Nanobubbles (NBs) are very small gas-filled cavities in solvents, and when their sizes reach diameters around 200 nm, they remain in solution for extended periods of time, featuring special chemical and physical properties. Here, we investigate the application of NBs in electrospray ionization. We show that the addition of CO2 or N2 NBs into spray solvents significantly improves signal responses of small molecules in both positive and negative modes during ESI-MS. The magnitude of the increase depends on analyte identity, solvent system, NB gas composition, and the method for preparing the NBs. When NBs are used to analyze proteins, both signal intensities and charge states increase. This is tentatively attributed to NB's increasing the total area of the hydrophobic gas-liquid interface, on which proteins can unfold, and improved transport of analytes to the droplet surface. This increase in the interface is likely also a contributing factor in the further enhancement of the rate at which reagents are converted into products when NBs are present compared to those measured from accelerated reactions from microdroplets during reactive-ESI experiments. Moreover, NBs can overcome solubility barriers when one of the reagents is gaseous and, thus, can be incorporated into an NB. This was demonstrated in the reaction between N,N-dibutyl-1,3-propane diamine and carbon dioxide, where the reaction conversion rate could be significantly improved when N2 NBs were present in solution, but even more so when the bubbles were composed of CO2.

A previous comparative study between helium and nitrogen as nebulizing and desolvation gases in electrospray ionization (ESI) and desorption electrospray ionization (DESI) found that the signal responses of compounds of varying sizes and polarities were improved. Here, an expanded selection of nebulizing gases was evaluated to investigate mechanisms of improvement. The set of nebulizing gases included hydrogen, helium, nitrogen, argon, and carbon dioxide. Results indicate that the signal enhancements are achieved by gases lighter than nitrogen and that the previously described helium effects can be improved by using the more economical and sustainable hydrogen as a nebulizing gas. Additionally, H2 and He reduce the desorption footprint, which could be potentially useful in increasing the resolution of chemical imaging microscopy, especially since, despite the smaller footprint obtained using helium and hydrogen, higher signals are obtained compared to nitrogen.

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.

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 in Escherichia coli were 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 clarified E. coli cell 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.

We introduce a new method, helium assisted desorption ionization, that uses helium as nebulizing gas in desorption electrospray ionization (DESI). We also investigate Helium Assisted Spray Ionization, when helium is used with a micro-electrospray ionization source similar to electrosonic spray ionization (ESSI). In both methods, analyte ion signals are improved, while the undesirable effects of species created by the breakdown products of atmospheric gases and helium are suppressed. Restriction provided by the extended ion inlet capillary reduces the influx of helium, preventing a deleterious increase in operating pressure in the intermediate ion transfer region. We demonstrate significant improvements in signal intensities for analytes of different sizes and polarities, including xanthine alkaloids, lipids, and proteins. Compared to regular DESI, ion intensities for both protein and small molecules increased with helium addition, while less-polar small molecules were improved to a greater extent. The improvements in signal response with helium nebulization generally followed the same trend using either helium assisted desorption or spray ionization, although the extent of improvement was more substantial in the desorption experiments. This observation suggests that desorption, ionization, and/or ion transport processes could be positively influenced when helium is used as nebulizing gas. Nebulization with helium also produces a smaller desorption footprint compared to nitrogen, promising benefits for chemical microscopy and other applications of DESI-MS.

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.

Exposure of electrospray droplets to organic vapors was shown to dramatically reduce alkali-metal adduction on protein ions and shift protein charge states. Since DESI-MS is affected by similar adduct species as ESI-MS and shares similar ionization mechanisms, polar organic vapor additives should likewise also improve the DESI-MS analysis of proteins. Here the DESI spray was exposed to a variety of polar organic vapor additives. Head space vapors of polar organic solvents were entrained in nitrogen gas and delivered to the atmosphere inside a semi-enclosed plastic enclosure surrounding the spray plume. The vapors of acetone, acetonitrile, ethyl acetate, methanol, and water were investigated. Vapor dependent effects were observed with respect to changes in protein charge state distributions and signal intensities. With ethyl acetate vapor addition, the signal intensities of all proteins investigated were significantly increased, including proteins larger than 25 kDa such as carbonic anhydrase II and bovine serum albumin.

