Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Chemistry

Supervisor

Dr. Lars Konermann

Abstract

Electrospray ionization mass spectrometry (ESI-MS) is a powerful technique for investigating protein structures, conformations, and interactions. Despite its widespread use, many fundamental aspects of ESI remain poorly understood. In this thesis, we use a combination of molecular dynamics (MD) simulations and experiments to gain insights into the hidden complexities of ESI-MS.

Chapter 2 discusses the topic of salt-induced protein signal degradation. Salts such as NaCl, CsCl, and tetrabutyl ammonium chloride (NBu4Cl) interfere with MS data acquisition, leading to adduct formation and signal suppression. MD simulations provide an explanation for these salt interferences. Signal suppression can be broken down into two effects, i.e., i) peak splitting due to adduction, ii) “genuine” signal suppression. The results obtained may be helpful to anticipate solution conditions for improved protein analyses by ESI-MS.

The two subsequent Chapters examine the mechanism of native protein supercharging, which represents a highly contentious topic. Chapter 3 uses MD simulations along with ion mobility mass spectrometry (IMS/MS). Holo-myoglobin (hMb) serves as a model protein, along with the two most common supercharging agents (SCAs), sulfolane and m-nitrobenzyl alcohol (m-NBA). Our data show that supercharging is caused by ‘charge trapping’ that arises from solvent segregation in the droplets, resulting in the formation of SCA-enriched surface layer and an aqueous core. The key factor to charge trapping is the differential solubility of charge carriers (such as Na+ or NH4+) in water compared to the exterior SCA layer. After complete water evaporation, residual SCA molecules impede charge carrier release from the droplet, and any remaining charge carriers will bind to the protein. Slow SCA evaporation eventually releases a highly charged protein into the gas phase that may undergo Coloumbic unfolding. These findings represent the first atomistic view of protein supercharging.

In Chapter 4, we explore the mechanism of native protein supercharging from a different perspective using a crown ether (18C6). 18C6 selectively binds Na+/NH4+ and enhances their solubility in the SCA layer. This facilitates the release of 18C6-bound charge carriers from the droplet. As a result, 18C6 suppressed supercharging effect, as confirmed both in MD simulations and experimentally. These data support the proposed charge trapping mechanism for both proteins and dendrimers.

A chain ejection model (CEM) has been proposed to account for the protein ESI behavior under such non-native conditions. The CEM envisions that unfolded proteins are driven to the droplet surface by hydrophobic and electrostatic factors, followed by gradual ejection via intermediates where droplets carry extended protein tails. Thus far it has not been possible to support the CEM through MD simulations. In Chapter 5 we overcome these difficulties and use MD simulations along with ion mobility experiments to confirm CEM as an ejection mechanism for unfolded proteins. Overall, the modeling and experimental work in this thesis provides unprecedented insights into the mechanism of protein charging and supercharging during ESI.

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