Extended Data Fig. 1: Optimization and characterization of the decryptE workflow for the profiling of drug induced expression changes at scale.

a) Boxplot showing the distribution of drug effect at 10,000 nM for all three measures of cell fitness. Each dot represents a drug (n = 144). Horizontal lines, boxes and whiskers of the boxplot depict the median, the range between the second and the third quartile and the 1.5-fold interquartile range. b) Dose-response curves of DHFR following treatment with Methotrexate for different times. c) Same as panel b) but for TYMS. d) Same as panel b) but for PLK1. e) Distribution of the relative number of identified protein groups from Jurkat cells as a function of the applied FAIMS compensation voltage (CV). f) Heatmap comparing the number and overlap of identified protein groups between any combination of two CVs (same data as in panel e). g) Number of identified protein groups from Jurkat cells as a function of the total LC-MS/MS time used per sample. h) Far left panel: schematic representation of the experimental design for testing the robustness of the micro-flow LC-FAIMS-MS/MS method. Colors represent the different sample types and the size of the ring segment is relative to the number of analyses in each segment (total of 250 samples analysed). Middle left panel: bar plot summarizing the number of proteins identified for each sample type. Error bars represent mean ± standard deviation (SD, n = 25 technical replicates for each sample type). Middle right panel: number of identified protein groups plotted as a function of the consecutive order in which the samples were analysed. Far right panel: Cumulative density plot summarizing the precision with which proteins were quantified across replicate experiments. Dotted lines indicate the respective fraction of proteins (50% and 90%) that were quantified with the given coefficient of variation (CoV). i) Left panel: Bar plot showing the number of identified proteins by single shot micro-flow LC MS/MS with or without FAIMS installed or by micro-flow LC-MS/MS after fractionation using high pH reversed phase chromatography (4 or 6 fractions) and using the specified amount of analysis time. Data are average values ± SD from n = 4 technical replicates. Right panel: same as panel h (far right, but for the data shown in the left panel of i).