Extended Data Fig. 1: Peptide disambiguation with PIGEON.
From: Site-resolved energetic information from HX–MS experiments
a, HX–MS peptides are frequently degenerate in mass. b, The number of degenerate peptides in E. coli DHFR (ecDHFR) given measurement uncertainty (Δm/z ~0.001 within experiments, ~0.01 between experiments). c, ecDHFR peptides are duplicatively matched to the same peak. d, Disambiguation of peptides in panel c using tandem MS fragment information. The natural isotopic distribution corresponding to a single MS compound is shown, as extracted from the marked portion of the base peak intensity chromatogram. MS2 ions from this compound are compared to theoretical ions for the matched peptides, and the matches scored based on the degree of fragment ion support. e,(i), MS2 data are pooled for all replicates and matched to theoretical peptides for each protein in the dataset. e(ii), Systematic mass errors are corrected by fitting a trendline to the highest scoring matches and re-thresholding around the trendline. All but the best peak for each peptide are discarded. e(iii), Each peptide for which there exists an identical match and which is not adequately supported by MS fragments is dropped. e(iv), Each charge state for which there exists an identical match (as in iii.) but which has fragment support is either kept (‘KEEP’), or discarded (‘DROP’). These peptides co-elute and both contribute to the signal. e(v), The final cleaned dataset contains peptides with scores distributed over the full range. f, Initial score and m/z error distribution for a representative pooled dataset (as in E, i). g, A duplicate match where the fragment ions support one peptide but not the other. h, Impact of each PIGEON step (systematic error correction and disambiguation) and peptide selection mode for four ecDHFR HX–MS datasets (Supplementary Tables 1 and 8). The use of multiple proteases increases peptide coverage: fungal protease (FP), pepsin, nepenthesin (NP), and alanyl aminopeptidase (AP). The m/z and score distribution for one resulting peptide pool (red box) are shown.
