6.2.1. Mass spectrometry analysis for the relative
quantification of acetylated lysins
Mass spectrometry is not inherently quantitative because proteolytic
peptides have different physiochemical properties (size, charge,
hydrophobicity, and more), which produce variations in the MS/MS
spectra. Therefore, for accurate quantification, it is generally
required to compare each peptide between experiments (Bantscheff et al.,
2007). For this reason, different labeling techniques coupled to LC-MS
have been developed: stable-isotope labeling with amino acids in cell
culture (SILAC), isotope-coded affinity tags (ICAT), tandem mass tag
(TMT), iTRAQ, multiplex isobaric tags, and heavy peptide AQUA are some
examples (Gingras et al., 2007; Lindemann et al., 2017; Zhang & Elias,
2017). Isotope labels can be introduced into amino acids metabolically,
chemically, or enzymatically. The labeled peptides are chemically
identical to the corresponding native peptide, and therefore the
difference in mass between the light and heavy peptides can be measured
in the mass spectrometer. So, the quantification is achieved by
comparing their respective signal intensities (Bantscheff et al., 2007;
Zhang & Elias, 2017). However, these methods have some limitations as
increased time and sample preparation complexity, high protein
concentration is required, the reagents used are expensive, incomplete
labeling, and the requirement for specific quantification software. So
far, only TMT and iTRAQ allow the comparison of multiple samples
simultaneously (Zhu et al., 2009).
An alternative strategy is the label-free quantification method for
analyzing two or more experiments. The relative quantification can be
made by comparing the direct mass spectrometric signal intensity for any
given peptide or counting the number of peptide-to-spectrum matches
(PSMs; spectral counting) obtained for each protein, as more abundant
proteins are more likely to be observed in peptide spectra (Bantscheff
et al., 2007; Lindemann et al., 2017; Zhu et al., 2009).
As shown in Table 4, using different labeling strategies, it has been
possible to quantify acetylated sites and proteins robustly and
precisely, which has allowed to elucidate the role of N-acetylation in
processes such as biofilm formation and pathogenesis, determine how is
the dynamics of acetylation during bacterial growth and if the carbon
source influences the PTM rate. For example, the quantitative lysine
acetylome analysis of the pathogen bacterium Bacillus nematocidaB16 revealed that during pathogenesis proteins involved in the synthesis
of
nematodeĀ attractants
and the secretion of the main virulence factors of B16 were acetylated
and that the acetylation levels of different lysine sites were regulated
significantly differently in the presence of nematodes. The results
suggested that lysine acetylation may play a role in regulating
B16-C. elegans interaction (Sun et al., 2018). For E.
coli , this analysis showed that many acetylated lysine residues are
regulated in an acetyl phosphate (acP)-dependent manner, demonstrating
that chemical Nε-lysine acetylation is a viable mechanism (Kuhn et al.,
2014). Gaviard et al. (2018) evidenced the importance of carrying out a
quantitative study since, in the analysis of P. aeruginosaproteome, it was found that the number of acetylated peptides varies
depending on the carbon source. However, the quantification of
acetylated peptides did not show a significant abundance difference.