Performance of the spectral assay library with different liquid chromatography gradients by DIA/SWATH-MS. (a) Number of unique peptides and (b) protein groups identified with chromatography gradients of different length. An approximately 20% increase in peptide and protein group identifications was observed in the 90 minutes gradient compared to the 15 minutes gradient length. The error bars indicate the variability within five replicates represented as standard error of the mean. These are calculated as the ratio of standard deviation of the number of quantified peptides or proteins observed in each gradient replicate to the square-root of the sample size (n = 5). The small yellow dots denote the number of identifications in each replicate. (c) The plot shows all identified protein groups ranked according to their abundance, highlighting the dynamic range of proteins that can be quantified with liquid chromatography of different gradient length. All gradients resulted in protein quantification across five orders of magnitude, with exception of the 15 min gradient which covered four orders of magnitude. (d) Pearson correlation of protein intensity values obtained from 1,483 proteotypic proteins that were quantified in all five technical replicates by both, 15 minutes and 90 minutes gradients. The high positive correlation indicates quantitative robustness between the gradient methods. (e) Distribution of the coefficient of variation (CV) of proteins identified in all five replicates at 1% protein FDR estimated by Spectronaut. The median CV of 10% (90 minutes gradient) to 11% (15 minutes gradient) correlates well with the expected technical variation. The first and third quartile are marked by a box with whisker marking a minimum/maximum value ranging to 12 interquartile and the median depicted as solid line. (f) Distribution of data points per elution peak for the different gradient methods estimated by Spectronaut. The first and third quartile are marked by a box with whisker marking a minimum/maximum value ranging to 3 interquartile and the median depicted as solid line.