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Introduction Previous work has shown that exposure of electrospray droplets to ethyl acetate produce spectra with more intense protein signal, as well as protein envelopes shifted toward higher charge states . This is of specific interest when carrying out DESI-MS analysis, as the technique struggles to analyze proteins larger than 25 kDa in size due to poor dissolution and adduction. . The mechanism by which ethyl acetate improves responses was studied by analyzing protein molecules in atmospheres modified with ethyl acetate and related polar organic compounds, and an analogue series of esters with increasing chain lengths. Methods All spectra were collected using a Thermo LTQ XL mass spectrometer. ESI samples were 2.5 μM cytochrome C, myoglobin or lysozyme in 80% methanol with 0.1% formic acid or in aqueous 100mM ammonium acetate. A polypropylene enclosure for introduction of additive to the atmospheric region around the ion source and transfer tube was constructed . Liquid additives were introduced at a controlled, continuous, flow rate of 70 μL/min onto a flash chromatography pad acting as a reservoir inside of the enclosure. The additive was allowed to evaporate from the reservoir pad to saturate the ionization region. Relative changes in signals upon vapor additions were reported as the ratio to enclosed signal without vapor modification. Preliminary Data Our results indicate dependence on the alkyl chain length on either side of the ester functional group. By increasing the alkyl chain length of alcohol and carboxylic acid precursors, the hydrophobicity of esters also increases. Longer alkyl chains proton affinities of these molecules. Proteins were analyzed from denaturing conditions and ionization is believed to occur through the chain ejection model (CEM). We suggest that the increasing hydrophobicity of the esters may increasingly aid in lowering the energy barrier of transfer of the denatured protein from the solvated state in bulk droplet to the gas phase. This might be a consequence of the formation of a condensed ester on the evaporative cooled microdroplet, or a gas phase interaction. The degree of improvement when modifying the ionization region with esters initially shows little to no increase when using those with smaller alkyl chains (C3, C4). With longer chains (C7), however, dramatic improvements in protein signal can be observed. This is particularly evident when analyzing higher charge state peaks corresponding to more unfolded protein populations, such peaks corresponding to more unfolded protein populations. This effect may be due to competition between vapor pressure of the atmospheric modifiers and hydrophobic interactions between the modifier and ejecting protein. Vapor pressure drastically decreases between ethyl acetate to longer chain esters such as butyl acetate. Additional heating of the pad will be investigated to compensate for this trade-off. . With even longer chain esters, such as ethyl heptanoate, we see a greater improvement, indicating that their increased hydrophobic character and resultant analyte interactions is more favorable. As well, the improved signal observed for higher charge state peaks consequently increases the protein average charge state. Novel Aspect Vapor addition of esters with increasing chain lengths to the atmospheric ionization region improve protein detection by electrospray-based methods 

Introduction The effects of the introduction of ambient gases into the region of the electrospray ionization (ESI) source have been investigated only in a very limited way. Dopant gases added to the ambient environment of the ESI Taylor cone can influence a variety of parameters associated with the ESI process. Even in the absence of covalent attachment to analytes, ambient gases can influence ESI characteristics such as the onset potential for electrospray, as well as mass spectral features such as the charge state distribution. In this study, a new model has been developed to account for modified onset potentials observed in response to analyte protein characteristics, and as a consequence of the presence of dopant gas in the ESI source region. Methods All experiments were performed on a SolariX ® Fourier Transform-Ion Cyclotron Resonance (FT-ICR) mass spectrometer (Bruker, Bremen, Germany) fitted with a CaptiveSpray ® ion source equipped with a nanoBooster ® (Bruker). This combined accessory is an enclosed chamber that allows the low-pressure addition of volatile dopant gas into the region surrounding the ESI Taylor cone. A manually operated toggle valve switches the arrival of pressurized N 2 gas (default mode) to that of the gas contained in the headspace of an attached solvent-containing glass bottle (the nanoBooster ®). Thus, the opening of the toggle valve introduces solvent vapor into the immediate vicinity of the ESI emitter. This solvent vapor will interact with the liquid stream undergoing ESI, thus acting as a dopant. Preliminary Data A new model has been developed to explain adsorption behavior at the electrospray emitter based upon data obtained using a series of dopant ambient gases in the nanoBooster ® headspace chamber. Four volatile dopants (acetone, acetonitrile, ethyl acetate, methanol) were added sequentially into the CaptiveSpray chamber prompting interactions with aqueous solutions containing three separate proteins (cytochrome c, lysozyme, myoglobin). The isoelectric point of the protein was found to exert a significant influence on the observed onset potential for ESI. Lysozyme, the most basic of the three tested proteins, consistently afforded the lowest onset potential, regardless of which dopant was present in the spray chamber. Myoglobin, the least basic of the three proteins, always yielded a higher onset potential than lysozyme, whereas the intermediate basicity cytochrome c gave variable rankings. These results build upon complementary findings that considered the effects of physical parameters of the employed dopants (dielectric constant, dipole moment, proton affinity, surface tension) on ESI behavior. Of these investigated parameters, the dopant proton affinity was found to exert the clearest influence on ESI onset potential, with lower onset potentials observed for higher proton affinity dopants. These converging trends related to the basicity of the dissolved protein undergoing ESI, and the proton affinity of the added dopant in the spray chamber, are presented here for the first time. This data indicates the importance of surface protons in attracting dopant gases whose arrival thereby modifies the surface of the solution undergoing ESI. A new model to explain the complementary nature of these converging trends is proposed and presented in a visual depiction. These results have implications that can enable a means to increase spray stability when performing ESI using purely aqueous solutions. Novel Aspect A new model has been formulated to explain gaseous dopant-dissolved analyte behavior in the ESI chamber. 

