Substrate specificity and product pattern of MLL3 protein variants. H3K4 unmethylated (H3; 1 to 19) and monomethylated H3 (1 to 17) peptides were methylated by MLL3-SET wild-type and MLL3-SET Y4884C mutant protein in the presence of complex members using unlabeled AdoMet. The samples were collected at different time points, and methylation was analyzed by mass spectrometry using the relative areas of the corresponding unmethylated and methylated peaks. (A) Matrix-assisted laser desorption/ionization (MALDI) spectra of the methylation of the H3K4me0 peptide with MLL3 wild-type (upper panel) and Y4884C (lower panel). The masses of the corresponding peptides are 2,423.4 Da (H3K4me0), 2,437.4 Da (H3K4me1), 2,451.4 Da (H3K4me2), and 2,465.4 Da (H3K4me3). (B) MALDI spectra of the methylation of the H3K4me1 peptide with MLL3 wild-type (upper panel) and Y4884C (lower panel). The masses of the corresponding peptides are 2,181.2 Da (H3K4me1), 2,195.2 Da (H3K4me2), and 2,209.2 Da (H3K4me3).