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Development of an integrated microfluidic mixing-electrospray platform for microsecond hydrogen-deuterium exchange mass spectrometry Neha Srikumar
- Format:
- Book
- Thesis/Dissertation
- Author/Creator:
- Srikumar, Neha, author.
- Language:
- English
- Subjects (All):
- Biochemistry.
- Bioengineering.
- Materials science.
- Engineering.
- Nanotechnology.
- 0487.
- 0202.
- 0794.
- 0652.
- 0537.
- Local Subjects:
- Biochemistry.
- Bioengineering.
- Materials science.
- Engineering.
- Nanotechnology.
- 0487.
- 0202.
- 0794.
- 0652.
- 0537.
- Genre:
- Academic theses
- Physical Description:
- 1 online resource (135 pages)
- Contained In:
- Dissertations Abstracts International 87-12B
- Place of Publication:
- Ann Arbor : ProQuest Dissertations and Theses, 2026
- Language Note:
- English
- Summary:
- While gold standard structural techniques such as X-ray crystallography, nuclear magnetic resonance (NMR), and cryo-electron microscopy have provided the basis for understanding ordered protein structures, much is not known about far more dynamic, disordered protein structures. Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) within proteins have been tied to a multitude of neurodegenerative disorders, diabetes, and cardiovascular disease which necessitates the study of their unconventional structure. Several secondary techniques exist which can probe the fast time scales (ns-µs) at which disordered proteins can move to form transient conformations, but none of these techniques can currently localize the positions of these movements down to single amino acid residues. One technique, hydrogen-deuterium exchange mass spectrometry (HDX-MS) shows some promise of solving this problem, as the technique can probe hydrogen exchange dynamics in proteins, thereby providing indirect information on protein structure. This is done by mixing protein with deuterated solvents, and measuring the time it takes for deuterium to exchange with hydrogens in the protein's structure. HDX-MS also allows for localization of the positions of the hydrogens being studied at sub amino acid resolution. However, hydrogens exchanges along the protein backbone of disordered regions and in all amino acid side chains happen in microsecond times, which currently cannot be effectively studied due to the lack of temporal resolution of HDX-MS instrumentation.In this work, a microsecond microfluidic mixer is integrated into an electrospray ionization (ESI) interface to be able to study microsecond hydrogen exchange dynamics with mass spectrometry in physiologically relevant conditions. The mixer is the first of its kind to attempt microsecond reactions at the lowest flow rates ever reported for any mixer design, and effectively mixes analytes with deuterated solvents for HDX. Additionally, embedding the mixer into an electrospray ionization orifice allows for limited dead time between HDX reaction conclusion and analysis with mass spectrometry. Exchange rates for small molecules that were tested with the system are within range of previously reported numbers for microsecond HDX reactions of similar analytes measured by NMR as well. This platform thus provides a potential solution for studying the dynamics of disordered proteins at high spatial and temporal resolution
- Notes:
- Source: Dissertations Abstracts International, Volume: 87-12, Section: B.
- Advisors: Issadore, David A.; Garcia, Benjamin A. Committee members: Sharp, Kim; Gupta, Kushol; Glasgow, Anum; Lee, Daeyeon
- Ph.D. University of Pennsylvania 2026
- Vendor supplied data
- Local Notes:
- School code: 0175
- ISBN:
- 9798247981374
- Access Restriction:
- Restricted for use by site license
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