Signal Response of Coexisting Protein Conformers in Electrospray Mass Spectrometry

Abstract

Electrospray ionization mass spectrometry (ESI-MS) is a commonly used tool for characterizing conformational changes of proteins in solution. Different conformations can be distinguished on the basis of their ESI charge state distributions. ESI-MS studies carried out under semidenaturing conditions result in bi- or multimodal distributions that reflect the presence of coexisting conformers. This study explores whether the concentration ratios of these species in solution are reflected in the measured ion intensities. Experiments on two model proteins, lysozyme and myoglobin, reveal that non-native polypeptide chains tend to result in a much stronger signal response than natively folded species. The measured ion intensity ratios can differ from the actual concentration ratios by as much as 2 orders of magnitude. It is proposed that the higher ionization efficiency of unfolded proteins is due to their partially hydrophobic character, which results in a larger surface activity and facilitates protein transfer into ion-producing progeny droplets. Conversely, natively folded proteins have a lower affinity for the air/liquid interface, such that ionization of these conformers is suppressed. The extent of ion suppression is strongly dependent on the experimental conditions such as flow rate and protein concentration, which determine if ESI occurs in a charge deficient or a charge surplus regime. These aspects should be taken into account for the design of ESI-MS-based protein folding experiments and for studies that use ion intensity ratios for the determination of protein-ligand binding affinities.

Do ionic charges in ESI MS provide useful information on macromolecular structure?

“…As a consequence, these species are much more likely to be located at the solvent/air interface in the charged droplets produced by electrospray and will have a much higher probability to be transferred to the ion-producing progeny droplets during Coulomb explosion events. Fortunately, the study also found that suppression of the ionic signal corresponding to the natively folded protein species can be minimized if the experiments are carried out in a charge surplus regime [11].…”

“…Many early studies implicitly assumed that the answer to this question is "yes," although some reservations were also expressed [9]. A recent elegant study by Konermann and coworkers explored the relationship between the fractional concentrations of different conformers and their respective ionic signals [11]. Convincing evidence was presented that non-native polypeptide chains generate higher signal response compared to the natively folded species.…”

Monte carlo simulation of macromolecular ionization by nanoelectrospray

“…However, tracking the evaporation and fission of all progeny droplets formed from all original droplets would be too computationally intense for a practical model, as a single ESI droplet will give rise to a multitude of progeny droplets during the evaporation/fission process. As an alternative, analyte ionization from progeny droplets was determined by whether analytes were one of two theoretical analyte types: cluster prone surface inactive (CP) or surface active (SA) [22]. CP analytes were modeled as particles that would cluster in progeny droplets and did not form gas-phase ions unless there was only one analyte in a progeny droplet.…”

“…McLuckey and coworkers [21] measured the signal intensities (related directly to the ionization efficiency) of analyte ions from protein mixtures electrosprayed at various pH values and deter-mined that the main parameter influencing ionization efficiency was the protein surface charge. Likewise, Kuprowski and Konermann [22] showed that the signal intensity of ions from ESI of denatured proteins with higher surface charge is greater than the signal intensity of ions from the ESI native state proteins. Thus far, however, a model of the ESI process for macromolecules has not been developed.…”

A simple model for the disintegration of highly charged solvent droplets during electrospray ionization

“…In contrast, compounds with low surface affinity accumulate in the charge-depleted residual parent, from where ionization is inefficient [21,22]. Two limiting cases are commonly discussed for the final step by which ions are released into the gas phase from nanometer-sized progeny droplets.…”