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 a-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 reliance on chromatography techniques