Rapid glycomic analysis of serum EVs reveals altered N-glycosylation patterns in ASD

Objective laboratory diagnostics for autism spectrum disorder (ASD) are lacking, necessitating rapid clinical screening tools. Because serum extracellular vesicle (EV) N-glycosylation captures critical neurodevelopmental signatures, we developed a fast, biologically interpretable diagnostic strategy. EVs from ASD patients with language impairment and neurotypical controls were isolated using a rapid extra-polyethylene glycol precipitation/filtration (EPF) workflow, benchmarked against ultracentrifugation. Following MALDI-TOF/MS profiling, machine learning was re-evaluated using repeated nested cross-validation to reduce optimistic bias and potential information leakage. Among five classifiers, Random Forest (RF) showed the best overall balance across discrimination, calibration, and classification metrics. RF-based SHAP analysis provided transparent interpretation, highlighting key discriminative glycans, including H4N3S1F1, H5N5S1F1, and H3N5F1. To elucidate molecular mechanisms, we integrated public EV transcriptomic data. This revealed significant dysregulation of N-glycosylation machinery genes (e.g., MAN1A1, NEU1, OSTC, RPN2), whose expression directionally aligned with observed glycan shifts in synaptic pathways. Collectively, this rapid serum EV N-glycomic workflow, combined with leakage-controlled RF-based interpretation, provides a promising foundation for non-invasive ASD biomarker discovery and future multicenter validation.