Profiling constitutive proteolytic events in vivo.

Area of Science:

• Proteomics
• Molecular Biology
• Biochemistry

Background:

• Proteases are essential for cellular functions, mediating constitutive proteolytic events in all known organisms.
• Understanding in vivo proteolysis is crucial for deciphering cellular mechanisms and disease pathologies.
• Current methods often struggle to differentiate true in vivo proteolytic events from artifacts or in vitro predictions.

Purpose of the Study:

• To develop and validate a robust method for defining constitutive proteolytic events in proteomes.
• To apply this method across diverse organisms, from bacteria to humans, to reveal conserved and specific processing events.
• To establish a reliable approach for distinguishing in vivo proteolysis from in vitro experimental outcomes.

Main Methods:

• Utilized N-terminal biotinylation of protein samples to specifically label newly synthesized or processed proteins.
• Employed affinity enrichment techniques to isolate biotinylated peptides from complex proteomes.
• Performed liquid chromatography-tandem mass spectrometry (LC-MS/MS) for sensitive and high-throughput identification and quantification of proteolytic events.

Main Results:

• Successfully profiled proteolytic events in Escherichia coli, yeast, mouse, and human proteomes.
• Demonstrated the in vivo specificity of methionine aminopeptidase in E. coli.
• Identified unexpected processing of mitochondrial transit peptides in yeast, mouse, and human samples, highlighting conserved regulatory mechanisms.

Conclusions:

• The developed N-terminal biotinylation and LC-MS/MS method is a simple, accessible, and broadly applicable tool for studying in vivo proteolysis.
• The findings provide critical insights into the specificity of protease action and protein processing in vivo.
• This approach enables accurate discrimination between genuine in vivo proteolytic events and those predicted or observed in vitro.