Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article


Doctor of Philosophy




Konermann, Lars


Electrospray ionization (ESI) mass spectrometry (MS) is widely used for the detection and characterization of various analytes. However, many fundamental aspects of the ESI process remain poorly understood. Using molecular dynamics (MD) simulations, MS, and ion mobility spectrometry (IMS), this thesis sheds light on the mechanisms whereby gaseous analyte ions are formed from highly charged ESI nanodroplets. After a general introduction (Chapter 1), Chapter 2 focuses on the ion evaporation mechanism (IEM), i.e., the ejection of analyte ions from the droplet surface. The IEM is well established for low MW compounds, but it has remained contentious whether this pathway is also viable for larger analytes. We examined this question using the 8.5 kDa protein ubiquitin. The structural stability of ubiquitin allows its charge in solution to be controlled via pH without triggering unfolding. Our results showed that ESI for small droplets proceeded via the charged residue mechanism (CRM). Surprisingly, MD runs on larger droplets culminated in IEM ejection of ubiquitin, as long as the protein carried a sufficiently large positive solution charge. Thus, our results reveal that the IEM is viable for intact folded proteins that are highly charged in solution, and for droplets in a suitable size regime. Chapter 3 provides insights into the nonspecific ESI clustering of proteins, a process that can be prevalent in experiments and that complicates the interpretation of mass spectra. We demonstrated how the entrapment of more than one protein molecule in an ESI droplet can generate nonspecific gaseous cluster ions via the CRM. Unexpectedly, data on cytochrome c uncovered an alternative mechanism, i.e., the formation of nonspecific complexes within ESI droplets, followed by the cluster IEM. In all cases, protein clusters were stabilized by intermolecular salt bridges. These data show that ESI-induced protein clustering does not follow a tightly orchestrated pathway, but can proceed via different avenues. Chapter 4 focuses on the ESI mechanism of peptides, which represent the most common analytes for proteomics applications. Typical peptides carry a net positive charge in solution for typically used acidic solvent mixtures. Traditional views suggest that this charge (along with the low molecular weight of peptides) should favor IEM behavior. This expectation is at odds with recent peptide MD investigations from other laboratories that showed CRM behavior. We resolved this conundrum by focusing on the 1 kDa peptide bradykinin. We found that small droplets predominantly release peptide ions via the CRM, while larger iii droplets favor IEM behavior. The prevalence of one over the other mechanism depends on the droplet size distribution in the ESI plume.

Summary for Lay Audience

Proteins are biological macromolecules that carry out critical functions in all living cells. Electrospray ionization (ESI) mass spectrometry (MS) has opened up new avenues for studying proteins, from fundamental biophysical investigations to applications in the biopharmaceutical industry. For every MS analysis, proteins have to be converted from solution-phase species into gaseous ions. The ESI process is driven by a high voltage that is being applied to the ESI capillary into which the protein solution is introduced. The mechanisms whereby gaseous ions are released from charged ESI nanodroplets remain unclear. Different models have been proposed. The focus of this thesis is on two possible mechanisms; i.e., the charged residue model (CRM) and the ion evaporation model (IEM). According to the CRM, gaseous proteins are released upon solvent evaporation to dryness. This model is widely accepted for large globular proteins. The IEM is a competing mechanism that is widely believed to describe the ESI behavior of small precharged molecules. For this second mechanism, the analyte is desorbed from the droplet surface due to electrostatic repulsion between the protein charge and other charge carriers within the droplet. In this work, molecular dynamic simulations are used to study the mechanism of protein and peptide ESI in more detail. The results of the Chapter 2 demonstrate for the first time that the CRM is not the only viable mechanism for native-like proteins. Instead, it is demonstrated that the IEM can be operative for proteins such as ubiquitin that retain a tightly folded conformation during ESI. Chapter 3 examines the formation of nonspecific complexes during ESI at high protein concentrations. The simulations revealed that the entrapment of multiple protein molecules within a charged droplet can generate different protein complexes following both IEM and CRM events. Chapter 4 reconciles the traditional belief that peptides generally show IEM behaviour with earlier simulation reports that found CRM behaviour. It is demonstrated that both scenarios are possible, depending on the peptide charge state and composition, as well as solvent properties, including the solution pH, and droplets dimensions. Overall, the results of this thesis demonstrate that both IEM and CRM events are possible for a wide range of analyte molecules, thereby contributing to a better general understanding of the ESI process.