Fig. 3

Workflow to obtain histone proteoform data from brown adipose tissue and liver. (A) The general workflow includes tissue homogenization, nuclei isolation, acid extraction, HPLC separation of histone families, and LC–MS/MS data acquisition. HPLC separation of histones shows a clean separation of histone families and H3 variants for (B) BAT and (C) liver. (D) MS1 average spectra of histone H4 extracted from BAT from 40 to 80 min show clear + 16 to + 9 charge states matching the m/z of histone H4. (E) The MS1 spectrum of 11,193.8565 Da species (761.2961 m/z, charge + 15) matches a mass of H4 with N-terminal acetylation + 0 methylations + 3 acetylations + 0 phosphorylations with less than 10 ppm error. (F) The ion map from MS2 fragmentation of species from (E) shows unambiguous localization information for the proteoform H4 < N-acK12acK16acK31ac > from a mass change at less than 10 ppm error. (G) Annotated MS2 spectrum shows most abundant peaks are from the same proteoform, < N-acK12acK16acK31ac > , with less than 10 ppm error