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.

Desalting protein ions in native mass spectrometry using supercharging reagents.

Effects of the supercharging reagents m-NBA and sulfolane on sodium ion adduction to protein ions formed using native mass spectrometry were investigated. There is extensive sodium adduction on protein ions formed by electrospray ionization from aqueous solutions containing millimolar concentrations of NaCl, which can lower sensitivity by distributing the signal of a given charge state over multiple adducted ions and can reduce mass measuring accuracy for large proteins and non-covalent complexes for which individual adducts cannot be resolved. The average number of sodium ions adducted to the most abundant ion formed from ten small (8.6-29 kDa) proteins for which adducts can be resolved is reduced by 58% or 80% on average, respectively, when 1.5% m-NBA or 2.5% sulfolane are added to aqueous solutions containing sodium compared to without the supercharging reagent. Sulfolane is more effective than m-NBA at reducing sodium ion adduction and at preserving non-covalent protein-ligand and protein-protein interactions. Desalting with 2.5% sulfolane enables detection of several glycosylated forms of 79.7 kDa holo-transferrin and NADH bound to the 146 kDa homotetramer LDH, which are otherwise unresolved due to peak broadening from extensive sodium adduction. Although sulfolane is more effective than m-NBA at protein ion desalting, m-NBA reduces salt clusters at high m/z and can increase the signal-to-noise ratios of protein ions by reducing chemical noise. Desalting is likely a result of these supercharging reagents binding sodium ions in solution, thereby reducing the sodium available to adduct to protein ions.

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.

Loss of siloxane monolayers from GaN surfaces in water.

Gallium nitride, with a thin passivating layer of Ga2O3, has been functionalized with octadecyltrichlorosilane (OTS) and aminopropyltriethoxysilane (APTES) self-assembled monolayers (SAMs). Water contact angles, atomic force microscopy, and X-ray photoelectron spectroscopy were used for characterization of the bare and functionalized surfaces. The SAMs are stable in acetonitrile, but both the hydrophobic OTS SAM and the hydrophilic APTES SAM completely desorb after 1-24 h of immersion in water and common buffers. The concentration of gallium in solution after a clean GaN chip is immersed in water is consistent with dissolution of roughly one monolayer of interfacial gallium oxide. Dissolution of this oxide layer could account for the loss of SAMs from GaN surfaces.

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.

Electrothermal supercharging of proteins in native electrospray ionization.

The formation of high charge-state protein ions with nanoelectrospray ionization (nESI) from purely aqueous ammonium bicarbonate solutions at neutral pH, where the proteins have native or native-like conformations prior to ESI droplet formation, is demonstrated. This "electrothermal" supercharging method depends on the temperature of the instrument entrance capillary, the nESI spray potential, and the solution ionic strength and buffer, although other factors almost certainly contribute. Mass spectra obtained with electrothermal supercharging appear similar to those obtained from denaturing solutions where charging beyond the total number of basic sites can be achieved. For example, a 17+ ion of bovine ubiquitin was formed by nESI of a 100 mM ammonium bicarbonate, pH 7.0, solution, which is three more charges than the total number of basic amino acids plus the N-terminus. Heating of the ESI droplets in the vacuum/atmosphere interface and the concomitant denaturation of the protein in the ESI droplets prior to ion formation appears to be the primary origin of the very high charge-state ions formed from these purely aqueous, buffered solutions. nESI mass spectra resembling those obtained under traditional native or denaturing conditions can be reversibly obtained simply by toggling the spray voltage between low and high values.

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.

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.

Supercharging protein complexes from aqueous solution disrupts their native conformations.

The effects of aqueous solution supercharging on the solution- and gas-phase structures of two protein complexes were investigated using traveling-wave ion mobility-mass spectrometry (TWIMS-MS). Low initial concentrations of m-nitrobenzyl alcohol (m-NBA) in the electrospray ionization (ESI) solution can effectively increase the charge of concanavalin A dimers and tetramers, but at higher m-NBA concentrations, the increases in charge are accompanied by solution-phase dissociation of the dimers and up to a ~22% increase in the collision cross section (CCS) of the tetramers. With just 0.8% m-NBA added to the ESI solution of a ~630 kDa anthrax toxin octamer complex, the average charge is increased by only ~4% compared with the "native" complex, but it is sufficiently destabilized so that extensive gas-phase fragmentation occurs in the relatively high pressure regions of the TWIMS device. Anthrax toxin complexes exist in either a prechannel or a transmembrane channel state. With m-NBA, the prechannel state of the complex has the same CCS/charge ratio in the gas phase as the transmembrane channel state of the same complex formed without m-NBA, yet undergoes extensive dissociation, indicating that destabilization from supercharging occurs in the ESI droplet prior to ion formation and is not a result of Coulombic destabilization in the gas phase as a result of higher charging. These results demonstrate that the supercharging of large protein complexes is the result of conformational changes induced by the reagents in the ESI droplets, where enrichment of the supercharging reagent during droplet evaporation occurs.

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.

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.