Top‐Down CSF Peptidomics Reveals Disease‐Associated Peptide Signatures and Oxidative Modifications in Multiple Sclerosis and Neuromyelitis Optica Spectrum Disorder

Multiple sclerosis and neuromyelitis optica spectrum disorder (NMOSD) are inflammatory demyelinating diseases of the CNS whose diagnosis is mainly based on clinical evidence, magnetic resonance imaging, and the analysis of cerebrospinal fluid (CSF). Current approaches provide only partial insight into disease-related biological processes, thus the need for deeper characterization of CNS-derived molecular signatures has emerged. Here, we applied a high-resolution top-down peptidomic approach in CSF from treatment naïve patients with CIS, relapsing-remitting multiple sclerosis (RRMS), NMOSD, and non-inflammatory neurological controls. The goal was to characterize their CSF endopeptidome, unravel insights into disease-associated proteolytic remodeling and molecular signatures not accessible through conventional proteomic approaches. 381 endogenous peptides were identified, including a substantial proportion of previously unreported sequences and oxidative PTMs. Quantitative analyses revealed disease-associated alteration of the CSF endopeptidome. A newly characterized C-terminal peptide derived from secretogranin-5 progressively decreased from CIS to RRMS and represented the most informative feature for discriminating patients using random forest (RF) analysis. In contrast, NMOSD patients exhibited a distinct profile characterized by widespread peptide reduction and decreased level of oxidation. Overall, these findings support the CSF endopeptidome as a biologically informative molecular layer associated with secretory pathway alteration, PTMs, and intrathecal inflammation. SIGNIFICANCE OF THE STUDY: Top-down peptidomics directly captures endogenous peptides and their native PTMs, providing information on proteolytic processing, that is lost in conventional digestion-based workflows or immune-based targeted approaches. In this top-down exploratory study, we characterized for the first time the CSF endopeptidome of treatment-naïve patients with CIS, RRMS stratified by oligoclonal-band (OCBs) status, NMOSD, and non-inflammatory neurological controls. The results revealed disease-associated patterns of granin-, osteopontin-, proSAAS-, and fibrinogen-derived peptides, especially for oxidative derivatives. A newly characterized C-terminal secretogranin-5 peptide showed a progressive cross-sectional decrease across the CIS and RRMS groups and contributed strongly to group classification, whereas NMOSD was characterized by widespread peptide depletion and a lower representation of oxidized peptide species. These findings support the CSF endopeptidome as a complementary molecular layer for investigating regulated proteolysis and redox-dependent alteration in inflammatory demyelinating disorders and identify peptide candidates for future validation in independent cohorts.