Oxazolone mediated peptide chain extension and homochirality in aqueous microdroplets.

Peptide formation from amino acids is thermodynamically unfavorable but a recent study provided evidence that the reaction occurs at the air/solution interfaces of aqueous microdroplets. Here, we show that i) the suggested amino acid complex in microdroplets undergoes dehydration to form oxazolone; ii) addition of water to oxazolone forms the dipeptide; and iii) reaction of oxazolone with other amino acids forms tripeptides. Furthermore, the chirality of the reacting amino acids is preserved in the oxazolone product, and strong chiral selectivity is observed when converting the oxazolone to tripeptide. This last fact ensures that optically impure amino acids will undergo chain extension to generate pure homochiral peptides. Peptide formation in bulk by wet-dry cycling shares a common pathway with the microdroplet reaction, both involving the oxazolone intermediate.

Rapid detection of IDH mutations in gliomas by intraoperative mass spectrometry.

The development and performance of two mass spectrometry (MS) workflows for the intraoperative diagnosis of isocitrate dehydrogenase (IDH) mutations in glioma is implemented by independent teams at Mayo Clinic, Jacksonville, and Huashan Hospital, Shanghai. The infiltrative nature of gliomas makes rapid diagnosis necessary to guide the extent of surgical resection of central nervous system (CNS) tumors. The combination of tissue biopsy and MS analysis used here satisfies this requirement. The key feature of both described methods is the use of tandem MS to measure the oncometabolite 2-hydroxyglutarate (2HG) relative to endogenous glutamate (Glu) to characterize the presence of mutant tumor. The experiments i) provide IDH mutation status for individual patients and ii) demonstrate a strong correlation of 2HG signals with tumor infiltration. The measured ratio of 2HG to Glu correlates with IDH-mutant (IDH-mut) glioma (P < 0.0001) in the tumor core data of both teams. Despite using different ionization methods and different mass spectrometers, comparable performance in determining IDH mutations from core tumor biopsies was achieved with sensitivities, specificities, and accuracies all at 100%. None of the 31 patients at Mayo Clinic or the 74 patients at Huashan Hospital were misclassified when analyzing tumor core biopsies. Robustness of the methodology was evaluated by postoperative re-examination of samples. Both teams noted the presence of high concentrations of 2HG at surgical margins, supporting future use of intraoperative MS to monitor for clean surgical margins. The power of MS diagnostics is shown in resolving contradictory clinical features, e.g., in distinguishing gliosis from IDH-mut glioma.

Aqueous microdroplets enable abiotic synthesis and chain extension of unique peptide isomers from free amino acids.

Amide bond formation, the essential condensation reaction underlying peptide synthesis, is hindered in aqueous systems by the thermodynamic constraints associated with dehydration. This represents a key difficulty for the widely held view that prebiotic chemical evolution leading to the formation of the first biomolecules occurred in an oceanic environment. Recent evidence for the acceleration of chemical reactions at droplet interfaces led us to explore aqueous amino acid droplet chemistry. We report the formation of dipeptide isomer ions from free glycine or L-alanine at the air-water interface of aqueous microdroplets emanating from a single spray source (with or without applied potential) during their flight toward the inlet of a mass spectrometer. The proposed isomeric dipeptide ion is an oxazolidinone that takes fully covalent and ion-neutral complex forms. This structure is consistent with observed fragmentation patterns and its conversion to authentic dipeptide ions upon gentle collisions and for its formation from authentic dipeptides at ultra-low concentrations. It also rationalizes the results of droplet fusion experiments that show that the dipeptide isomer facilitates additional amide bond formation events, yielding authentic tri- through hexapeptides. We propose that the interface of aqueous microdroplets serves as a drying surface that shifts the equilibrium between free amino acids in favor of dehydration via stabilization of the dipeptide isomers. These findings offer a possible solution to the water paradox of biopolymer synthesis in prebiotic chemistry.

Spontaneous Oxidation in Aqueous Microdroplets: Water Radical Cation as Primary Oxidizing Agent.

