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 nematode 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.