Defense Date
2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Pharmaceutical Sciences
First Advisor
Adam Hawkridge
Second Advisor
Glen Kellogg
Abstract
The chemoproteomic methods of activity-based protein profiling (ABPP) and photoaffinity labeling (PAL) can facilitate target protein discovery for bio-active compounds in the early stages of drug discovery research. These methods elicit target proteins using small molecule chemical probes that structurally mimic the drug candidate. Mass spectrometry (MS) is the preferred detection method, and is the only analytical technique that can provide protein sequence identity and direct molecular detection of the protein-probe conjugate to enable binding site localization and confident discovery of target proteins. However, the detection and identification of unknown, low abundant, non-template modified peptides using the traditional approach of liquid chromatography-tandem mass spectrometry (LC-MS/MS) “shotgun proteomics” poses several technical challenges. To address these challenges, two research objectives were pursued in this dissertation. Firstly, a robust tagging strategy was developed, which capitalizes on the natural abundance of the boron isotopes embedded in BODIPY, a commercially available fluorescent tag. A scripting tool was developed to computationally identify the isotopically-encoded chemoproteomics-labeled peptides from within complex LC-MS/MS datasets, and this strategy achieved three times the sensitivity compared with commercial peptide sequencing software. Screening for the boron isotopes effectively decouples the discovery of labeled peptides from the sequencing step to circumvent issues with exact mass matching and abundance bias, and can be used in any chemoproteomic investigation for target discovery. Furthermore, the unique signatures from the BODIPY tag enabled localization of the amino acid residue that was labeled in a cysteine-functionalized protein standard. In the second study, the glutathione (GSH)-glutathione S-transferase (GST) binding interaction was investigated for use as a potential photolabeling positive control. GSH was functionalized with a photo-reactive diazirine probe and demonstrated similar binding affinity to GST compared with the native ligand, and ability to covalently label GST in a gel read-out. These results indicate potential for the GSH-probe and GST reagents to serve as a model system for chemoproteomic standardization.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
8-5-2024