Catherine A. Cassou

Protein Conformation and Supercharging with DMSO from Aqueous Solution

The efficacy of dimethyl sulfoxide (DMSO) as a supercharging reagent for protein ions formed by electrospray ionization from aqueous solution and the mechanism for supercharging were investigated. Addition of small amounts of DMSO to aqueous solutions containing hen egg white lysozyme or equine myoglobin results in a lowering of charge, whereas a significant increase in charge occurs at higher concentrations. Results from both near-UV circular dichroism spectroscopy and solution-phase hydrogen/deuterium exchange mass spectrometry indicate that DMSO causes a compaction of the native structure of these proteins at low concentration, but significant unfolding occurs at ~63% and ~43% DMSO for lysozyme and myoglobin, respectively. The DMSO concentrations required to denature these two proteins in bulk solution are ~3–5 times higher than the concentrations required for the onset of supercharging, consistent with a significantly increased concentration of this high boiling point supercharging reagent in the ESI droplet as preferential evaporation of water occurs. DMSO is slightly more basic than m-nitrobenzyl alcohol and sulfolane, two other supercharging reagents, based on calculated proton affinity and gas-phase basicity values both at the B3LYP and MP2 levels of theory, and all three of these supercharging reagents are significantly more basic than water. These results provide additional evidence that the origin of supercharging from aqueous solution is the result of chemical and/or thermal denaturation that occurs in the ESI droplet as the concentration of these supercharging reagents increases, and that proton transfer reactivity does not play a significant role in the charge enhancement observed.

The role of conformational flexibility on protein supercharging in native electrospray ionization

Effects of covalent intramolecular bonds, either native disulfide bridges or chemical crosslinks, on ESI supercharging of proteins from aqueous solutions were investigated. Chemically modifying cytochrome c with up to seven crosslinks or ubiquitin with up to two crosslinks did not affect the average or maximum charge states of these proteins in the absence of m-nitrobenzyl alcohol (m-NBA), but the extent of supercharging induced by m-NBA increased with decreasing numbers of crosslinks. For the model random coil polypeptide reduced/alkylated RNase A, a decrease in charging with increasing m-NBA concentration attributable to reduced surface tension of the ESI droplet was observed, whereas native RNase A electrosprayed from these same solutions exhibited enhanced charging. The inverse relationship between the extent of supercharging and the number of intramolecular crosslinks for folded proteins, as well as the absence of supercharging for proteins that are random coils in aqueous solution, indicate that conformational restrictions induced by the crosslinks reduce the extent of supercharging. These results provide additional evidence that protein and protein complex supercharging from aqueous solution is primarily due to partial or significant unfolding that occurs as a result of chemical and/or thermal denaturation induced by the supercharging reagent late in the ESI droplet lifetime.

Electrothermal supercharging in mass spectrometry and tandem mass spectrometry of native proteins.

Electrothermal supercharging of protein ions formed by electrospray ionization from buffered aqueous solutions results in significant increases to both the maximum and average charge states compared to native mass spectrometry in which ions are formed from the same solutions but with lower spray potentials. For eight of the nine proteins investigated, the maximum charge states of protonated ions formed from native solutions with electrothermal supercharging is greater than those obtained from conventional denaturing solutions consisting of water/methanol/acid, although the average charging is slightly lower owing to contributions of small populations of more folded low charge-state structures. Under these conditions, electrothermal supercharging is slightly less effective for anions than for cations. Equivalent sequence coverage (80%) is obtained with electron transfer dissociation of the same high charge-state ion of cytochrome c formed by electrothermal supercharging from native solutions and from denaturing solutions. Electrothermal supercharging should be advantageous for combining structural studies of proteins in native environments with mass spectrometers that have limited high m/z capabilities and for significantly improving tandem mass spectrometry performance for protein ions formed from solutions in which the molecules have native structures and activities.

