Quantitation of EZH2 wild-type and EZH2 Y641F (MutF) protein expression by mass spectrometry. (A) Tandem MS spectra of recombinant EZH2 wild-type and MutF peptides (residues 635-654) used in the MRM assay. Peptide fragments (y and b ions) identified are labeled, and transitions used in the MRM assay are circled in red (supplemental Figure 2). (B) Extracted ion chromatograms of MRM signals for EZH2 from the gel fractions containing total EZH2 from the DOHH2 and WSU-DLCL2 samples. Traces correspond to the wild-type (green), MutF (red), and common (blue, residues 442-456) peptides. For MRM quantification, peak areas for each coeluting transition were summed for each peptide. (C) External standard curve of mixtures of recombinant EZH2^WT and EZH2^Y641F proteins, in triplicate. The wild-type and MutF peptides show different inherent ionization ability as illustrated by the difference in heights and areas of their specific peaks, thus necessitating the use of an external calibration curve to determine the corresponding percentage of EZH2^WT and EZH2^Y641F proteins in the sample. The equation fit to the curve used for the calculations is shown. Points A and B on the graph represent 2 biologic replicates of total endogenous EZH2 immunoprecipitated from the WSU-DLCL2 cell line. An independent 50:50 mixture of EZH2^WT/EZH2^Y641F recombinant proteins was analyzed (labeled as point “1:1”) to validate the accuracy of the procedure.