Search for: All records

Abstract This review presents progress made in the ambient analysis of proteins, in particular by desorption electrospray ionization‐mass spectrometry (DESI‐MS). Related ambient ionization techniques are discussed in comparison to DESI‐MS only to illustrate the larger context of protein analysis by ambient ionization mass spectrometry. The review describes early and current approaches for the analysis of undigested proteins, native proteins, tryptic digests, and indirect protein determination through reporter molecules. Applications to mass spectrometry imaging for protein spatial distributions, the identification of posttranslational modifications, determination of binding stoichiometries, and enzymatic transformations are discussed. The analytical capabilities of other ambient ionization techniques such as LESA and nano‐DESI currently exceed those of DESI‐MS for in situ surface sampling of intact proteins from tissues. This review shows, however, that despite its many limitations, DESI‐MS is making valuable contributions to protein analysis. The challenges in sensitivity, spatial resolution, and mass range are surmountable obstacles and further development and improvements to DESI‐MS is justified. 

RationalePurification of recombinant proteins is a necessary step for functional or structural studies and other applications. Immobilized metal affinity chromatography is a common recombinant protein purification method. Mass spectrometry (MS) allows for confirmation of identity of expressed proteins and unambiguous detection of enzymatic substrates and reaction products. We demonstrate the detection of enzymes purified on immobilized metal affinity surfaces by direct or ambient ionization MS, and follow their enzymatic reactions by direct electrospray ionization (ESI) or desorption electrospray ionization (DESI). MethodsA protein standard, His‐Ubq, and two recombinant proteins, His‐SHAN and His‐CS, expressed inEscherichia coliwere immobilized on two immobilized metal affinity systems, Cu–nitriloacetic acid (Cu‐NTA) and Ni‐NTA. The proteins were purified on surface, and released in the ESI spray solvent for direct infusion, when using the 96‐well plate form factor, or analyzed directly from immobilized metal affinity‐coated microscope slides by DESI‐MS. Enzyme activity was followed by incubating the substrates in wells or by depositing substrate on immobilized protein on coated slides for analysis. ResultsSmall proteins (His‐Ubq) and medium proteins (His‐SAHN) could readily be detected from 96‐well plates by direct infusion ESI, or from microscope slides by DESI‐MS after purification on surface from clarifiedE. colicell lysate. Protein oxidation was observed for immobilized proteins on both Cu‐NTA and Ni‐NTA; however, this did not hamper the enzymatic reactions of these proteins. Both the nucleosidase reaction products for His‐SAHN and the methylation product of His‐CS (theobromine to caffeine) were detected. ConclusionsThe immobilization, purification, release and detection of His‐tagged recombinant proteins using immobilized metal affinity surfaces for direct infusion ESI‐MS or ambient DESI‐MS analyses were successfully demonstrated. Recombinant proteins were purified to allow identification directly out of clarified cell lysate. Biological activities of the recombinant proteins were preserved allowing the investigation of enzymatic activity via MS. 

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. 

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