6.2. Mass spectrometry analysis for the identification of acetylated lysins
Liquid chromatography coupled to mass spectrometry (LC-MS/MS) systems have become the method of choice for the sequencing and identification of thousands of proteins and also is essential, for the study PTMs, due to it is sensitivity, precision, and accuracy. Despite lysine acetylation emerging as a frequently occurring PTM, its low abundance compared to non-acetylated proteins requires enrichment steps before the detection in high-throughput proteomics. Protein affinity purification and prefractionation by immunoprecipitation are commonly used to reduce sample complexity and enrich acetylated proteins/peptides (Mischerikow & Heck, 2011). As demonstrated by Choudhary et al. (2009), the affinity enrichment of acetylated peptides allowed to increase in the number of acetylation sites identified since, in a separate experiment without affinity enrichment, the number of acetylation sites was 60-fold lower (Choudhary et al., 2009).
Most of the published studies on prokaryotic organisms use purification systems to the enrichment of acetylated peptides for the global characterization of protein acetylation (Table 3). For example, an integrated approach combining pan antibodies for protein enrichment with OrbitrapTM mass spectrometry (MS/MS) for comprehensive profiling of lysine acetylation in E. coli was developed by Zhang et al. (2013). This study identified 1070 acetylation sites on 349 proteins, along with the identification of peptide sequences and the assignment of acetylated sites. With this approach, not only previously reported acetylated proteins were identified, but also new target proteins were found. For example, the analysis identified isocitrate lyase as a novel acetylated protein. Additionally, many novel acetylation sites were discovered (Zhang et al., 2013). The acetylome study of Vibrio parahemolyticus , S. eriocheiris, and Bacillusamyloliquefaciens , showed that the combination of immunoaffinity enrichment of lysine-acetylated peptides with LC-MS/MS (liquid chromatography–mass spectrometry), allows the identification of a greater number of acetylated proteins, which represent 13.6%, 44.69% and 32.9% of the total proteins in each bacterium (Lui et al., 2016; Meng et al., 2016; Pan et al., 2014).
As shown by Nakayasu and coworkers, protein enrichment is not an essential step in proteomics studies since the global proteome coverage of 48 organisms totaled 73,656 proteins (902,937 peptides) and 9,107 acetylated proteins identified, averaging ~190 per organism (24,397 total acetylated peptides, ~508 per organism). These numbers are substantially more extensive than several previous studies (Nakayasu et al., 2017).
Thus, mass spectrometry analysis is a powerful method to identify specific acetylated peptides and sites in a mixture of acetylated and non-acetylated peptides with a high confidence level. Also, it is possible to quantitatively characterize the acetylome and determine how the relative abundance of protein acetylation changes in response to different conditions (Diallo et al., 2019).