Previous studies have suggested that the loss in sensitivity of DESI-MS for large molecules such as proteins is due to the poor dissolution during the short time scale of desorption and ionization. An investigation into the effect of serine as a solvent additive leads to the interesting observation that there is a concentration-dependent improvement in protein signal intensity when micromolar to low millimolar concentrations of serine is combined with a suitable co-additive in DESI spray. This effect, however, was not observed during similar ESI-MS experiments, where the same solvents and proteins were sprayed directly into the MS inlet. This suggests that the mechanism of signal improvement in DESI is associated with the desorption step of proteins, possibly by facilitating dissolution or improving solubility of proteins on the surface in the solvent micro-layer formed during DESI. Other than poor dissolution, cation adduction such as by sodium ions is also a major contributing factor to the mass-dependent loss in sensitivity in both ESI and DESI, leading to an increase in limits of detection for larger proteins. The adduction becomes a more pressing issue in native-state studies of proteins, as lower charge states are more susceptible to adduction. Previous studies have shown that addition of amino acids to the working spray solution during ESI-MS reduces sodium adduction and can help in stabilization of native-state proteins. Similar to the observed reduction in sodium adducts during native-state ESI-MS, when serine is added to the desorbing spray in DESI-MS, the removal of up to 10 mM NaCl is shown. A selection of proteins with high and low pI and molecular weights was analyzed to investigate the effects of serine on signal intensity by improvements in protein solubility and adduct removal. Graphical Abstract

It is frequently said that DESI-MS follows a similar ionization mechanism as ESI because of similarities usually observed in their respective mass spectra. However, practical use of DESI-MS for protein analysis is limited to proteins with lower molecular weights (< 25 kDa) due to a mass-dependent loss in signal intensity. Here we investigated commonly used volatile acids and their ammonium salt buffers for DESI-MS analysis of protein. We noticed that, surprisingly, some additives influence the analysis differently in DESI compared to ESI. Improved signal intensities with both DESI and ESI were obtained when acetic and formic acid were added into aqueous methanol spray solvents with both DESI and ESI. On the other hand, while with ESI the addition of ammonium salts into spray solutions strongly reduced both signal and S/N , with DESI signal intensities and S/N were improved dramatically. Ammonium bicarbonate when used with DESI reduced the total amount of adduction and delivered excellent signal-to-noise ratios with high intensity; however, it also denatures protein. When native state protein mass spectra are preferred, ammonium acetate would also deliver reasonable adduct removal and improved S / N . The amount of total adduction of individual adducting species and of all species could not be correlated with differences in either solutions pH values or with proton affinities of the anions. An obvious difference between DESI and ESI mass spectrometry is the effects of protein solubility during droplet pickup (desorption), but differences in the sizes, velocities, and composition of ionizing droplets were also discussed as important factors. Graphical Abstract ᅟ

The analysis of protein by desorption electrospray ionization mass spectrometry (DESI-MS) is considered impractical due to a mass-dependent loss in sensitivity with increase in protein molecular weights. With the addition of ammonium bicarbonate to the DESI-MS analysis the sensitivity towards proteins by DESI was improved. The signal to noise ratio (S/N) improvement for a variety of proteins increased between 2- to 3-fold relative to solvent systems containing formic acid and more than seven times relative to aqueous methanol spray solvents. Three methods for ammonium bicarbonate addition during DESI-MS were investigated. The additive delivered improvements in S/N whether it was mixed with the analyte prior to sample deposition, applied over pre-prepared samples, or simply added to the desorption spray solvent. The improvement correlated well with protein pI but not with protein size. Other ammonium or bicarbonate salts did not produce similar improvements in S/N, nor was this improvement in S/N observed for ESI of the same samples. As was previously described for ESI, DESI also caused extensive protein unfolding upon the addition of ammonium bicarbonate. Graphical Abstract ᅟ.

A review of ambient ionization mass spectrometry highlighting the central role of sample preparation immediate to and during sample analysis.

Ionic liquids (ILs) are highly polar solvents with unique physicochemical properties that make them promising green alternatives to volatile organic solvents. Since ILs can be toxic to organisms, the development of methods to degrade ILs into harmless molecules prior to disposal is critical to enhancing their green properties. In this study, metabolites generated during the biodegradation of 1-butyl-3-methylimidazolium chloride (BMIMCl) by an enriched, activated sludge microbial community were investigated. Biodegradation of BMIM and the metabolic products released into the growth media were examined using 1H-NMR spectroscopy and mass spectrometry. To the best of our knowledge, this is the first reported complete primary catabolism of the biodegradation-resistant BMIMCl ionic liquid. The bacterial community responsible for degradation was analyzed using a 16S-rRNA amplicon approach. Although the community was diverse, Bacteroidetes was the predominant phylum. The study provides a greater insight into imidazolium-based IL biodegradability and a means to proactively prevent the ecotoxicity of the BMIM cation and its metabolites, by complete primary biodegradation of the cation and removal of most resulting metabolites, prior to release into aquatic waste streams.

