Graduation Semester and Year

2019

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Chemistry

Department

Chemistry and Biochemistry

First Advisor

Saiful M Chowdhury

Abstract

Mass spectrometry (MS)-based chemical cross-linking technique is a potential tool for understanding the protein structure and protein-protein interactions. However, low abundances of cross-linked products and the data complexity are the major challenges in this field. New strategies are required to pinpoint cross-linked peptides with high confidence. Our lab designed and synthesized a novel cross-linker with two differential MS-cleavable bonds, namely DUCCT. DUCCT-cross-linked peptide generates different diagnostic fragment ions in Collision Induced Dissociation (CID) and Electron Transfer Dissociation (ETD) (tandem mass spectrometric technique) which helps in the unambiguous identification of cross-linked peptide. We demonstrate the efficiency of DUCCT technology with model peptides and proteins. Moreover, we found that DUCCT showed better labeling efficiency compared to BS3 (commercial cross-linker) in an immune cell called macrophage. The low abundance of inter-crosslinked peptides in a mixture of high abundance of unmodified and dead-end peptides hinders the data analysis. To address this limitation, our lab developed a next-generation DUCCT cross-linker with enrichment functionality (PC-DUCCT-Biotin). This technique can enrich the cross-linked products from the complex biological sample, and the tandem mass spectrometry technique provides confidence in the identification of cross-linked peptides by generating diagnostic fragments. DUCCT-treated cross-linked protein samples were automatically analyzed by the in-house developed bio-informatics tool, Cleave-XL. We also demonstrated the capability of Cleave-XL in searching the cross-linked products from protein complexes. Currently, the software can efficiently search the small or medium size protein complexes, containing 5 to 10 proteins. The improvement process of the Cleave-XL is underway, and we believe that it will be an efficient bio-informatics tool for protein structure and interactome study in the bio-medical field. As a continuation of cross-linking development, we designed another compact MS-cleavable cross-linker, HI-ETD-XL. The signature fragmentation pattern of HI-ETD-XL during electron transfer dissociation (ETD) will contribute significantly to the structural proteomics field. Currently, we are working with Toll-like Receptors (TLRs) signaling cascades using different generations of cleavable cross-linkers. We believe our newly developed cross-linking approaches will significantly contribute to protein interactions and structural proteomics research.

Keywords

Cross-linking, Mass Spectrometry

Disciplines

Chemistry | Physical Sciences and Mathematics

Comments

Degree granted by The University of Texas at Arlington

Included in

Chemistry Commons

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