Fig. 1: Screening of Nε-lysines-modified histone H3(1–21) peptides as KDM3A substrates.
From: KDM3A catalyses the oxidation of acetyl-lysine to hydroxyacetyl-lysine on histone H3K9
a,b, KDM3A-catalysed demethylation of Nε-methylated lysine 9 (mass shift: −14 Da) (a) and acetyl-hydroxylation of Nε-acetylated lysine 9 (mass shift: +16 Da) (b) on histone H3. Each two-electron oxidation is coupled to the conversion of O2/2OG to succinate/CO2. c–h, KDM3A was incubated with histone H3(1–21) peptides containing Nε-modified lysines and analysed by MALDI–TOF MS. c–g, Representative MS spectra of H3(1–21)K9me1 (c), H3(1–21)K9me2 (d), H3(1–21)K9me3 (e), H3(1–21)K9ac (f) and H3(1–21)K9ac-[2H3] (g). A −14-Da mass shift indicates loss of one methyl group; a +16-Da shift is consistent with hydroxylation. Black: t = 0 min; red: t = 60 min. h, KDM3A-catalysed hydroxylation of H3(1–21)K9ac in the absence of assay components. Conditions: KDM3A, 0.5 µM; histone peptide, 10 µM; ascorbate (Asc), 500 µM; Fe(II), 50 µM; 2OG, 100 µM; TCEP, 500 µM; 60 min (37 °C). Data are presented as mean ± s.d. (n = 3 independent assays). 2OG, 2-oxoglutarate; Asc, sodium l-ascorbate; TCEP, tris(2-carboxyethyl)-phosphine.
