5.3. Identification, Localization, and Quantitative Analysis of Acetylated Proteins

Edman degradation, MS, and NMR are common methods used for the identification, localization, and quantitation of acetylated proteins. Edman degradation is commonly used to sequence unmodified peptides and has also been used to identify acetylated lysine. However, Edman degradation requires a large number of purified samples and can only detect 20–25 residues, which is time-consuming. Hence, this is not an optimal method for studying protein acetylation [19].

MS is an important method for the study of protein acetylation, and it can detect acetylated proteins or peptides, identify acetylated sites, and quantify acetylated proteins. In general, the analysis of protein acetylation is carried out by a continuous process that includes the digestion of proteins; the enrichment or fractionation of acetylated peptides; MS or MS/MS; and bioinformatic analysis. The quality of peptides is critical for the accuracy and precision of the MS results. Ahmed et al. improved the content and purity of peptides through methods including in-gel digestion and filter-aided sample preparation (FASP) [60].

NMR is a non-destructive technique used to study acetylation. Selective isotope labeling, high-resolution NMR, and 2-D heteronuclear NMR (¹H-¹⁵N and ¹H-¹³C NMR) are commonly used to identify acetylation [80,81,82]. However, NMR is used only for analyzing acetylated peptides, not proteins or complex mixtures, because overlapping peaks may impact the protein analysis results.