The “PepSAVI-MS” Pipeline for Natural Product Bioactive Peptide Discovery

Abstract

The recent increase in extensively drug-resistant bacterial pathogens and the associated increase of morbidity and mortality demonstrate the immediate need for new antibiotic backbones with novel mechanisms of action. Here, we report the development of the PepSAVI-MS pipeline for bioactive peptide discovery. This highly versatile platform employs mass spectrometry and statistics to identify bioactive peptide targets from complex biological samples. We validate the use of this platform through the successful identification of known bioactive peptides from a botanical species, Viola odorata. Using this pipeline, we have widened the known antimicrobial spectrum for V. odorata cyclotides, including antibacterial activity of cycloviolacin O2 against A. baumannii. We further demonstrate the broad applicability of the platform through the identification of novel anticancer activities for cycloviolacins by their cytotoxicity against ovarian, breast, and prostate cancer cell lines.

Exploring bioactive peptides from bacterial secretomes using PepSAVI‐MS: identification and characterization of Bac‐21 from Enterococcus faecalis pPD1

“…PepSAVI‐MS is amenable to a variety of bioassay formats and can be modified to accommodate any target pathogen. High‐throughput microtitre‐based assays and agar diffusion‐based assays have been previously demonstrated with PepSAVI‐MS (Kirkpatrick et al ., , ). While many bacterial species bode well in high throughput 96‐well assays (as presented in the original implementation of the PepSAVI‐MS pipeline), this format is not amenable to some bacterial species that fail to grow to high density (e.g.…”

“…Retention time filters were selected to eliminate background ions, mass filters to select for the common mass range of bioactive peptides and charge state filters to eliminate unwanted small molecules. Peak-picked data were binned and filtered using the previously established workflow-informed criteria (Kirkpatrick et al, 2017). Briefly, binning was performed with a 0.05 Da window of features with identical charge states and filtering required a maximum abundance inside the bioactivity area of interest (fractions 35-39) with < 1% of that abundance outside of the chosen window.…”

“…PepSAVI-MS (Statistically guided bioactive peptides prioritized via mass spectrometry) was developed to expedite the search for botanical bioactive peptides while addressing limitations of bioassay-guided fractionation and genome mining approaches (Kirkpatrick et al, 2017). To enhance the search for potent and effective antimicrobial peptides (AMPs), we now extend this pipeline to bacterial secretomes, a rich source of AMPs with potentially novel mechanisms of action (MOAs) created and refined by extreme inter-and intra-species competition (Riley and Wertz, 2002;Golneshin et al, 2015;Kommineni et al, 2015).…”

Mining for Microbial Gems: Integrating Proteomics in the Postgenomic Natural Product Discovery Pipeline

“…Thus, the complexity of the tandem MS data is greatly reduced, and the peptide tags can be efficiently generated for data mining. Another proteomics‐based PNP discovery pipeline is PepSAVI‐MS . This pipeline starts with the fractionation of small peptides from biological samples, and then analyzes the bioactivity of these fractions in a high‐throughput manner.…”

Development of novel analytical methods for the study of ethnobotanical diabetes treatments and diabetes biomarkers

“…These skills will be essential in translating my research to a usable format within Specify.Advancement of Plant KnowledgeAs described in the 'Nature of Project' section, appending this important chemical and ethnobotanical information will have far-reaching and multitudinous impacts within current plant research: Chemotaxonomy studies can utilize metabolomics profiles to correlate phylogeny to metabolic diversity117,118 . Drug discovery efforts are very recently refocusing on plant-derived molecules of all types2,36,119 . Chemical ecology studies often rely on plant metabolite diversity for clues to species interaction 120 .…”