Quantitation of Reactive Acyl-CoA Species MediatedProtein Acylation by HPLC–MS/MS

Recently discovered acylation by reactive acyl-CoA species is considered a novel regulatory mechanism in epigenetics and metabolism. Established analytical methods like Western blotting and proteomics fail to detect the plethora of acylation structures in a single analysis and lack the ability of absolute quantitation. In this paper, we developed an HPLC–MS/MS method for the simultaneous detection and quantitation of 14 acylated lysine species in biological samples. Extensive effort was invested into method validation resulting in recovery rates between 75 and 93% and levels of detection in the nanomolar range. Thus, we were able to quantitate 8 acylation structures in mouse liver, kidney, heart, and brain. Further enrichment by repetitive HPLC fractionation resulted in the quantitation of 6 additional acylation structures including 4 novel modifications: N6-acetoacetyl lysine, N6-isovaleryl lysine, N6-(2-methylbutyryl) lysine, and N6-tiglyl lysine.