Formation of Gaseous Proteins via the Ion Evaporation Model (IEM) in Electrospray Mass Spectrometry.

The mechanisms whereby protein ions are released into the gas phase from charged droplets during electrospray ionization (ESI) continue to be controversial. Several pathways have been proposed. For native ESI the charged residue model (CRM) is favored; it entails the liberation of proteins via solvent evaporation to dryness. Unfolded proteins likely follow the chain ejection model (CEM), which involves the gradual expulsion of stretched-out chains from the droplet. According to the ion evaporation model (IEM) ions undergo electrostatically driven desorption from the droplet surface. The IEM is well supported for small precharged species such as Na+. However, it is unclear whether proteins can show IEM behavior as well. We examined this question using molecular dynamics (MD) simulations, mass spectrometry (MS), and ion mobility spectrometry (IMS) in positive ion mode. Ubiquitin was chosen as the model protein because of its structural stability which allows the protein charge in solution to be controlled via pH adjustment without changing the protein conformation. MD simulations on small ESI droplets (3 nm radius) showed CRM behavior regardless of the protein charge in solution. Surprisingly, many MD runs on larger droplets (5.5 nm radius) culminated in IEM ejection of ubiquitin, as long as the protein carried a sufficiently large positive solution charge. MD simulations predicted that nonspecific salt adducts are less prevalent for IEM-generated protein ions than for CRM products. This prediction was confirmed experimentally. Also, collision cross sections of MD structures were in good agreement with IMS data. Overall, this work reveals that the CRM, CEM, and IEM all represent viable pathways for generating gaseous protein ions during ESI. The IEM is favored for proteins that are tightly folded and highly charged in solution and for droplets in a suitable size regime

Atomistic Insights into the Formation of Nonspecific Protein Complexes during Electrospray Ionization.

Native electrospray ionization (ESI)-mass spectrometry (MS) is widely used for the detection and characterization of multi-protein complexes. A well-known problem with this approach is the possible occurrence of nonspecific protein clustering in the ESI plume. This effect can distort the results of binding affinity measurements, and it can even generate gas-phase complexes from proteins that are strictly monomeric in bulk solution. By combining experiments and molecular dynamics (MD) simulations, the current work for the first time provides detailed insights into the ESI clustering of proteins. Using ubiquitin as a model system, we demonstrate how the entrapment of more than one protein molecule in an ESI droplet can generate nonspecific clusters (e.g., dimers or trimers) via solvent evaporation to dryness. These events are in line with earlier proposals, according to which protein clustering is associated with the charged residue model (CRM). MD simulations on cytochrom

Investigating the Mechanism of Protein and Peptide Electrospray Ionization

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

Mobile Protons Limit the Stability of Salt Bridges in the Gas Phase: Implications for the Structures of Electrosprayed Protein Ions.

Electrosprayed protein ions can retain native-like conformations. The intramolecular contacts that stabilize these compact gas-phase structures remain poorly understood. Recent work has uncovered abundant salt bridges in electrosprayed proteins. Salt bridges are zwitterionic BH+/A- contacts. The low dielectric constant in the vacuum strengthens electrostatic interactions, suggesting that salt bridges could be a key contributor to the retention of compact protein structures. A problem with this assertion is that H+ are mobile, such that H+ transfer can convert salt bridges into neutral B0/HA0 contacts. This possible salt bridge annihilation puts into question the role of zwitterionic motifs in the gas phase, and it calls for a detailed analysis of BH+/A- versus B0/HA0 interactions. Here, we investigate this issue using molecular dynamics (MD) simulations and electrospray experiments. MD data for short model peptides revealed that salt bridges with static H+ have dissociation energies around 700 kJ mol-1. The corresponding B0/HA0 contacts are 1 order of magnitude weaker. When considering the effects of mobile H+, BH+/A- bond energies were found to be between these two extremes, confirming that H+ migration can significantly weaken salt bridges. Next, we examined the protein ubiquitin under collision-induced unfolding (CIU) conditions. CIU simulations were conducted using three different MD models: (i) Positive-only runs with static H+ did not allow for salt bridge formation and produced highly expanded CIU structures. (ii) Zwitterionic runs with static H+ resulted in abundant salt bridges, culminating in much more compact CIU structures. (iii) Mobile H+ simulations allowed for the dynamic formation/annihilation of salt bridges, generating CIU structures intermediate between scenarios (i) and (ii). Our results uncover that mobile H+ limit the stabilizing effects of salt bridges in the gas phase. Failure to consider the effects of mobile H+ in MD simulations will result in unrealistic outcomes under CIU conditions

Formation of Gaseous Peptide Ions from Electrospray Droplets: Competition between the Ion Evaporation Mechanism and Charged Residue Mechanism.

The transfer of peptide ions from solution into the gas phase by electrospray ionization (ESI) is an integral component of mass spectrometry (MS)-based proteomics. The mechanisms whereby gaseous peptide ions are released from charged ESI nanodroplets remain unclear. This is in contrast to intact protein ESI, which has been the focus of detailed investigations using molecular dynamics (MD) simulations and other methods. Under acidic liquid chromatography/MS conditions, many peptides carry a solution charge of 3+ or 2+. Because of this pre-existing charge and their relatively small size, prevailing views suggest that peptides follow the ion evaporation mechanism (IEM). The IEM entails analyte ejection from ESI droplets, driven by electrostatic repulsion between the analyte and droplet. Surprisingly, recent peptide MD investigations reported a different behavior, that is, the release of peptide ion