Illuminating the dark space of bioactive peptides with mass spectrometry

Natural product peptides embody a suite of inherent bioactivities and serve as a template to inspire new chemistries and molecular scaffolds in drug discovery and agrotechnology. Mapping the vast and diverse bioactive peptidome, however, is largely obfuscated by unpredictable molecular transformations in both non-ribosomal sequences and highly post-translationally modified ribosomal protein products. Mass spectrometry is a powerful analytical technique with modern instrumentation achieving unprecedented resolving power, rapid and sensitive gas-phase separations, and versatile multistage fragmentation techniques. As such, mass spectrometry can be (1) leveraged to characterize traditionally difficult-to-sequence natural product peptide modifications <i>via</i> enhanced gas-phase technologies and (2) coupled with complementary 'Omics' approaches to predict peptide structure through transcripts, motifs, biosynthetic pathways, and the biomolecular machinery involved in peptide biogenesis. Herein, the challenges of and recent innovations in mass spectrometry towards the discovery and characterization of natural product bioactive peptides are profiled.