ABSTRACT
Significant advances in directed evolution have allowed for the exploration of many new and important non-natural transformations; however, many of these ground-breaking reactions still rely on the use of low-throughput chromatography-based methods to sift through variants. To remove this limitation, we adopted a high-throughput screening strategy to evaluate libraries of enzyme variants for improved activities. This method, SAMDI-MS, has previously been used to extensively profile enzymatic activity both in situ and from complex lysates, while permitting the analysis of thousands of samples per hour and 105 experiments per day. This talk will first detail the development of SAMDI and its ability to analyze chemical reactions, including enzyme activities. I will then discuss how we use this platform to identify improved catalysts for non-natural C–H alkylation from random libraries of cytochrome P411s. Unpurified reaction products are immobilized to a self-assembled monolayer and analyzed by mass spectrometry, allowing for the direct evaluation of variants in a thousandth of the time required for chromatographic-based methods. The alkylation reaction was chosen specifically because it cannot make use of current high-throughput techniques and its reaction products are among the most challenging to detect using conventional methods. The technique may be tailored to assay enzymatic activity for both natural and non-natural transformations where higher throughput is desired.
BIOGRAPHY
Adam J. Pluchinsky is a Ph.D. candidate at Northwestern University in the Department of Biomedical Engineering. He attended both Fordham University and Columbia University, earning his first BS degree in Chemistry and a second in Biomedical Engineering, respectively, in 2016. He currently conducts his doctoral thesis research under the mentorship of Milan Mrksich, Ph.D. and in collaboration with Frances Arnold, Ph.D. at Caltech. He is honored as a recipient of National Science Fellowship’s Graduate Research Fellowship Program in addition to being appointed a fellow through the Northwestern Center for Leadership. He serves on an advisory board for his university’s career advancement center and has held positions in student organizations where he organized events for hundreds of members both on campus and at various Chicago venues. He has also served as a leadership coach for a variety of organizations on campus.
In his research Adam develops techniques to assay enzyme activities in high throughput to identify improved activity for important and highly challenging biosynthetic transformations. Most notably, he leads a team of researchers who use this approach to both speed-up and enable directed evolution for a variety of applications. These methods are based in SAMDI mass spectrometry, a technology developed in the Mrksich Group. His work is devoted to shifting the limitations in this field.
