Acetylated and methylated peptides, in common with other PTM peptides, are typically present at substoichiometric levels relative to nonmodified peptides. As such, they are generally not detected by MS without specific enrichment. The classical biochemical method of immunoprecipitation has been widely employed. Naturally occurring, PTM-specific binding “reader” domains provide an alternative capture reagent with potential for customization via protein engineering for optimal pan recognition of acetylated peptides or for those within specific amino acid or PTM context [93]. Chemistry-based approaches, such as variant of the biotin switch technique, are unbiased with respect to consensus site motifs for acetylation and as such provide a useful addition to the analytical toolkit [94].

These are described later and can be used in isolation or combined with prior biochemical separations based on alterations in chemical and physical characteristics of the post-translationally modified protein, relative to unmodified forms. These properties include charge, pi, and hydrophobicity. Examples of fractionation techniques include isoelectric focusing [95] and different chromatography separations: basic reversed-phase HPLC [19, 96], HILIC and strong cation exchange (SCX) [97], and WCX/HILIC [25].

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