Variability in label-free SRM coupled to Anubis automated analysis. Proteins are divided into ribosomal (RIB), fatty-acid synthesis (FAS), and virulence-associated proteins (Virulome), which are respectively high-, medium-, and low-abundant. (a) Median CVs across the peptides in 3 protein groups, for 6 × 10 technical replicates and 9 biological replicates. Error bars show the interquartile range. (b) Median CV ranges for the 6 × 10 technical replicates compared to median CV ranges 2 sets separately prepared replicates (A2.1apr + A2.2apr and A2.1jun + A2.2jun). (c) Median CV ranges for the 6 × 10 technical replicates compared to median CV ranges of 2 sets replicates analyzed at different times (A2.1apr + A2.1jun and A2.2apr + A2.2jun). (d) Median CVs with interquartile ranges for the technical replicate sets compared to all second batch replicates (A2.1apr, A2.2apr, A2.1jun and A2.2jun), with and without normalization. (e) Illustration of normalization by ribosomal housekeeping proteins (RS10_STRA1, RL22_STRP1, RL1_STRP1, and RS17_STRA1) in one biological replicate. Each row represents a peptide, and each column a replicate – grouped into 5 sets (). Color denotes the quantity of the peptide in that replicate compared to its average across all replicates. From comparing A2.1apr + A2.2apr to A2.1jun + A2.2jun it is clear that the time of analysis greatly affects the measured quantity. After normalization these differences are removed, allowing joining of the replicates.