Extended Data Fig. 5: Isoproterenol-induced change in Rad detected by mass spectrometry.

a, Right, the MS² spectrum (top) and TMT quantification parameters (bottom) for a Rad peptide changed upon treatment of murine hearts with isoproterenol. Shown is the MS² spectrum that was used to identify the Rad peptide IFGGIEDGPEAEAAGHTYDR. Left, m/z ratios for b and y ions identified in the spectrum and their deviation from theoretical m/z ratios. We measured the precursor mass as 778.71 Da with a charge of +3. Peptide modifications were +229.16 Da for TMT on the peptide N terminus and lysine residues, +57.02 Da for cysteine alkylation and +15.99 for methionine oxidation. Shown are ion injection times, isolation specificity, sum of signal-to-noise (SN) over all TMT channels, TMT raw intensities, adjusted intensities and final SN intensities used for relative quantification, as well as synchronous precursor selection (SPS) ion m/z ratios (isolated in the ion trap with Notch filtering; ‘Notch mz’ denotes the ion m/z ratio of individual isolated SPS ions prior to HCD fragmentation and MS³). b, Table showing gene names of proteins with P < 0.05 for the three approaches: cardiomyocytes isolated from α_1C–APEX and β_2B–APEX mice, and α_1C–APEX hearts. Genes in yellow are common to all groups, but note that for Mast2, the fold change is not consistent. Data are mean fold changes for five pairs of biologically independent pairs of α_1C–APEX2 cardiomyocyte samples, three pairs of biologically independent pairs of β_2B–APEX cardiomyocyte samples, and ten α_1C–APEX2 hearts, five without isoproterenol and five with isoproterenol. Non-adjusted unpaired two-tailed t-test. c, Venn diagram showing the data from b. Rrad, Rad; Prkaca, PKA catalytic subunit; Acss1, acyl-CoA synthetase short-chain family member 1. Rad is the only protein that is consistently changed amongst the three approaches.