Solution Additives that Desalt Protein Ions in Native Mass Spectrometry

The presence of many salts, such as sodium chloride, can adversely affect the performance of native electrospray ionization mass spectrometry for the analysis of proteins and protein complexes by reducing the overall molecular ion abundances and distributing signal for any given charge state into many cationized forms with various numbers of adducts attached. Several solution additives, such as ammonium bromide, ammonium iodide, and NaSbF(6), can significantly lower the extent of sodium ion adduction to the molecular ions of proteins and protein complexes. For ubiquitin, addition of 25 mM ammonium bromide or ammonium iodide into aqueous solutions also containing 1.0 mM NaCl results in a factor of 72 and 56 increase, respectively, in the relative abundances of the fully protonated molecular ions compared to when these additives are not present. The effectiveness of this method for reducing sodium ion adduction is related to the low proton affinity (PA) values of the anions. Anions with very low PA also have a propensity to adduct as an acid molecule, but these adducts can be readily dissociated from the molecular ions either by activation in the source or subsequently by collisional activation in the mass spectrometer. This method of reducing sodium ion adduction to proteins is simple and requires no experimental modifications, making it an attractive alternative to other methods for desalting proteins prior to mass spectrometry analysis.

Anions in electrothermal supercharging of proteins with electrospray ionization follow a reverse Hofmeister series.

The effects of different anions on the extent of electrothermal supercharging of proteins from aqueous ammonium and sodium salt solutions were investigated. Sulfate and hydrogen phosphate are the most effective anions at producing high charge state protein ions from buffered aqueous solution, whereas iodide and perchlorate are ineffective with electrothermal supercharging. The propensity for these anions to produce high charge state protein ions follows the following trend: sulfate > hydrogen phosphate > thiocyanate > bicarbonate > chloride > formate ≈ bromide > acetate > iodide > perchlorate. This trend correlates with the reverse Hofmeister series over a wide range of salt concentrations (1 mM to 2 M) and with several physical properties, including solvent surface tension, anion viscosity B-coefficient, and anion surface/bulk partitioning coefficient, all of which are related to the Hofmeister series. The effectiveness of electrothermal supercharging does not depend on bubble formation, either from thermal degradation of the buffer or from coalescence of dissolved gas. These results provide evidence that the effect of different ions in the formation of high charge state ions by electrothermal supercharging is largely a result of Hofmeister effects on protein stability leading to protein unfolding in the heated ESI droplet.

High-Frequency AC Electrospray Ionization Source for Mass Spectrometry of Biomolecules

A novel high-frequency alternating current (AC) electrospray ionization (ESI) source has been developed for applications in mass spectrometry. The AC ESI source operates in a conical meniscus mode, analogous to the cone-jet mode of direct current (DC) electrosprays but with significant physical and mechanistic differences. In this stable conical-meniscus mode at frequencies greater than 50 kHz, the low mobility ions, which can either be cations or anions, are entrained within the liquid cone and ejected as droplets that eventually form molecular ions, thus making AC ESI a viable tool for both negative and positive mode mass spectrometry. The performance of the AC ESI source is qualitatively shown to be frequency-dependent and, for larger bio-molecules, the AC ESI source produced an ion signal intensity that is an order of magnitude higher than its DC counterpart.

Frequency Dependence of Alternating Current Electrospray Ionization Mass Spectrometry

The novel effects resulting from the entrainment of low mobility ions during alternating current (ac) electrospray ionization are examined through mass spectrometry and voltage/current measurements. Curious phenomena such as pH modulation at high frequencies (>150 kHz) of an applied ac electric field are revealed and explained using simple mechanistic arguments. Current measurements are utilized to supplement these observations, and a simplified one-dimensional transient diffusion model for charge transport is used to arrive at a scaling law that provides better insight into the ac electrospray ionization process. Moreover, because of the different pathway for ion formation in comparison to direct current (dc) electrospray, ac electrospray (at frequencies >250 kHz) is shown to reduce the effects of ionization suppression in a mixture of two molecules with different surface activities.