Dissertation/Thesis Abstract

Profiling the substrate specificity of lysine deacetylases using peptide arrays and SAMDI mass spectrometry
by Gurard-Levin, Zachary A., Ph.D., The University of Chicago, 2010, 188; 3432724
Abstract (Summary)

Assays of enzyme activities are fundamental to biological research, drug discovery, and in the clinic. The development and application of bioanalytical methods continue to be dominant themes in analytical chemistry driven by a motivation for small sample volumes, parallel assays, and label-free detection formats. In this dissertation I describe the evolution and application of a label-free assay that combines self-assembled monolayers of alkanethiolates on gold with matrix assisted laser desorption/ionization (MALDI) mass spectrometry—a technique that we have termed SAMDI—to study the lysine deacetylase (KDAC) enzymes. The KDAC enzyme family regulates many cellular activities such as gene expression, cell cycle, differentiation, and diseases including cancer, yet remain poorly understood due to a lack of suitable reagents, including selective substrates and inhibitors, capable of differentiating their activities and roles in the cell. The benefits of the SAMDI assay are evident with each application described herein such as the characterization of the activity of the KDAC8 isoform on peptides derived from the H4 histone tail, in combination with high-content peptide arrays to profile the specificity of distinct KDAC isoforms, identifying selective substrates capable of measuring endogenous activities, and in high-throughput screens to identify small molecule inhibitors. This dissertation concludes with a description of ongoing and future work to continue to apply the SAMDI technique to investigate the network of acetyltransferases and KDACs—enzymes having opposing activities—as a method to begin to understand how the various states of the acetylome are achieved and maintained in the cell.

Indexing (document details)
Advisor: Mrksich, Milan
Commitee: Kozmin, Sergey A., Piccirilli, Joseph A.
School: The University of Chicago
Department: Chemistry
School Location: United States -- Illinois
Source: DAI-B 72/02, Dissertation Abstracts International
Source Type: DISSERTATION
Subjects: Analytical chemistry, Biochemistry, Organic chemistry
Keywords: Epigenetics, High-throughput screening, Histone code, Lysine deacetylases, Peptide arrays, Posttranslational modifications, Substrate specificity
Publication Number: 3432724
ISBN: 9781124376677
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