High-Frequency Microfluidic Fractionation for Compound-Resolved Bioactivity-Based Metabolomics

Specialized metabolites represent a prolific source of potential drug candidates. However, the process from detecting bioactivity in a crude metabolite extract to unambiguously identifying the active agent is a tedious and expensive endeavor. Speeding up this procedure is crucial, as new drugs, such as antibiotics, are urgently needed. Furthermore, the systematic functional assessment of complex metabolome samples represents a key bottleneck in nontargeted metabolomics, which once solved, holds the potential to fundamentally advance our systematic understanding of biology. To tackle this central bioanalytical challenge, we developed a compound-resolved bioactivity-based metabolomics workflow that combines nontargeted liquid chromatography tandem mass spectrometry (LC-MS/MS), high frequency fractionation on microfluidic devices and subsequent readout with luminescent bioreporter strains. Central for this workflow is a custom high-speed (∼1 Hz frequency) fractionation device that spots the mobile phase onto a microfluidic paper-analytical device (μPAD) in parallel to MS/MS data acquisition. Subsequently, the μPAD can be overlaid with a bioreporter strain, which displays cellular stress by expressing luciferase. The luminescence signal can then be correlated to MS signals through their chromatographic profiles. We evaluated five different luciferase-expressing bioreporter strains which provide information about different antibacterial modes of action, and tested the workflow with different antibiotic standards and mixtures thereof, as well as crude extracts from the known antibiotic producer Saccharopolyspora erythraea. Our results demonstrated high sensitivity (up to 1 ng/spot, depending on compound and bioreporter) and the rapid identification of multiple antimicrobial compounds out of crude extracts, highlighting the practicality and high-throughput capability of this compound-resolved bioactivity-based metabolomics approach.