Date of Award
12-2021
Degree Name
Doctor of Philosophy
Department
Chemistry
First Advisor
Dr. Andre R. Venter
Second Advisor
Dr. Todd J. Barkman
Third Advisor
Dr. David L. Huffman
Fourth Advisor
Dr. Kelly A. Teske
Keywords
Mass spectrometry, DESI, protein, electrospray ionization, additives, amino acids
Abstract
Electrospray ionization mass spectrometry (ESI-MS) is a soft ionization technique that allows detection of macromolecules, such as intact proteins, by the formation of multiply charged ions from solutions. Desorption electrospray ionization mass spectrometry (DESI-MS) is an ambient ionization technique that directly samples analyte from a surface during ESI-MS analysis. Although DESI-MS is highly accomplished at the analyses of metabolites, lipids, and other small molecules, it is far more limited when it comes to protein analysis. While most of the field in ambient ionization MS has moved towards primarily applications, our approach has been to explore the use of DESI-MS and direct ESI-MS to answer fundamental scientific questions. Understanding the mechanisms by which proteins are analyzed with these techniques provides essential insight into protein behavior and enables improving these techniques even further.
The presented work focuses on improving DESI-based protein analysis via solution- phase and gas-phase additives and understanding the underlying mechanisms by which these additives improve protein signal. DESI-MS and complementary direct ESI-MS experiments were used to (1) investigate the effect of amino acid additives on protein signal, (2) understand the mechanism by which amino acid additives improve protein signal during DESI-MS, (3) investigate the effect of organic solvent vapors on protein signal, and (4) incorporate these techniques and findings into developing a novel method for rapid analysis of immobilized His- tagged proteins.
As a result, we were able to successfully improve protein analysis by DESI-MS through the addition of L-serine to the desorption solvent. Serine was shown to act as a solubility enhancing additive through improving dissolution of unfolding proteins during the extraction/desorption step of DESI-MS, potentially by inhibiting aggregation. Exposing the DESI-MS sampling region to ethyl acetate vapors also improved the signal intensity of proteins similar to previously reported ESI-MS observations. Finally, the potential application of direct ESI-MS and DESI-MS for rapid analysis of immobilized recombinant His-tagged proteins from Ni-NTA and Cu-NTA coated surfaces was evaluated. We successfully demonstrated the capture and release of recombinant His-tagged human ubiquitin from Ni-NTA and Cu-NTA surfaces by DESI-MS. Furthermore, we show the detection of His-tagged recombinant protein directly out of complex solutions containing the total protein fraction of the E. coli expression system and the lysis buffer, after purifying on Ni- and Cu-NTA plates. This work demonstrated the potential of direct ESI-MS and DESI-MS for rapid analysis of recombinant His-tagged proteins from crude bacterial cell lysate.
Access Setting
Dissertation-Open Access
Recommended Citation
Javanshad, Roshan, "Mechanisms and Applications of Improved Protein Analysis by Desorption Electrospray Ionization Mass Spectrometry (DESI-MS)" (2021). Dissertations. 3780.
https://scholarworks.wmich.edu/dissertations/3780
Javanshad SWAA