Mass Spectrometric Protein Footprinting of Conformational Changes Induced by Chromatographic Surfaces

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Focke, K. (2022). Mass Spectrometric Protein Footprinting of Conformational Changes Induced by Chromatographic Surfaces. Unc Charlotte Electronic Theses And Dissertations.
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Abstract

Reversed-phase liquid chromatography (RPLC) of intact proteins induces conformational changes due to the denaturing effects of both the hydrophobic stationary phase and the high-organic mobile phase, which can lead to poor recovery and badly distorted or artifactual chromatographic peaks. A surprising additional consequence is that the length of the column does not matter. We are investigating these structural changes using mass spectrometric protein footprinting, which gives information about changes in the solvent accessibility of modifiable sites. Differences in site accessibility following adsorption to and desorption from chromatographic surfaces indicate regions of structural alteration in proteins. We seek to determine if bovine serum albumin (BSA) adsorbs in a preferred binding orientation, if denaturation increases with surface residence time, and if the conformation in high-organic solvent depends on whether the protein was first adsorbed. Glutamate and aspartate groups of BSA are amidated with methylamine and EDAC. Tryptic digests of the modified proteins are analyzed using LC-MS/MS. Peptides are identified with tandem mass spectrometry, and modification sites are located through mass shifts in the tandem mass spectra. Quantification is performed by comparing relative peak areas of modified and unmodified peptides in extracted ion chromatograms for their respective precursors. While results remain somewhat ambiguous in the absence of instrumental replicates of individual samples and modeling, our research presents a method for footprinting proteins adsorbed to chromatographic surfaces and confirms the apparent contribution of both the hydrophobic stationary phase and the high-organic mobile phase to conformational changes observed during RPLC.

Details
Author

Focke, Kimberly

Title

Mass Spectrometric Protein Footprinting of Conformational Changes Induced by Chromatographic Surfaces

Physical Description

1 online resource (83 pages) : PDF

Date

2022

Degree Granting Institution

University of North Carolina at Charlotte

Abstract

Reversed-phase liquid chromatography (RPLC) of intact proteins induces conformational changes due to the denaturing effects of both the hydrophobic stationary phase and the high-organic mobile phase, which can lead to poor recovery and badly distorted or artifactual chromatographic peaks. A surprising additional consequence is that the length of the column does not matter. We are investigating these structural changes using mass spectrometric protein footprinting, which gives information about changes in the solvent accessibility of modifiable sites. Differences in site accessibility following adsorption to and desorption from chromatographic surfaces indicate regions of structural alteration in proteins. We seek to determine if bovine serum albumin (BSA) adsorbs in a preferred binding orientation, if denaturation increases with surface residence time, and if the conformation in high-organic solvent depends on whether the protein was first adsorbed. Glutamate and aspartate groups of BSA are amidated with methylamine and EDAC. Tryptic digests of the modified proteins are analyzed using LC-MS/MS. Peptides are identified with tandem mass spectrometry, and modification sites are located through mass shifts in the tandem mass spectra. Quantification is performed by comparing relative peak areas of modified and unmodified peptides in extracted ion chromatograms for their respective precursors. While results remain somewhat ambiguous in the absence of instrumental replicates of individual samples and modeling, our research presents a method for footprinting proteins adsorbed to chromatographic surfaces and confirms the apparent contribution of both the hydrophobic stationary phase and the high-organic mobile phase to conformational changes observed during RPLC.

Genre

masters theses

Subjects--Topics

Analytical chemistry
Biochemistry

Degree

M.S.

Keywords

Chromatographic Surfaces
Covalent Labeling
Protein Adsorption
Protein Footprinting
Rplc
Structural Mass Spectrometry

Subject Area

Chemistry

Advisor(s)

Cooper, Brian

Committee Members

Poler, Jordan
Troutman, Jerry
Nesmelov, Yuri

Degree Note

Thesis (M.S.)--University of North Carolina at Charlotte, 2022.

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Rights Holder Information

Copyright is held by the author unless otherwise indicated.

Identifier

Focke_uncc_0694N_13221

Permalink

http://hdl.handle.net/20.500.13093/etd:3146