High-Throughput Screening of Isomeric Reaction Products by Droplet Microfluidics Coupled to Cyclic Ion Mobility-Mass...

Engineered biocatalysts enable highly selective chemical transformations with low environmental impact. Development of biocatalysts by directed evolution requires screening many enzyme variants for improved catalytic properties. The low throughput of commonly used label-free screening methods, e.g., liquid chromatography-mass spectrometry (LC-MS), becomes the rate-limiting step in biocatalyst development, limiting the coverage of protein sequence space explored. Direct MS methods have been applied to biocatalyst screening; however, these methods cannot be used to evaluate isomer selectivity. Ion mobility spectrometry is a separation technique readily combined with MS, facilitating isomer differentiation on the millisecond time scale. Here, we present a droplet microfluidic system coupled to cyclic ion mobility-mass spectrometry (cIM-MS) to enable the screening of isomeric products. The system was applied to the separation of biaryl benzofuran dimers formed by the fungal cytochrome P450 KtnC. The isomeric 5,7'-bibenzofuran and 7,7'-bibenzofuran products from KtnC variants were baseline-resolved within 32 ms by cIM. By infusing biocatalytic reaction mixtures as 5 nL droplets, an analysis throughput of 1.2 s/droplet was achieved using cIM-MS. Droplet cIM-MS was used to quantify standards in the reaction matrix with high agreement to actual concentrations of each isomer (i.e., R² of 0.97 and 0.98 for total bibenzofuran content and fractional 5,7'-bibenzofuran content, respectively). For samples with enzymatically formed product isomers, droplet cIM-MS identified the same active variants and had comparable reproducibility (RSD of 10-15%) to analysis by LC-MS and produced this data 128 times faster. The method is expected to be suitable for improving the rate of biocatalyst development for isomer-selective reactions.