Native surface mass spectrometry via static liquid extraction and laser ablation sampling for structural characterization of macromolecules in gas phase.

Native Surface Mass Spectrometry is an emerging field in biochemical sciences that aims to combine non-denaturing macromolecular proteomics with structural biology and biophysics to elucidate protein functions. By sampling endogenous proteins from their intact biological matrix, proteins' higher-order structure, reaction stoichiometry, and structural motifs can be evaluated as close to "in-vivo" conditions as possible. Integration of ion-mobility spectrometry and mass spectrometry enhances the extent of biophysical characterization for protein and protein complexes. In this dissertation, an online extraction-ionization platform, using either a solid-liquid microextraction or laser ablation microextraction sampling, was coupled to a commercial mass spectrometer equipped with traveling wave-ion mobility for in depth characterization of proteins' "nativeness" under native surface MS conditions. Multiplexed collision induced unfolding (CIU) results from ion mobility mass spectrometry (IM-MS) data showed that unfolding pathway dynamics for proteins sampled via solid-liquid microextraction and laser ablation capture were analogous. Although the presence of adducts and limited mass resolving power at high masses (> 100 kDa) can severely constrain the identification capabilities of direct native surface MS, by separating the detection and microextraction events, concurrent tryptic digest and LC-IM-MS/MS analyses provided valuable complementary data. Moreover, a bioinformatics pathway was developed to correlate the observed peaks from native IM-MS and CIU experiments to protein identities from bottom-up proteomics as well as the number of observable unfolding intermediates. Collectively, our findings suggest that further laser ablation experiments, in conjunction with bottom-up and IM-MS proteomics, could be refined to enable spatially resolved characterization of protein complexes and non-covalent interactions.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Doctoral

Grantor

Baylor University.

Year dc:date.issued

2024

Author dc:creator

• Villacob, Raul A., 1992-

Advisor dc:contributor.advisor

• Solouki, Touradj, 1963-

• Ion mobility.
• Gas-phase structural biology.
• Proteomics.
• Ionization sources.

Statement dc:rights

• Baylor University works are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. Contact libraryquestions@baylor.edu for inquiries about permission.

Language dc:language.iso

en

Handle dc:identifier.uri

https://hdl.handle.net/2104/12986

OAI identifier oai:identifier

oai:baylor-ir.tdl.org:2104/12986