Self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS) is a platform that combines self-assembled monolayers of alkanethiolates on gold and MALDI mass spectrometry analysis to report mass changes resulting from surface reactions.  The synthetic flexibility of the monolayer and the use of mass spectrometry as a generalizable readout method enables SAMDI-MS characterization of a broad spectrum of biochemical interactions.  Furthermore, SAMDI-MS is compatible with high-throughput sample preparation and processing and finds important applications in high-throughput screening and experimentation.  In this thesis, I will present the development and application of three unique SAMDI-MS assays.  In Chapter 2, I will describe the development of a SAMDI-MS assay for the cancer-associated enzyme isocitrate dehydrogenase 1 (IDH1). The assay uses a hydrazone formation reaction to immobilize IDH1’s αKG product to the surface, where it can be quantified as a measure of the enzyme’s activity or expression level.  This work reports a valuable tool in the assessment of IDH1 inhibitor or knockdown agent candidates and serves as an important early example of SAMDI-MS’s ability to assay metabolic enzymes with molecular substrates and products in complex sample environments.  In Chapter 3, I will describe the application of the protein interaction by SAMDI (PI-SAMDI) assay, which measures protein-ligand interactions through binding-induced enhancement in the activity of a reporter enzyme, to explore the ligand preferences of the PHD1 reader domain of histone demethylase KDM5A.  This works reveals new insights into permissible PHD1-H3 binding interactions and establishes PI-SAMDI as a useful preliminary screening tool in comprehensive studies of protein-ligand binding.  In Chapter 4, I will describe the development of a SAMDI affinity selection mass spectrometry (ASMS) assay, in which surface-bound biomarker is detected upon interaction with an immobilized aptamer, to characterize aptamer-biomarker binding interactions for biomarker sensing.  Characterizing the binding of the small molecule streptomycin to an oligonucleotide aptamer and the binding of the protein neuropeptide Y to a peptide aptamer yielded biomarker detection, but failed to report specific, high-affinity interactions.  This work validates the fundamental design principle of SAMDI ASMS and reveals important criteria in the selection of an appropriate aptamer system for the assay.

Creator

• Anderson , Sarah Elizabeth

DOI

• https://doi.org/10.21985/n2-2gk2-r645

Subject

• Chemistry

Language

• en

Alternate Identifier

• etdadmin_upload_812568
• http://dissertations.umi.com/northwestern:15516

Date created

• 2021-01-01

Resource type

• Dissertation

Rights statement

• In Copyright