Exploration of the unique chemical properties of interfaces can unlock new understanding. A striking example is the finding of accelerated reactions, particularly spontaneous oxidation reactions, that occur without assistance of catalysts or external oxidants at the air interface of both aqueous and organic solutions (provided they contain some water). This finding opened a new area of interfacial chemistry but also caused heated debate regarding the primary chemical species responsible for the observed oxidation. An overview of the literature covering oxidation in microdroplets with air interfaces is provided, together with a critical examination of previous findings and hypotheses. The water radical cation/radical anion pair, formed spontaneously and responsible for the electric field at or near the droplet/air interface, is suggested to constitute the primary redox species. Mechanisms of accelerated microdroplet reactions are critically discussed and it is shown that hydroxyl radical/hydrogen peroxide formation in microdroplets does not require that these species be the primary oxidant. Instead, we suggest that hydroxyl radical and hydrogen peroxide are the products of water radical cation decay in water. The importance of microdroplet chemistry in the prebiotic environment is sketched briefly and the role of partial solvation in reaction acceleration is noted.

Machine-Learning Classification of Bacteria Using Two-Dimensional Tandem Mass Spectrometry.

Biothreat detection has continued to gain attention. Samples suspected to fall into any of the CDC's biothreat categories require identification by processes that require specialized expertise and facilities. Recent developments in analytical instrumentation and machine learning algorithms offer rapid and accurate classification of Gram-positive and Gram-negative bacterial species. This is achieved by analyzing the negative ions generated from bacterial cell extracts with a modified linear quadrupole ion-trap mass spectrometer fitted with two-dimensional tandem mass spectrometry capabilities (2D MS/MS). The 2D MS/MS data domain of a bacterial cell extract is recorded within five s using a five-scan average after sample preparation by a simple extraction. Bacteria were classified at the species level by their lipid profiles using the random forest, k-nearest neighbor, and multilayer perceptron machine learning models. 2D MS/MS data can also be treated as image data for use with image recognition algorithms such as convolutional neural networks. The classification accuracy of all models tested was greater than 99%. Adding to previously published work on the 2D MS/MS analysis of bacterial growth and the profiling of sporulating bacteria, this study demonstrates the utility and information-rich nature of 2D MS/MS in the identification of bacterial pathogens at the species level when coupled with machine learning.

Miniature mass spectrometer-based point-of-care assay for measuring phosphatidylethanol in blood.

We demonstrate proof-of-concept for point-of-care assessment of long-term alcohol consumption by measuring phosphatidylethanol in blood/dried blood spots with nano-electrospray ionization and MS/MS using a miniature mass spectrometer. 'Abstinence', 'moderate', and 'chronic' consumption could be distinguished rapidly for both sample types, and quantitative performance was obtained with blood (LoQ-100 ng mL-1).

Re-Imagining Drug Discovery using Mass Spectrometry.

It is argued that each of the three key steps in drug discovery, (i) reaction screening to find successful routes to desired drug candidates, (ii) scale up of the synthesis to produce amounts adequate for testing, and (iii) bioactivity assessment of the candidate compounds, can all be performed using mass spectrometry (MS) in a sequential fashion. The particular ionization method of choice, desorption electrospray ionization (DESI), is both an analytical technique and a procedure for small-scale synthesis. It is also highly compatible with automation, providing for high throughput in both synthesis and analysis. Moreover, because accelerated reactions take place in the secondary DESI microdroplets generated from individual reaction mixtures, this allows either online analysis by MS or collection of the synthetic products by droplet deposition. DESI also has the unique advantage, amongst spray-based MS ionization methods, that complex buffered biological solutions can be analyzed directly, without concern for capillary blockage. Here, all these capabilities are illustrated, the unique chemistry at droplet interfaces is presented, and the possible future implementation of DESI-MS based drug discovery is discussed.

High-Throughput Diversification of Complex Bioactive Molecules by Accelerated Synthesis in Microdroplets.

