A method to determine lysine acetylation stoichiometries

Area of Science:

• Biochemistry
• Proteomics
• Molecular Biology

Background:

• Lysine acetylation is a crucial post-translational modification regulating diverse biological processes.
• Quantifying the extent of lysine acetylation (stoichiometry) is challenging, hindering functional studies.

Purpose of the Study:

• To develop and validate a mass spectrometry-based method for accurate measurement of protein lysine acetylation stoichiometry.
• To assess acetylation stoichiometry across different cellular compartments in mammalian cells.

Main Methods:

• A mass spectrometry approach combining isotope labeling and diagnostic fragment ion detection was employed.
• Stoichiometry of ~750 acetylated peptides from mammalian cell lysates was determined.
• Histone H4 acetylation was cross-validated using sodium butyrate treatment and immunoblotting.

Main Results:

• The method successfully quantified lysine acetylation stoichiometry for numerous peptides.
• Histone H4 acetylation levels showed expected changes upon deacetylase inhibition.
• Acetylation stoichiometry was found to be significantly higher in nuclear proteins compared to mitochondrial and cytosolic proteins.

Conclusions:

• The developed mass spectrometry method provides a robust tool for measuring lysine acetylation stoichiometry.
• This technique facilitates detailed investigation into the functional implications of acetylation levels.
• Cellular compartment-specific differences in acetylation stoichiometry were highlighted, suggesting distinct regulatory mechanisms.