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Full text release has been delayed at the author's request until August 04, 2027

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Pairing Covalent Labeling and Native Mass Spectrometry to Study Protein Conformation

Whitford, Evan Nathaniel
http://orcid.org/0009-0005-1120-0889
http://rave.ohiolink.edu/etdc/view?acc_num=osu1714958974527209

Abstract Details

2024, Master of Science, Ohio State University, Chemistry.
The function of a protein is closely linked to its 3D structure, highlighting the significance of studying the structural arrangement of a protein to understand its function, interactions, and reactivity with other biomolecules better. Mass spectrometry (MS) enables the investigation of protein structure to gain insight into structural features such as stoichiometry and conformation. MS, when paired with covalent labeling (CL), utilizes reagents, such as diethylpyrocarbonate (DEPC), to modify solvent accessible amino acids. These modifications add a known mass tag that minimally perturbs the native structure. Modified residues are detected by tandem MS of peptides generated by enzymatic digestion. CL data provides insight into the topology of a protein, as information regarding the solvent accessibility, intrinsic reactivity of specific amino acids, and higher order structure effects can be observed in labeling results. While protein prediction software can be adapted to apply CL-MS data to enhance protein tertiary structure prediction, very little experimental labeling data of proteins with an intrinsically disordered region (IDR) is available. We aim to optimize a procedure for labeling proteins with an IDR with the objective of gathering data from experimental DEPC labeling and combining it with computational modeling. This integrated approach will enhance the prediction of protein structures that contain an IDR. Data presented in this thesis show that native mass spectrometry (nMS) enables the examination of protein reactivity with the label and the investigation of protein integrity after a label has been added. Ion mobility and collision induced unfolding experiments enable the detection of subtle structural effects from labeling experiments. Bottom-up proteomics localizes and quantifies where labels are added to proteins. The illustrated strategy of combining nMS and CL-MS ensures accurate characterization of labeled proteins and validates DEPC as a suitable label, particularly for proteins containing an IDR like MEIN1.
Vicki Wysocki (Advisor)
Steffen Lindert (Committee Member)
Amanda Hummon (Committee Member)
45 p.
Chemistry
Covalent labeling; native mass spectrometry

Recommended Citations

Citations

  • Whitford, E. N. (2024). Pairing Covalent Labeling and Native Mass Spectrometry to Study Protein Conformation [Master's thesis, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1714958974527209

    APA Style (7th edition)

  • Whitford, Evan. Pairing Covalent Labeling and Native Mass Spectrometry to Study Protein Conformation . 2024. Ohio State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1714958974527209.

    MLA Style (8th edition)

  • Whitford, Evan. "Pairing Covalent Labeling and Native Mass Spectrometry to Study Protein Conformation ." Master's thesis, Ohio State University, 2024. http://rave.ohiolink.edu/etdc/view?acc_num=osu1714958974527209

    Chicago Manual of Style (17th edition)

osu1714958974527209
© 2024, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.
Release 3.2.12