Late-stage diversification of drug molecules is an important strategy in drug discovery that can be facilitated by reaction screening using high-throughput experimentation. Here we present a rapid method for functionalizing bioactive molecules based on accelerated reactions in microdroplets. Reaction mixtures are nebulized at throughputs better than 1 reaction/second and the accelerated reactions occurring in the microdroplets are followed by desorption electrospray ionization mass spectrometry (DESI-MS). Because the accelerated reactions occur on the millisecond timescale, they allow an overall screening throughput of 1 Hz working at the low nanogram scale. Using this approach, an opioid agonist (PZM21) and an antagonist (naloxone) were diversified using three reactions important in medicinal chemistry: sulfur fluoride exchange (SuFEx) click reactions, imine formation reactions, and ene-type click reactions. Some 269 functionalized analogs of naloxone and PZM21 were generated and characterized by tandem mass spectrometry (MS/MS) after screening over 500 reactions.

Quantitative Determination of Water in Organic Liquids by Ambient Mass Spectrometry.

This study uses a rapid tandem mass-spectrometry method to determine water content in complex organic solutions. Emphasis is placed on trace-water analysis by a fast and accurate alternative to the Karl-Fischer method. In this new method, water is captured by a charge-labeled molecular probe. Water binds strongly with high specificity to the strongly electrophilic aldehyde site in a charge-labelled molecule (N-methylpyridinium); competitive binding by other analytes is effectively discriminated against in the mass-measurement step. Quantitative determinations are made over a wide concentration range, 0.001 % (10 ppm) to 99 %, with better than 10 % relative standard deviation, along with short (1 min) analysis times using small sample volumes (several µL). Applications include water measurement in simple organic solvents, for example, deuterated solvents, as well as in complex mixtures, for example, organic reaction mixtures. Additionally, this method allows for water monitoring in levitated droplets. Mechanistic investigations into the impact of water on important chemical processes in organic synthesis and environmental science are reported.

Metabolomic and Lipidomic Profiling of Bacillus Using Two-Dimensional Tandem Mass Spectrometry.

Lipidomic and metabolomic profiles of sporulated and vegetative Bacillus subtilis and Bacillus thuringiensis from irradiated lysates were recorded using a quadrupole ion trap mass spectrometer modified to perform two-dimensional tandem mass spectrometry (2D MS/MS). The 2D MS/MS data domains, acquired using a 1.2 s scan of negative ions generated by nanoelectrospray ionization of microwave irradiated spores, showed the presence of dipicolinic acid (DPA) as well as various lipids. Aside from microwave radiation to extract DPA and lipids from spores, sample preparation was minimal. Characteristic lipid and metabolic profiles were observed using 107─108 cells of the two Bacillus species. Major features of the lipid profiles observed for the vegetative states included sets of phosphatidylglycerol (PG) lipids. Product ion spectra were extracted from the 2D MS/MS data, and they provided structural information on the fatty acid components of the PG lipids. The study demonstrates the flexibility, speed, and informative power of metabolomic and lipidomic fingerprinting for identifying the presence of spore-forming biological agents using 2D MS/MS as a rapid profiling screening method.

Bacterial growth monitored by two-dimensional tandem mass spectrometry.

The growth of the bacterium E. coli was monitored by targeting the phospholipid constituents through desorption electrospray ionization and characterizing individual sets of isomers by recording the full 2D MS/MS data domain in a single scan of a modified quadrupole ion trap mass spectrometer. The experiments tested the applicability of the new instrumental capabilities which include sample interrogation at the molecular level for multiple components at speeds of <10 seconds/sample. The major lipids observed were phosphatidylethanolamines and phosphatidylglycerols and the growth experiment showed fatty acid chain modification from alkene to cyclopropyl groups over time. Notably, these novel MS scans were also performed using desorption electrospray ionization (DESI) to quickly sample complex mixtures without pre-separation. This demonstration experiment has implications for the value of ambient ionization mass spectrometry for monitoring biological systems on physiologically relevant timescales.

Miniature mass spectrometer-based point-of-care assay for cabotegravir and rilpivirine in whole blood.

