Application of the CIRAD mass spectrometry approach for lysine acetylation site discovery

Mass spectrometry (MS)-based methods typically assess acetylation by detection of a diagnostic ion at 126.1 m/z, corresponding to the immonium ion of acetyl-lysine -NH(3), which is generated by collisionally induced dissociation. A novel implementation of this approach, based on the accurate mass and retention time technique, couples high mass resolution measurement with rapid cycling between low and elevated collision energies to generate intact and fragment high-resolution mass spectra. This allows acetyl lysine diagnostic ions at 126.1 m/z to be monitored and aligned to the precursor m/z based on retention time profile. The technique is termed Collisionally Induced Release of Acetyl Diagnostic. Sequence information is also obtained for acetylation site assignment. This technique to identify acetylation species is information independent as it does not require the sequence of the protein/peptides to identify acetylation, and thus complementary to data-dependent methods. It is suitable for analysis of acetylated peptides, or proteins enriched by immunoprecipitation with acetyl lysine-specific antibodies.