CSP biomarkers for future immunoassays in CSF and serum, as previously suggested by us22 and others30. To test this hypothesis, we longitudinally assessed CSP expression in brain tissue from EAE mice. Confirmation of autoimmune T-cell response and CSP expression trajectories was achieved with cytokine ELISPOT and ELISA immunoassays, respectively, for selected CSPs. Importantly, M2 proteomics revealed characteristic CSP expression waves that preceded the onset of clinical EAE symptoms.