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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