Identifying Differentially Expressed Proteins via Plasma Exosomal Proteomics.

Extracellular vesicle (EV) proteomics can be used to study intercellular communication and find biomarkers of non-invasive diseases. Traditional separation methods (ultracentrifugation, size exclusion chromatography) and data-dependent acquisition (DDA) mass spectrometry usually have the drawbacks of copurification of pollutants, poor reproducibility, and insufficient sampling of low-abundance peptides. This protocol describes a workflow for label-free extracellular vesicle proteomics, which combines size exclusion chromatography for vesicle separation, data-independent acquisition (DIA) for deep discovery, and parallel reaction monitoring (PRM) for targeted verification. Plasma-derived extracellular vesicles are treated by standardized lysis, digestion, and LC-MS/MS procedures, so that the protein group of extracellular vesicles can be identified and quantified robustly. The main advantages of this scheme are that it can achieve high quantitative reproducibility, unbiased detection of low-intensity peptides, and seamless transition from discovery to targeted verification, while also being suitable for liquid biopsy samples and various cardiovascular diseases. Key features • Combines size exclusion chromatography (SEC)-based EV isolation with label-free data-independent acquisition (DIA) for deep, unbiased discovery-phase extracellular vesicle proteomics. • Employs parallel reaction monitoring (PRM) for orthogonal, highly sensitive targeted validation of candidate biomarkers discovered by DIA. • PRM records the entire, high-resolution fragment ion spectrum for the targeted peptide. • Developed for atrial fibrillation; adaptable to other cardiovascular diseases, therapy monitoring, and liquid biopsies.