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

Area of Science:

• Biochemistry
• Cell Biology
• Proteomics

Background:

• N-terminal acetylation (Nt-acetylation) is a widespread post-translational modification in eukaryotes, impacting protein fate.
• The role of Nt-acetylation in extracellular vesicles (EVs), key signaling mediators, is largely unknown.
• Extracellular vesicles (EVs) are critical for intercellular communication and hold potential as biomarkers.

Purpose of the Study:

• To develop a high-throughput workflow (evPNAS) for quantifying protein Nt-acetylation stoichiometry in EVs.
• To characterize the Nt-acetylation landscape in EVs derived from different cell types.
• To investigate the regulation and functional implications of Nt-acetylation in EVs.

Main Methods:

• High-purity isolation of EVs using functionalized magnetic beads.
• On-bead SP3 sample preparation for mass spectrometry.
• Optimized mass spectrometry data acquisition including singly charged peptides for comprehensive N-terminomics.

Main Results:

• Successfully quantified over 500 unique acetylated N-termini in HEK293 EVs with high reproducibility.
• Revealed a conserved Nt-acetylation landscape in EVs from H4 neuroglioma cells, enriched for NatA and NatB substrates.
• Demonstrated that Nt-acetylation is largely cotranslational and preserved during EV biogenesis.
• Showed that NatA activity impacts EV Nt-acetylation stoichiometry and that autophagy inhibition alters EV Nt-acetylation levels.

Conclusions:

• Nt-acetylation is a significant molecular signature in EVs, reflecting cellular enzymatic activity.
• The evPNAS platform enables comprehensive EV N-terminomics for biomarker discovery.
• Nt-acetylation patterns in EVs offer insights into EV biogenesis and cargo regulation.
• This work opens new avenues for understanding EV function and developing EV-based diagnostics.