Quantitating the Specificity and Selectivity of Gcn5-Mediated Acetylation of Histone H3
Figure 5
Multiple views of Gcn5-mediated H3 acetylation kinetics from bottom-up MS analysis, when [H3] = 12 μM, [Gcn5] = 180 nM, and [acetyl-CoA] = 200 μM.
(A) Changes of modifications on KSTGGKAPR: KaSTGGKaAPR (open circle), KaSTGGKpAPR (open square), KpSTGGKaAPR (open triangle), and KpSTGGKpAPR (solid reverse triangle). (B) Changes of modifications on KQLATKAAR: KaQLATKaAAR (solid circle), KaQLATKpAAR (solid square), KpQLATKaAAR (solid triangle), and KpQLATKpAAR (open reverse triangle). The data of (A) and (B) were directly obtained from MS SRM analysis. (C) Kinetics of fractions of acetylated K9 (solid circle), K14 (solid square), K18 (solid triangle), and K23 (open reverse triangle). (D) Kinetic of total acetylated lysine concentration on H3. The plots of (C) was generated from the calculation of (A) and (B). Apparently, K14 is the primary acetylation lysine by Gcn5 catalysis. While only the total or multiple acetylation is monitored, the acetylation amount from minor acetylation sites could be neglected, especially at short time points.