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.