Abstract
Antimicrobial peptides (AMPs) are host defense peptides with a range of functions/activities/modes of action that are ubiquitously expressed across all forms of life. Continued discovery of novel AMPs presents exciting opportunities to address evolving resistance to existing treatments in multiple fields, including agricultural pathogens/pests as well as antimicrobial and chemotherapeutics for human health. However, typical discovery methods including bioassay-guided fractionation and genome mining generally lack the capacity for robust AMP discovery in non-model/unsequenced organisms. PepSAVI-MS (Statistically guided bioactive peptides prioritized via mass spectrometry) was developed as an AMP discovery approach that addresses some of the limitations associated with these standard methods. PepSAVI-MS is a multi-pronged pipeline that includes peptide library creation, bioactivity screening, LC-MS analysis, and statistical modeling for putative AMP identification. The original implementation of PepSAVI-MS outlined strategies for the fractionation of plant extracts with strong cation exchange chromatography (SCX). Herein, we elaborate on recent improvements to peptide library creation through the use of orthogonal fractionation methods, specifically crude SCX chromatography and reversed-phase liquid chromatography (RPLC). This optimization of the "peptide library creation" step has demonstrated improvements for processing and AMP identifications via PepSAVI-MS.