6.2.2. Stoichiometry of site-specific lysine acetylation
Although relative quantification is a powerful technique that allows to establish if there are variations in the acetylation levels in different conditions, it is limited by the fact that the changes are relative to the total protein abundance, which can vary from one condition to another, giving misinterpretations about the physiological significance of this PTM. Analysis of acetylation stoichiometry or occupancy can allow us to identify the critical acetylation sites whose changes in abundance are physiologically more important.
However, determining the acetylation stoichiometry is a complex task, mainly because the ionization efficiency of modified and unmodified peptides in a mass spectrometer is different. For this reason, various working groups have reported methods and workflows for the precise quantification of site-specific protein acetylation occupancy, which are based on the comparison of the proportion of endogenously acetylated lysine versus chemically labeled lysine that is not endogenously acetylated (Baeza et al., 2014; Gil et al., 2017; Miyagi, 2017; Weinert et al., 2014; Weinert et al., 2017; Wei et al., 2018).
The first protocol developed for directly quantifying stoichiometric of site-specific acetylation in bacteria was based on chemical acetylation of free lysine residues with isotopic acetic anhydride, followed by trypsin cleavage and MS analysis. The method was applied to analyze the entire proteome of E. coli , specifically to determine the role of deacetylases, CobB, on both site-specific and global acetylation (Baeza et al., 2014). In a similar study, Weiner et al. (2017) determined the absolute acetylation stoichiometry but used a serial dilution of SILAC-labeled peptides (SDSILAC). Although the methodologies differ in the way of labeling the peptides, with both approaches, it was shown that sirtuin deacetylase deficiency affects central metabolism and leads to both site-specific and global changes in protein acetylation stoichiometry (Baeza et al., 2014; Weiner et al., 2017).
Exploring the relationship of chemical acetylation and how it affects the enzymatic activity of glycolytic proteins, it was found that possibly a maximum of 10% of non-enzymatically acetylated proteins reach a stoichiometry that could inhibit their activity and that enzymes such as GapA and GpmA are acetylated at high stoichiometry (Schastnaya et al., 2023). The authors suggest that AcP-acetylation is specific and may exert control over metabolism.
Knowing the stoichiometry of acetylation can help to establish how it changes and whether it exerts a regulatory effect or only has a constitutive function necessary for protein folding or stable interaction.