Fig. 2: Protein detection and quantification performance.

a Number of quantified proteins for different injection amounts and instrument settings. Numbers of proteins detected in 1, 2, or all 3 injection replicates for each dataset (nanoflow 25% duty cycle scheme and standard scheme; Evosep 200, 100, and 60 samples per day (SPD) methods) are shown with different color shades, average numbers are indicated. Numbers reported by the original dia-PASEF workflow are shown in gray¹⁴. The numbers of proteins detected by both workflows are indicated with dashed horizontal lines. b Coefficients of variation (CV) distributions for the same datasets. The boxes correspond to the interquartile range, with the median indicated, and the whiskers extend to the 5–95% percentiles. c Quantification accuracy of dia-PASEF data analyzed with the new software workflow. We reanalyzed previously recorded data¹⁴, generated by spiking a yeast digest into a HeLa digest (200 ng) in different proportions (A, 45 ng, and B, 15 ng) and analyzed in triplicates using a 90-min nanoLC gradient. The runs were processed using a spectral library created with FragPipe. Horizontal lines indicate the expected ratios. On the boxplot, the boxes correspond to the interquartile range, with the median indicated, and the whiskers extend by a 1.5× interquartile range. Expected ratios are indicated with gray lines. d Analysis of a dilution series acquired on timsTOF Pro 2, a second-generation dia-PASEF-capable mass spectrometer, using a 93-min 300 nL/min gradient and a pre-column (Methods). Average protein numbers for triplicate injections after filtering at 1% run-specific protein q-value are shown. e Comparison of the performance of DIA-NN (gray) and Spectronaut (orange) on the leukemia dataset²¹. Total numbers of precursors and proteins (top), protein ID numbers distributions, and consistency of protein detection (bottom) are compared. The y-axis on the histograms represents the counts.