Fig. 1: Isotopic arginine labeling and detection strategy for ATE1 substrates and arginylation sites.
From: An unbiased proteomic platform for ATE1-based arginylation profiling
a, Scheme of arginyl installation onto proteins by ribosomal synthesis (translational) and ATE1 (post-translational). b, Arginylation profiling platform for arginylation site and substrate discovery from biological samples. Lysate is labeled by isotopic arginine molecules (Arg10 and Arg0), respectively, using ATE1 assay, mixed and digested. Peptides are fractionated and analyzed by mass spectrometry in data-dependent acquisition mode. Proteomics data are searched to produce peptide identifications (IDs), among which peptide pairs modified by H and L Arg are further evaluated for MS1 isotopic features. c, Isotopic arginylation of a peptide ATE1 substrate. EICs were extracted using monoisotopic peaks based on calculated m/z values. EIC in black indicates chromatography of the unmodified peptide. d, Isotopic feature in MS1 spectra and their summary (upper, center and lower: 75, 50 and 25 percentiles, respectively). The first monoisotopic peaks in technical MS1 scans (n = 81) from a representative LC–MS run is set to 1 for normalization. Relative intensities of other isotopic peaks (n = 81, 80, 61, 28, 8, 81, 81, 79, 53, 25 and 3 technical scans) are displayed. A 10-ppm error is set for all MS1 isotopic peaks. e, Ratio summary of MS1 pairs in four replicates (n = 4) using doublet, quartet and sextet peaks. Detailed ratios from the replicates are provided. The numbers of pairs in quartet and sextet are normalized to the numbers of pairs in doublet from respective replicate. Nle, norleucine.
