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

Abstract

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.

Protein Secondary Structure Affects Glycan Clustering in Native Mass Spectrometry

“…However, the non-linear glycan clustering behaviour of Ubq hints at ionization following another model, as previously suggested by MD simulations. According to these simulations small proteins can also be ionized through the ion evaporation model (IEM) [24], which would explain the peculiar behaviour of Ubq and may also play a role for other small proteins. The cyt c clustering patterns have a non-Gaussian character, which could be caused by ISD or varying ESI efficiencies.…”

Protein secondary structure affects glycan clustering in native mass spectrometry

“…However, the non-linear glycan clustering behaviour of Ubq hints at ionization following another model as previously suggested by MD simulations. According to these simulations small proteins can also be ionized through the ion evaporation model (IEM) [29], which would explain the peculiar behaviour of Ubq and may also play a role for other small proteins. The cyt c clustering patterns have non-Gaussian character, which could be caused by ISD or varying ESI efficiencies.…”

Direct observation of ion emission from charged aqueous nanodrops: effects on gaseous macromolecular charging

“…4,5,20,[22][23][24] Molecular dynamics (MD) simulations support ion evaporation of Na + and other small ions from nanodrops with diameters <5.5 nm. [22][23][24] For droplets that contain larger analytes, results from MD simulations suggest the possibility of several different mechanisms. Simulations by Consta and co-workers indicate that droplets that contain a DNA or RNA ion can distort into "star" morphologies in the late stages of desiccation.…”

“…25,26 Recent simulations by Aliyari and Konermann suggest an ion emission-like process may also occur for small native proteins that carry a sufficiently high charge in solution. 24 Following on early pioneering work by Gamero-Castaño and Fernández de la Mora, 27 Hogan et al 28 proposed a hybrid model that combines both the charge residue and eld emission models (combined charge residue and eld emission model or CCRFEM). In this model, the charge states of proteins and other macromolecules are not determined solely by the Rayleigh limit of a droplet of similar size, as would the be case for just the CRM, but rather the charge is also limited by eld-induced emission of small charged solute ions and clusters from the protein-containing nanodroplet (an IEM process).…”

“… 25,26 Recent simulations by Aliyari and Konermann suggest an ion emission-like process may also occur for small native proteins that carry a sufficiently high charge in solution. 24 …”