Fig. 3: Deconvolution of chimeric DIA spectra.

a, Precursors quantified by CHIMERYS, DIA-NN and Spectronaut in at least one (orange) or two (gray) out of three replicate 2-h DIA single-shot measurements of two conditions (n = 6). Data taken from the LFQbench-type dataset and acquired on an Orbitrap QE HF-X with 8-Th isolation windows. Identifications are filtered at 1% run-specific precursor-level FDR or additionally at 1% run-specific precursor-level eFDR (Supplementary Methods). b, Apex intensities for precursors surviving (gray) or not surviving (red) 1% run-specific precursor-level eFDR for the same data as in a, analyzed by CHIMERYS, DIA-NN and Spectronaut. c, Number of fragment ions used for the quantification of precursors by CHIMERYS, DIA-NN and Spectronaut for the same data as in a. Identifications are filtered at 1% run-specific precursor-level FDR and colored by whether they also survive 1% run-specific precursor-level eFDR (Supplementary Methods). d, Example fragment ion XICs exported from Spectronaut for two high-scoring precursors with similar confidence (all six library fragments are shown). The top one is also identified by CHIMERYS, while the bottom one is not. Spectronaut identifies some precursors based entirely on fragment ions with intensities below or equal to 1 according to their report. The bottom panel shows the highest-scoring one of these precursors. The vertical dashed gray lines mark Spectronaut’s reported EG.StartRT and EG.EndRT for the corresponding precursors, with 105.88 min and 106.59 min for the top and 24.45 min and 24.74 min for the bottom XIC, respectively.