Parent award abstract. The controlled evolution of proteins in the laboratory is a valuable biomedical tool for accessing biomolecules for industrial, therapeutic and research applications. This process, also known as directed evolution, allows one to employ the specificity and selectivity that Nature imbues within its privileged biomolecules to construct unnatural products that would otherwise be inefficient or laborious to generate chemosynthetically. While this process is incredibly powerful, an existing bottleneck is the subsequent screening of the resulting variants for these high value products. The directed evolution process typically generates hundreds to thousands of mutants or library members for biochemical analysis. In some cases, fluorescent reporter systems or bioactivity assays can be employed as a general biochemical readout, however, this does not inform on specific chemical transformations towards diverse small molecule targets. When high value chemical products are the subject of these directed evolution experiments, researchers employ multiple orthogonal analytical techniques, including: high performance liquid chromatography (HPLC); gas chromatography (GC); mass spectrometry (MS); and nuclear magnetic resonance (NMR). This becomes time and infrastructure intensive when thousands of variants need to be evaluated; even if variants are pooled in curated groups, considerable effort is needed for chromatographic assessment. Additionally, many of these methodologies may not be sensitive or specific enough to necessitate detection of low titer production of the desired product(s). Based on these shortcomings of the screening platforms, we are proposing to leverage our labs’ existing strengths to develop a high-throughput, specific, and sensitive mass spectrometry platform to screen directed evolution libraries for bioactive chemical products without chromatographic separation. The McKinnie lab has expertise in synthetic chemistry and biochemistry and has specifically worked on the α- ketoglutarate-dependent dioxygenase enzyme to construct neuroactive kainic acid on the gram scale. The Sanchez lab has expertise in natural product discovery and mass spectrometry techniques such as imaging mass spectrometry and tandem mass spectrometry. These respective strengths will allow us to develop an innovative pipeline for screening thousands of directed evolution library members to prioritize variants that direct the chemistry towards kainoid-ring glutamate receptor agonists and antagonists. Our pipeline will allow for unprecedented measurements in chemical specificity and be broadly applicable for any groups looking to conduct directed evolution. · Current directed evolution screening platforms are time-consuming or low throughput · The combined expertise of our team is highly interdisciplinary · Mass spectrometry and trapped ion mobility spectrometry allow for high dimensionality measurements directly from mutant colonies without relianc...