Two novel sensors bearing rhodamine B and quinoline units have been synthesized. One of these, 1, allows sensitive and selective detection of Ni2+ and Cr3+ by forming non-fluorescent (1-Ni2+) and fluorescent (1-Cr3+) complexes respectively. Both metals trigger the formation of highly colored ring-open spirolactam. These form excellent probes for CN− which quenches the fluorescence of the 1-Cr3+ complex by extracting the Cr3+. Both Cr3+ and Cu2+ gave color changes with 2, but they are easily identified separately via the large fluorescence enhancement that occurs only with Cr3+.

The real-time in-line microlocalized-desorption sample processing that takes place during ambient ionization are of general analytical use, in addition to their utility during in direct analysis mass spectrometry. By decoupling the microlocalized-desorption sample-processing steps from direct analysis many benefits are realized, such as separate optimization capabilities for desorption and ionization. By using these novel sample-processing steps benefits are also realized over traditional sample-preparation procedures, such as solvent extraction, or swabbing for surface collection. The chapter illustrates how the decoupled desorption procedure can be used to obtain detailed information about the overall mechanism of the ambient ionization methods, illustrated by application to desorption electrospray ionization (DESI), as an example. It is shown that the desorption and ionization aspects of DESI respond differently to changes in operational conditions. This information will help practitioners of ambient ionization to select appropriate conditions for their analyses. In addition, applications of the spray desorption collection (SDC) technique are shown for analyses other than direct mass spectrometry.

The addition of certain reagents during the electrospray ionization mass spectrometry of proteins can shift the protein ion signal charge-state distributions (CSDs) to higher average charge states, a phenomenon known as 'supercharging'. The role of reagent gas-phase basicity (GB) during this process was investigated in both the negative and positive ion modes. Reagents with known or calculated GBs were added individually in equimolar amounts to protein solutions which were subsequently electrosprayed for mass spectrometry analysis. Shifts in the CSDs of the protein ion signals were monitored and related to the reagents' GBs. Trends for this data were evaluated for possible insights into a supercharging mechanism involving the direct interaction between supercharging reagent and protein ion. Reagent GB was confirmed to be directly related to the amount of supercharging observed in the negative ion mode. Supercharging in the positive ion mode, on the other hand, showed a maximal trend. Interestingly, a loss of signal and supercharging efficacy was observed for reagents with GBs intermediate within the investigated range, between ~800 and ~840 kJ mol(-1), at the 100 mM concentration used in the present study. The possibility of a direct interaction model for supercharging in the negative and positive ion modes dependent on the GBs of the protein ions and reagents is discussed. In the positive ion mode, supercharging appears to depend on the stability of a proton bridge formed between the reagent and a highly charged protein ion.

Facial traumas often occur as a result of accidents or injuries at workplace, aggression or accidental falling. Their assessment is made by means of Multislice Computed Tomography (CT) due to its high resolution, providing precise information on fracture routes, bone displacement, soft tissue injuries as well as the traumatic complications in the maxillofacial area.Material and Methods: We examined 214 patients aged between 7-79 with maxillofacial trauma and who underwent CT examination in CT Department of the County Hospital Oradea. Results. Out of the 214 patients surveyed, 92 (43%) showed single lesions, the remaining 122(57%) patients had multiple lesions on viscerocranium. A number of 47 (22%) patients had maxillofacial trauma within a multiple trauma. In single lesions (92 cases i.e. 43%), the distribution was as follows: mandibular lesions in 42 patients , nasal bones lesions in 22 patients, frontal/ maxillary sinuses lesions in 16 patients, and orbital lesions in 12 patients, respectively. We grouped the 42 patients with mandibular fractures by several criteria such as: histopathologic type, number of outbreaks, topography.Conclusions. Computer tomography is the reference technique in the detection and characterization of facial trauma. Fractures of mandibula were present in 46% of those patients undergoing a single trauma. It is necessary to perform multiplanar reconstructions especially coronary sections and 3D reconstructions in order to perform a correct classification.

ADVERTISEMENT RETURN TO ISSUEPREVReviewNEXTMechanisms of Real-Time, Proximal Sample Processing during Ambient Ionization Mass SpectrometryAndre R. Venter*†, Kevin A. Douglass†, Jacob T. Shelley‡, Gregg Hasman, Jr.†, and Elahe Honarvar†View Author Information† Department of Chemistry, Western Michigan University, Kalamazoo, Michigan 49008-5413, United States‡ Department of Chemistry and Biochemistry, Kent State University, Kent, Ohio 44242, United States*E-mail: [email protected]Cite this: Anal. Chem. 2014, 86, 1, 233–249Publication Date (Web):December 5, 2013Publication History Received27 November 2013Published online16 December 2013Published inissue 7 January 2014https://doi.org/10.1021/ac4038569Copyright © 2013 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views3464Altmetric-Citations123LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit Read OnlinePDF (2 MB) Get e-AlertsSUBJECTS:Desorption,Ionization,Ions,Liquids,Solvents Get e-Alerts