Extended Data Fig. 3: LC-MS/MS validation of H3Q5his on peptides and in cells.

a, Proposed structure of histaminylated glutamine. b, High resolution/high mass accuracy mass spectrum of the triply charged histaminylated H3 tail peptide (ARTKQTARKSTGGKA-NH2; +histamine/+TG2 condition). The difference between measured and expected mass of the amidated peptide was 1.5 ppm. c, Extracted m/z (5 ppm) ion traces of the 2+, 3+ and 4+ amidated H3 tail peptide with (right panels) and without (left panels) histaminylated glutamine. Top, middle and bottom panels show signals measured under –/–, –/+ and +/+ (histamine/TG2) conditions, respectively. Integrated areas under curve are shown next to the peaks. Based on extracted signals, the reaction is close to complete. d, Tandem mass spectrum (35,000 resolution) of the doubly charged glutamine 5 histaminylated H3 tail peptide (+histamine/+TG2 condition). Selected fragment ions (y and b) are labelled. Lowest mass was m/z 100. Vertical lines in red and blue within the peptide sequence are used to show matched peptide fragment ions. e, LC-MS/MS identification of endogenous H3Q5his in HEK293T cells transfected with TG2-WT following histamine treatments. The cellular MS/MS spectra were aligned to that of a synthetic H3Q5his peptide, and y+ ions are annotated. Right: relative intensities of the most abundant y fragment ions from the H3Q5his peptide in 293T cells expressing TG2-WT vs. TG2-C277A. All experiments repeated 2X.