SAMDI-MS (Self-Assembled Monolayers for MALDI-TOF Mass Spectrometry) couples the use of chemically-defined self-assembled monolayers of alkane thiolates on gold surfaces with MALDI-TOF mass spectrometry for rapid characterization of the surfaces. Reactions performed on the surfaces can be quantified directly by detection of the reaction substrates and products. This rapid detection by mass spectrometry allows SAMDI to offer a generalizable and quantitative platform for bioassay development that can easily be extended to a wide range of enzymatic reactions without the need for development of reaction-specific assays. In this work I present the extension of this SAMDI platform to the development and application of two novel bioassays. The first, is an assay for the detection of protein-ligand interactions. In the work I present here, this assay is applied to the characterization of adaptor domains. Adaptor domains are small, protein domains that bind to sites of post-translational modification. These protein modules do not affect a measurable mass change on their own. Instead, this assay takes advantage of the rate enhancement of enzyme mediated reactions on surfaces that occurs when the enzyme and substrate are colocalized to the surface. Potential ligand peptides are patterned on a monolayer with substrate for a reporter enzyme. Adaptor domains fused to reporter enzyme will localize the reporter to the surface, in a manner that is dependent on the affinity for the ligand, where the reporter will more rapidly convert substrate to product. This covalent record of the transient bind interaction can be quantified, allowing the adaptor-ligand interactions to be rapidly profiled and rank-ordered by affinity. I use this assay to profile chromodomains, which bind to methylated lysine, on biologically relevant ligands derived from the histone 3 protein. Additionally, this assay is applied to profiling SH2 domains, which bind to sites of phosphorylation on tyrosine, showing how it can be used to examine affinity of an adaptor domain to large libraries of potential ligands. The second assay I will present in this work is a general platform for the rapid characterization of enzyme reaction networks which use the Coenzyme A (CoA) cofactor. CoA is essential to cellular metabolism and is involved in a huge number of reactions, the products of which have a wide range of desirable applications in industrial and pharmaceutical settings. There is a great deal of interest in using these biological pathways to manufacture small molecules industrially, but this goal is hindered by a lack of analytical techniques for rapidly characterizing and optimizing these complex reaction networks. Here, I present an assay which couples bio-specific capture chemistry with rapid analysis by SAMDI to create a general platform for the detection and quantification of CoA metabolites. The assay works by using a chemical probe to selectively capture the CoA metabolites from lysates and complex reaction mixtures; these captured metabolites are then selectively immobilized on arrays of self-assembled monolayer and characterized by SAMDI. This strategy offers a significant increase in the throughput of reaction characterization by eliminating the need for lengthy purification techniques and development of metabolite-specific detection methods. In this work, I apply this assay to the optimization of a cell-free reaction system for the bio-synthesis of HMG-CoA, the metabolic precursor to isoprenoid metabolites.

Creator

• O'Kane, Patrick Thomas

DOI

• https://doi.org/10.21985/n2-88kv-cc25

Subject

• Analytical chemistry
• Materials Science
• Biochemistry

Language

• en

Alternate Identifier

• http://dissertations.umi.com/northwestern:14615
• etdadmin_upload_661860

Keyword

• SAMDI
• mass spectrometry
• bioassay
• chromodomains
• cell-free reactions
• microfluidics

Date created

• 2019-01-01

Resource type

• Dissertation

Rights statement

• In Copyright