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.