Searle et. al.; Using Data Independent Acquisition (DIA) to Model High-responding Peptides for Targeted Proteomics Experiments; Mol Cell Proteomics. 2015 Sep;14(9):2331-40. doi: 10.1074/mcp.M115.051300. Epub 2015 Jun 22. [Publication]
Instrument: TSQ Vantage
Targeted mass spectrometry is an essential tool for detecting quantitative changes in low abundant proteins throughout the proteome. Although Selected Reaction Monitoring (SRM) is the preferred method for quantifying peptides in complex samples, the process of designing SRM assays is laborious. Peptides have widely varying signal responses dictated by sequence-specific physiochemical properties; one major challenge is in selecting representative peptides to target as a proxy for protein abundance. Here we present PREGO, a software tool that predicts high responding peptides for SRM experiments. PREGO predicts peptide responses with an artificial neural network trained using 11 minimally redundant, maximally relevant properties. Crucial to its success, PREGO is trained using fragment ion intensities of equimolar synthetic peptides extracted from data independent acquisition (DIA) experiments. Due to similarities in instrumentation and the nature of data collection, relative peptide responses from DIA experiments are a suitable substitute for SRM experiments because they both make quantitative measurements from integrated fragment ion chromatograms. Using an SRM experiment containing 12973 peptides from 724 synthetic proteins, PREGO exhibits a 40-85% improvement over previously published approaches at selecting high responding peptides. These results also represent a dramatic improvement over the rules-based peptide selection approaches commonly used in the literature.
An SRM training validation data set was constructed using the protocols presented in Stergachis et al. Briefly, clones for GST fusion proteins from the pANT7_cGST clone collection (https://dnasu.org/DNASU/Home.do) were synthesized in vitro using the Pierce 1-step Human Coupled in vitro protein synthesis kit (Thermo Scientific; Bremen, Germany). In instances where a cDNA clone was unavailable, recombinant proteins were simply purchased from a commercial source. GST tagged proteins were captured using glutathione sepharose 4B beads (GE Healthcare Life Sciences; Pittsburgh, PA), and iteratively washed to remove nonspecific binders. Bead bound GST fusion proteins were individually denatured with 5 mM dithiothreitol (DTT) for 30 minutes at 60 °C and alkylated with 15 mM iodoacetic acid (IAA) for 30 minutes at room temperature. Proteins were then digested 1 ug of sequencing grade modified porcine trypsin (Promega; Madison, WI) for 2 hours at 37°C.