Summary of the Anubis algorithm. (a) Example of data from measurement of a target peptide. (b) User-provided reference chromatograms of the target peptide. (c) From panel b the target pairwise fragment ratios are calculated. (d) In the data, every time point during which any pairwise fragment ratio is close enough to its target is marked as a possible peak of peptide elution. (e) Using wavelet analysis, p-values are estimated for each possible peak, and the most specific peak is chosen. (f) Again the target pairwise fragment ratios are used to remove any substantial interference, which gives the final peak. This is quantified as the summed integrals of the fragments. (g) We validated Anubis with a large SRM study of 8 laboratories, 10 peptides, and a 9-point dilution series. Using SIS references, Anubis achieves equal accuracy as the previously published manual analysis. (h) Label-free Anubis analysis gives still accurate, but slightly reduced, performance.

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.

Application of label-free SRM on S. pyogenes to study protein abundance differences upon growth with human plasma supplement. (a) Each row represents one peptide, with peptides grouped into proteins and separated by a white row. 0% plasma and 10% plasma columns display measured quantities in the 2 × 9 biological replicates grown with and without 10% plasma (). Wilcoxon and t test columns show the p-values of Wilcoxon rank sum tests and t tests between conditions, with light green indicating significance ≤0.05. The fold change column shows the means ratio between samples, with blue being down-regulated in plasma and red up-regulated. While the high-abundant ribosomes show almost no regulation, indicating that they are not affected by plasma, almost the entire FAS II pathway is significantly down-regulated in plasma by about 40%. (b) The virulome protein C5A peptidase shows consistent significant up-regulation in all peptides. (c) Streptopain is reliably detected in 0% plasma samples but not at all in 10% plasma samples, indicating down-regulation beyond our limit of detection. (d) d-Alanine–polyphosphoribitol ligase subunit 1 and 2 both show significant down-regulation in 10% plasma. All error bars represent standard deviation.