Graduation Semester and Year
Fall 2024
Language
English
Document Type
Dissertation
Degree Name
Doctor of Philosophy in Chemistry
Department
Chemistry and Biochemistry
First Advisor
Saiful M. Chowdhury
Abstract
Post-translational modifications (PTMs) play a crucial role in dictating the structure, stability, and function of proteins. This thesis focuses on advancing analytical approaches for studying key PTMs, particularly disulfide bonds, cysteine residues, and covalent labeling of tyrosine. Several innovative bioconjugation methods were developed using mass spectrometry-based methodologies.
Disulfide bonds are essential for maintaining protein structural integrity but are prone to scrambling under reducing conditions or when free cysteines are present. In chapter 2, a robust mass spectrometry workflow was demonstrated to analyze disulfide-bonded peptides, involving reduction of disulfide bonds with tris(2-carboxyethyl) phosphine (TCEP) and re-bridging via dibromomaleimide (DBM)-mediated crosslinking. A potential enrichment methodology of maleimide linked disulfide bonded peptides was demonstrated using copper catalyzed alkyne-azido click chemistry, which enables biotin-avidin purification of the labeled disulfide bonded peptides.
Chapter three introduced a targeted bioconjugation approach for tyrosine residues using 4-phenyl-3H-1,2,4-triazole-3,5(4H)-dione (PTAD). This reagent selectively reacts with tyrosine’s phenolic group under mild conditions, creating covalent modifications which detected by tandem mass spectrometry. The PTAD labeling technique expands the toolkit for studying surface exposed tyrosine amino acids in proteins that further helps to investigate protein structural dynamics.
Additionally, in Chapter 4, we discussed a fluorescent thiol tagging strategy for the sensitive detection and quantification of cysteinyl peptides. We utilized a reagent N-(9-acridinyl) maleimide (NAM), which fluoresces upon conjugation with thiols; the conjugated cysteinyl peptides were analyzed in biological samples using fluorescence spectroscopy and tandem mass spectrometry.
These innovative methodologies highlight significant progress in the field of proteomics, enhancing the sensitivity and specificity of PTM analysis. These strategies offer powerful tools for studying protein chemistry, biomarker discovery, therapeutic development, and structural proteomics.
Keywords
Proteomics, Mass Spectrometry, Bioconjugation, Disulfide bonds, LC-MS/MS
Disciplines
Analytical Chemistry
License
This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 4.0 International License.
Recommended Citation
Cornelius, Sharel, "BIOCONJUGATION AND MASS SPECTROMETRY METHODS FOR COMPREHENSIVE ANALYSIS OF PROTEIN STRUCTURES AND POST-TRANSLATIONAL MODIFICATIONS" (2024). Chemistry & Biochemistry Dissertations. 281.
https://mavmatrix.uta.edu/chemistry_dissertations/281