Selected reaction monitoring (SRM) MS is a highly selective and sensitive technique to quantify protein abundances in complex biological samples. To enhance the pace of SRM large studies, a validated, robust method to fully automate absolute quantification, and to substitute for interactive evaluation, would be valuable. To address this demand, we present Ariadne, a Matlab® software. To quantify monitored targets, Ariadne exploits metadata imported from the transition lists, and targets can be filtered according to mProphet output. Signal processing and statistical learning approaches are combined to compute peptide quantifications. To robustly estimate absolute abundances, the external calibration curve method is applied, ensuring linearity over the measured dynamic range. Ariadne was benchmarked against mProphet and Skyline by comparing its quantification performance on three different dilution series, featuring either noisy/smooth traces without background or smooth traces with complex background. Results, evaluated as efficiency, linearity, accuracy, and precision of quantification, showed that Ariadne’s performance is independent of data smoothness and complex background presence, and that Ariadne outperforms mProphet on the noisier dataset and improved 2 fold Skyline’s accuracy and precision for the lowest abundant dilution with complex background. Remarkably, Ariadne could statistically distinguish from each other all different abundances, discriminating dilutions as low as 0.1 and 0.2 fmol. These results suggest that Ariadne offers reliable and automated analysis of large-scale SRM differential expression studies.

To benchmark our software we selected 43 proteotypic peptides (PTPs) 39 covering 31 endogenous Drosophila melanogaster proteins (see supplementary file TableS1). These peptides were synthesized either as crude light peptides (JPT) or as heavy labeled AQUA 8 isotopes (Sigma) for absolute quantification. The heavy AQUA peptides were spiked into the crude peptide mix in known concentrations from 100 fmol to 0.1 fmol (100, 50, 20, 10, 4, 2, 0.5, 0.2, 0.1) absolute peptide amount. iRT-kit peptides (Biognosys) 40 were added to the samples at a 1:20 concentration to enable retention time normalization and re-alignment for optimal mProphet data evaluation. As biological matrix for the complex background dataset, total lysate from Drosophila melanogaster Kc 167 cells was used. Total lysate was prepared in 8 volumes of RIPA Buffer (150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 500 µM EDTA, 100 µM EGTA, 1.0% Triton X-100, 1% sodium deoxycholate, protease inhibitors). After precipitation with ice-cold acetone and two subsequent washing steps with 80 % acetone the extracted proteins were reduced with 5 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and treated with 10 mM iodoacetamide to modify cysteine residues prior to digestion with trypsin. Resulting peptides were cleaned up by reverse phase C18 chromatography (Finisterre SPE16 columns, WICOM). For mass spectrometry analysis, samples were diluted in buffer containing 5% acetonitrile, 0.2% formic acid, yielding a final concentration of background peptides of ~0.2microgram/microliter.