Applications of SAMDI Mass Spectrometry: Shifting Limits in Directed Evolution and the Development of Spatiotemporal Sequencing

Pluchinsky, Adam John

doi:https://doi.org/10.21985/n2-z2mp-b976

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Applications of SAMDI Mass Spectrometry: Shifting Limits in Directed Evolution and the Development of Spatiotemporal Sequencing

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SAMDI-MS, which stands for self-assembled monolayers (SAMs) for matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS), is a powerful tool that has enabled the development of novel high-throughput screening and experimentation methods for decades. SAMDI-MS works by immobilizing analytes to functionalized SAMs prior to MS analysis and is capable of studying enzymatic and chemical reactions performed in solution or directly on the surface. In this dissertation, I adopt the SAMDI platform for the development and application of unique bioassays for use in two major fields of research, directed evolution and protein sequencing. In these fields, I use the technology to remove a most persistent bottleneck and add a new dimension of analysis, respectively. I then further build out the technique’s flexibility to analyze molecules of interest. In the first chapter, I harness SAMDI-MS’s ability to rapidly screen thousands of cell-based reactions to develop a screening assay that is capable of screening reactions from libraries of enzyme variants significantly faster than state-of-the-art techniques. Suitable high-throughput and generalizable screening techniques are mandatory in directed evolution, as libraries often exceed several hundred enzyme variants; however, many directed evolution campaigns still rely on the use of low-throughput chromatography-based screening methods. Here, I present a high-throughput strategy for screening libraries of enzyme variants for improved activity. Unpurified reaction products are immobilized to a self-assembled monolayer and analyzed by mass spectrometry, allowing for direct evaluation of thousands of variants in under an hour. The method was demonstrated with libraries of randomly mutated cytochrome P411 variants to identify improved catalysts for a non-natural biochemical C–H alkylation reaction. This reaction was chosen because it is challenging to detect using traditional methods, demonstrating SAMDI-MS’s flexibility for a wide variety of reactions. The evolved catalyst may also find use in organic synthesis as its products are difficult to synthesize by chemical means otherwise. The technique may be tailored to evolve enzymatic activity for a variety of transformations where higher throughput is needed. It is with this research, in collaboration with the lab of Frances Arnold, the recipient of the 2018 Nobel Prize in chemistry, that we shift the attention of the field from this bottleneck to new challenges. In the second chapter, I use a recently developed extension of the SAMDI technique, imaging SAMDI (iSAMDI), that enables high resolution microfluidic sequencing of surface-bound peptides that is capable of resolving amino acids of identical mass. Techniques that offer single-residue resolution of amino acids are important in for proteomic research, especially where the precise sequence of a protein is not already known in a database or where disease related mutations have altered the sequence of a protein. Here, I immobilize peptides to the floor of a microfluidic flow cell and use exopeptidases to generate peptide ladders that span the channel. I then use iSAMDI to provide a record of the peptide ladders. While the difference in mass between the ladders, read by MALDI-MS, can be used to reveal the individual amino acids in the peptide, the difference in specificity the exopeptidases exhibit for each amino acid can also be used to distinguish between residues by a simple visual analysis of images of their kinetic signatures provided by iSAMDI. This extension allows for the resolution of isobaric amino acids—information that could not be obtained by MALDI-MS alone and generally difficult to obtain using modern sequencing techniques. The iSAMDI assay is shown with a variety of peptides and exopeptidases. Finally, I describe new developments and additional findings in immobilization techniques that may find use in future SAMDI-MS applications. I describe methods for capturing analytes to the surfaces via the azide-alkyne “click” cycloaddition using both copper and copper-free systems with multiple strategies. Having a large toolbox of capture chemistries is important for selecting the best system to analyze samples.

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