Graduation Semester and Year
Fall 2024
Language
English
Document Type
Dissertation
Degree Name
Doctor of Philosophy in Chemistry
Department
Chemistry and Biochemistry
First Advisor
Dr. Saiful M. Chowdhury
Abstract
The application of mass spectrometry (MS)-based chemical crosslinking techniques holds significant promise in elucidating protein structures and deciphering interactions between proteins. Nevertheless, the field faces significant hurdles due to the limited identification scope of crosslinked products and the complex nature of the resulting data. Innovations are necessary to accurately identify crosslinked peptides with enhanced confidence.
Our laboratory has developed a chemical crosslinking technology and synthesized an innovative crosslinking agent known as DUCCT (Dual cleavable chemical crosslinking technology). This unique crosslinker incorporates two distinct MS-cleavable bonds. DUCCT crosslinker produces distinct diagnostic fragment ions for the same precursor ion when subjected to collision-induced dissociation (CID) and Electron Transfer Dissociation (ETD) techniques in tandem mass spectrometry. This distinctive behavior significantly contributes to the unambiguous identification of crosslinked peptides. Through the utilization of model peptides and proteins, we successfully demonstrate the efficacy of the DUCCT technology. In this report, our study centers around a more in-depth exploration of the effectiveness of this novel crosslinker in examining the binding domain interaction between the cat allergen protein complex (Fel d 1) and the viral spike protein of the SARS-CoV-2 virus.
Additionally, understanding the functions of proteins requires identifying post-translational modifications (PTMs). We have also extended the scope of chemical labeling strategies by introducing a new reagent diphenyldiazomethane (DPDAM) to assess the identification efficacy of functional acidic residues in proteins using mass spectrometry. Furthermore, inter crosslinked peptides are scarce within mixtures dominated by unmodified and dead-end peptides, presenting significant challenges to effective data analysis. In response to this constraint, our laboratory has introduced an advanced enhancement-based crosslinker, referred to as Alkyne-DUCCT, which uses “click chemistry” incorporating an alkyne enrichment functionality. This innovative approach enables the selective enrichment of crosslinked products from complicated biological samples. Subsequent employment of tandem mass spectrometry techniques bolsters the confidence of crosslinked peptide identification through the generation of diagnostic fragments. We employed the in-house developed bioinformatics tool, Cleave-XL, to automate the analysis of DUCCT/Alkyne-DUCCT-treated crosslinked protein samples. Overall, we believe the utilization of dual cleavable crosslinking methodology holds the potential to greatly enhance the identification of crosslinks, and by labeling acidic residues, DPDAM provides valuable insights into protein structure and function, as well as potential therapeutic targets.
Keywords
Cat allergen, SARS CoV-2, Mass Spectrometry, Chemical Crosslinking
Disciplines
Analytical Chemistry
License
This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 4.0 International License.
Recommended Citation
Talukder, Akash, "Next-generation cleavable crosslinkers for high confidence in protein interactions data analysis" (2024). Chemistry & Biochemistry Dissertations. 279.
https://mavmatrix.uta.edu/chemistry_dissertations/279