The surprising formation of highly charged protein ions from aqueous ammonium bicarbonate solution is a fascinating phenomenon referred to as electrothermal supercharging (ETS). Although the precise mechanism involved is not clearly understood, previous studies predominantly suggest that ETS is due to native protein destabilization in the presence of bicarbonate anion inside the electrospray ionization droplets under high temperatures and spray voltages. To evaluate existing hypotheses surrounding the underlying mechanism of ETS, the effects of several additives on protein charging under ETS conditions were investigated. The changes in the protein charge state distributions were compared by measuring the ratios between the intensities of highest intensity charge states of native and unfolded protein envelopes and shifts in the lowest and highest observed charge states. This study demonstrated that source temperature plays a more important role in ETS compared to spray voltage, especially when using a nebulized microelectrospray ionization source. Moreover, the effect of amino acids on ETS were generally in good agreement with the extensive literature available on the stabilization or destabilization of proteins by these additives in bulk solution. Among the natural amino acids, protein supercharging was significantly reduced by proline and glycine; however, imidazole provided the highest degree of noncovalent complex stabilization against ETS, outperforming the amino acids. Overall, our study shows that the simple addition of stabilizing reagents such as proline and imidazole can reduce the extent of apparent protein unfolding and supercharging in ammonium bicarbonate solution and provide evidence against the roles of charge depletion and thermal unfolding during ETS. 

Naturally occurring amino acids have been broadly used as additives to improve protein solubility and inhibit aggregation. In this study, improvements in protein signal intensity obtained with the addition of l -serine, and structural analogs, to the desorption electrospray ionization mass spectrometry (DESI-MS) spray solvent were measured. The results were interpreted at the hand of proposed mechanisms of solution additive effects on protein solubility and dissolution. DESI-MS allows for these processes to be studied efficiently using dilute concentrations of additives and small amounts of proteins, advantages that represent real benefits compared to classical methods of studying protein stability and aggregation. We show that serine significantly increases the protein signal in DESI-MS when native proteins are undergoing unfolding during the dissolution process with an acidic solvent system ( p -value = 0.0001), or with ammonium bicarbonate under denaturing conditions for proteins with high isoelectric points ( p -value = 0.001). We establish that a similar increase in the protein signal cannot be observed with direct ESI-MS, and the observed increase is therefore not related to ionization processes or changes in the physical properties of the bulk solution. The importance of the presence of serine during protein conformational changes while undergoing dissolution is demonstrated through comparisons between the analyses of proteins deposited in native or unfolded states and by using native state-preserving and denaturing desorption solvents. We hypothesize that direct, non-covalent interactions involving all three functional groups of serine are involved in the beneficial effect on protein solubility and dissolution. Supporting evidence for a direct interaction include a reduction in efficacy with d -serine or the racemic mixture, indicating a non-bulk-solution physical property effect; insensitivity to the sample surface type or relative placement of serine addition; and a reduction in efficacy with any modifications to the serine structure, most notably the carboxyl functional group. An alternative hypothesis, also supported by some of our observations, could involve the role of serine clusters in the mechanism of solubility enhancement. Our study demonstrates the capability of DESI-MS together with complementary ESI-MS experiments as a novel tool for understanding protein solubility and dissolution and investigating the mechanism of action for solubility-enhancing additives.