The Effect of Small Cations on the Positive Electrospray Responses of Proteins at Low pH

Abstract

Solutions consisting of protein and small molecule mixtures have been subjected to electrospray ionization to study the influence of small molecule/cation components at high concentrations on the electrospray responses of proteins. Emphasis was placed on solutions consisting of equal parts methanol and water and containing 1 vol % acetic acid. The results, therefore, are relevant to low pH solutions with significant organic content, a commonly used set of conditions in electrospray ionization mass spectrometry that tends to denature proteins. A variety of small cations/molecules were selected to sample a range of chemical characteristics. For example, sodium and cesium cations were studied to represent metal ions, tetrabutylammonium and tetramethylammonium cations were studied to represent quaternary ammonium compounds with different surface activities, and octadecylamine and glycine were studied to represent species that compete for protons but have different surface activities. A methodology for measuring relative ion suppression efficiencies was developed and applied for protein ions derived from bovine cytochrome c. The form of the small cation (i.e., metal ion, quaternary ammonium ion, or protonated molecule) did not appear to be a factor in determining the efficiency with which protein ion signals were suppressed. The extent to which ions are expected to concentrate on the surface, however, was the major factor in determining the ion suppression efficiency. Itwas found that the ion suppression efficiency of the most surface active species in this study was comparable to that of a protein on another protein after normalization by charge. These results are particularly relevant to the development of mixture analysis strategies based on ionization and tandem mass spectrometry applied to mixtures of whole proteins.

Monte carlo simulation of macromolecular ionization by nanoelectrospray

“…For macromolecules and with nano-ESI, as were studied here, the effects of analyte clustering for both CP and SA analytes appear to be the limiting factor in ESI ionization, as efficiency decreases occurred in simulations for CP analyte concentrations around 10 M and SA analyte concentrations around 100 M. Although macromolecules would presumably be multiply charged [7], analyte clustering would likely be the factor limiting complete analyte ionization at these concentrations. However, for small, surface active analytes, such as the 2 kDa analyte simulated here, charge depletion would be limiting [48]. The influence of charge depletion on ESI efficiency would also be magnified if larger droplets (ESI rather than nano-ESI) were used.…”

“…The influence of charge depletion on ESI efficiency would also be magnified if larger droplets (ESI rather than nano-ESI) were used. Future modeling efforts will need to take into account both analyte clustering and charge depletion, the presence of SA and CP ions in the same droplets (true modeling of macromolecular mixture ESI), and the effects of the presence of small cations [7,48] to fully describe the limiting factor in ESI.…”

Advances in the analysis of proteins and peptides by capillary electrophoresis with matrix‐assisted laser desorption/ionization and electrospray‐mass spectrometry detection

“…By complete solvent evaporation the ion will be transferred to gas phase and excess droplet charge condenses on the analyte [82].…”

“…(ii) The "Iribarne and Thomson model" proposes that after droplet reduction to a specified size direct (analyte-)ion emission from the droplet surface takes place before the actual Raleigh limit is attained [82]. This process, better known as "ion evaporation model" (IEM), becomes dominant over Coulomb fission for droplets with radii 10 nm [72].…”

“…This process, better known as "ion evaporation model" (IEM), becomes dominant over Coulomb fission for droplets with radii 10 nm [72]. There is strong experimental evidence that small molecules undergo ion evaporation, whereas the CRM is relevant for macromolecules, such as proteins [72,82]. Nonetheless, ion evaporation could also become the dominant process for peptides/proteins with low M r [11].…”