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).