Liver-derived extracellular vesicle proteomics reveals mechanisms and potential biomarkers of metabolic dysfunction-associated steatotic liver disease

Metabolic dysfunction–associated steatotic liver disease (MASLD) involves disturbances in hepatic metabolism, inflammation, and extracellular matrix remodeling, yet minimally invasive biomarkers for early detection and monitoring remain limited.We profiled the proteome of putative liver-derived extracellular vesicles (L-EVs) isolated directly from plasma using ASGR1/CYP2E1 immunocapture in a clinically characterized cohort including lean controls, obesity control group, and individuals with obesity and MASLD undergoing weight-loss intervention. L-EV preparations were characterized and their hepatic association assessed using nanoparticle tracking analysis, high-resolution flow cytometry (hFCM), CD9 immunogold transmission electron microscopy (TEM), Western blotting, and mass-spectrometry–based proteomics.Exploratory analyses suggested L-EV proteomic differences associated with obesity and MASLD, affecting metabolic, inflammatory, extracellular matrix, and fibrogenic pathways. Several MASLD-associated proteins, such as cathepsin L (CTSL) and the tumor protein D52-like 2 (TPD52L2), correlated strongly with liver fat, insulin resistance, and fibrosis indices. Weight loss induced dynamic shifts in L-EV proteins, most notably between one and five months, accompanying metabolic improvement and changes in fibrosis-related biomarkers. Biomarker analysis identified two combinatorial protein panels with moderate discriminatory performance for MASLD (AUCs 0.702–0.724). Three candidate proteins, SerpinE1, GNA13, and CTSL, were also detected in plasma L-EVs using hFCM, supporting their potential as biomarkers of MASLD within the ASGR1-positive circulating EV population. Overall, L-EV proteomics provides molecular insight into MASLD and its changes during weight loss, with potential applications in biomarker discovery and longitudinal monitoring.