Investigating the Mechanism of Protein and Peptide Electrospray Ionization
Abstract
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.