Comprehensive Profiling of Protein N-Terminal Acetylation Stoichiometry in Extracellular Vesicles

N-terminal acetylation (Nt-acetylation) is one of the most abundant protein modifications in eukaryotes, influencing protein stability, localization, interactions, etc. However, its role in shaping the proteome of extracellular vesicles (EVs), which are critical mediators of intercellular signaling, remains poorly characterized. Here, we present a streamlined and robust workflow to measure protein Nt-acetylation stoichiometry in EVs (evPNAS) with high throughput. This approach integrates high-purity EV isolation on chemically functionalized magnetic beads, on-bead SP3 sample preparation, and optimized data acquisition, incorporating coverage of singly charged peptides. We measured the stoichiometry of over 500 unique acetylated N-termini from HEK293 EVs with a high reproducibility and accuracy. Application of our evPNAS to H4 neuroglioma cells and their secreted EVs revealed a strongly conserved Nt-acetylation landscape characterized by enrichment of NatA and NatB substrates. These findings suggest that Nt-acetylation is predominantly established cotranslationally and is preserved during EV biogenesis. Additionally, siRNA-mediated knockdown of NatA resulted in a significant, substrate-specific decrease in Nt-acetylation stoichiometry in both cells and EVs, validating the functional imprint of NAT activity in EVs. Furthermore, treatment with Bafilomycin A1 to inhibit cellular autophagy increased Nt-acetylation levels in EVs, indicating regulation of Nt-acetylation on EV cargo in response to cellular stress. Together, our findings establish Nt-acetylation as an important molecular signature in EVs, reflecting intracellular enzymatic activity, while revealing unique aspects of EV functions. This study provides the first platform for EV N-terminomics and offers new opportunities for investigating EV-based biomarker discovery.