Fig. (2) Histone demethylase assay formats. (A) Quantitative mass spectrometry measures demethylation of histone peptide substrates by identification and relative quantitation of peptides by MS/MS. Here, LSD1 demethylates dimethyl-H3K9 to consecutively generate mono-methylated and unmodified H3K9 peptides. (B) Activity of JmjC lysine demethylases may be measured by coupling formaldehyde production to NAD⁺ reduction using the coupling enzyme FDH and monitoring NADH formation. (C) Lysine demethylation catalyzed by LSD1 can be measured using the coupling enzymes HRP or FDH to quantitate hydrogen peroxide or formaldehyde products, respectively. FDH-coupled formaldehyde detection is also applicable to the testing of 2-OG–dependent JmjC-domain histone demethylases.