Identification of diagnostic biomarkers for relapsing-remitting multiple sclerosis in plasma by mass spectrometry-based proteomics Garcia-Silva, Victor Manuel Chicano-Galvez, Eduardo Garcia-Sanchez, María Isabel Peralbo-Molina, Ángela Hernández Valladares, María del Carmen Biomarker Extracellular vesicles Mass spectrometry This study received financial support from the University of Granada (Program 18 of the University’s own funding plan). The sponsor was not involved in the design, collection, analysis, or interpretation of data included in the study, in the writing of the manuscript, or in the decision to submit for publication. Funding for open access charge: Universidad de Granada / CBUA. In the absence of molecular biomarkers, the current diagnosis of multiple sclerosis is based on clinical assessment, neuroimaging, and detection of oligoclonal bands in cerebrospinal fluid. Early and rapid diagnosis using patient samples obtained by non-invasive methods would be a major advance in clinical management. We tested 5 different methods for preparation of the plasma proteome for liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. These were as follows: (1) single-pot, solid-phase-enhanced sample preparation (SP3), (2) iST and (3) ENRICH-iST of raw plasma, (4) SP3 of highly abundant-proteins depleted plasma (DEPL-SP3), and (5) SP3 of plasma extracellular vesicles (EV-SP3). DEPL-SP3 and EV-SP3 sample preparation workflows yielded the highest numbers of quantified plasma proteins. Using both methods, we analyzed the plasma proteome of 15 relapsing-remitting multiple sclerosis (RRMS) patients and 5 healthy controls. We found 54 and 35 regulated plasma proteins with DEPL-SP3 and EV-SP3 workflows, respectively. Among them, von Villebrand factor (VWF) was identified as a potential RRMS diagnostic biomarker. The use of sample preparation workflows for LC-MS/MS analysis that describe both the soluble and EV plasma proteomes might increase the likelihood of identifying new and robust RRMS biomarkers. 2026-01-07T08:35:01Z 2026-01-07T08:35:01Z 2025-12-22 journal article García-Silva VM, Chicano-Galvez E, García-Sánchez MI, Peralbo-Molina Á, Hernández-Valladares M. Identification of diagnostic biomarkers for relapsing-remitting multiple sclerosis in plasma by mass spectrometry-based proteomics. J Neuropathol Exp Neurol. 2025 Dec 22:nlaf145. doi: 10.1093/jnen/nlaf145. Epub ahead of print 0022-3069 1554-6578 https://hdl.handle.net/10481/109215 10.1093/jnen/nlaf145 eng http://creativecommons.org/licenses/by-nc-nd/3.0/ open access Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License Oxford University Press