HIV prevention and treatment with injectable cabotegravir and/or rilpivirine administered once every 4 to 8 weeks is an attractive alternative to daily therapy. Prescribed dosage and drug concentrations in plasma are based on patient data collected in clinical trials, but actual patients are expected to exhibit more variability in drug concentrations, which is important to quantify. Here, we demonstrate the first quantitative point-of-care assay with a miniature mass spectrometer to assess these drug concentrations in whole blood. Quantitative performance is obtained using paper spray ionization in combination with tandem mass spectrometry (MS/MS) in the clinically relevant concentration range of both drugs. Limits of quantitation (LoQs) of cabotegravir and rilpivirine are measured to be 750 ng/mL and 20 ng/mL, respectively. The assay turnaround time is < 4 min, and strong linear relationships are established between MS/MS responses and concentration, with percentage of relative standard deviations (RSDs) that are <15% at concentrations above the LoQs. The speed, portability, low power consumption, and specificity offered by the miniature instrument should make it an appropriate platform for measuring drug concentrations in a walk-in clinic using small volumes of patient blood.

Simultaneous and Spontaneous Oxidation and Reduction in Microdroplets by the Water Radical Cation/Anion Pair.

Microdroplets show unique chemistry, especially in their intrinsic redox properties, and to this we here add a case of simultaneous and spontaneous oxidation and reduction. We report the concurrent conversions of several phosphonates to phosphonic acids by reduction (R-P → H-P) and to pentavalent phosphoric acids by oxidation. The experimental results suggest that the active reagent is the water radical cation/anion pair. The water radical cation is observed directly as the ionized water dimer while the water radical anion is only seen indirectly though the spontaneous reduction of carbon dioxide to formate. The coexistence of oxidative and reductive species in turn supports the proposal of a double-layer structure at the microdroplet surface, where the water radical cation and radical anion are separated and accumulated.

Accelerated and Concerted Aza-Michael Addition and SuFEx Reaction in Microdroplets in Unitary and High-Throughput Formats.

The sulfur fluoride exchange (SuFEx) reaction is significant in drug discovery, materials science, and chemical biology. Conventionally, it involves installation of SO2 F followed by fluoride exchange by a catalyst. We report catalyst-free Aza-Michael addition to install SO2 F and then SuFEx reaction with amines, both occurring in concert, in microdroplets under ambient conditions. The microdroplet reaction is accelerated by a factor of ∼104 relative to the corresponding bulk reaction. We suggest that the superacidic microdroplet surface assists SuFEx reaction by protonating fluorine to create a good leaving group. The reaction scope was established by performing individual reactions in microdroplets of 18 amines in four solvents and confirmed using high-throughput desorption electrospray ionization experiments. The study demonstrates the value of microdroplet-assisted accelerated reactions in combination with high-throughput experimentation for characterization of reaction scope.

Collection and Characterization by Mass Spectrometry of the Neutral Serine Octamer Generated upon Sublimation.

Serine forms neutral octameric clusters during sublimation, as demonstrated by electrostatically deflecting thermally ionized serine species from the sublimate, then gently ionizing the remaining neutrals for examination by mass spectrometry (MS). The MS results demonstrate a strong homochiral preference in the neutral octamer (measured after its gentle ionization), while the smaller serine clusters are achiral. In the initial stages of its sublimation, nonracemic solid serine generates a neutral serine monomer as the principal species in the vapor phase, with a significant enantiomeric enrichment relative to the solid. The serine monomer, when the flux is sufficient, assembles into the octamer, which displays a much higher chiral purity than the monomer. The serine octamer is separated from other neutral clusters in the sublimate by a new method based on the different distances that the clusters travel in an inert gas stream before they condense in a cooled collector. The deposited octamer is subsequently dissolved, and the solution is investigated by MS. The spectrum confirms that the collected serine octamer has undergone chiral enrichment relative to the starting solid used in the sublimation. The chiral enrichment observed in going from the serine monomer to octamer can be accommodated using a chemical model, grounded on the homochiral preference of the neutral serine octamer. Using the enantiomeric excess (ee %) of the vapor-phase monomer as the input, the model output matches the experimental octamer ee % when subliming solid serine with various initial ee % values.

Novel Ion Trap Scan Modes to Develop Criteria for On-Site Detection of Sulfonamide Antibiotics.

Advances in ambient ionization techniques have facilitated the direct analysis of complex mixtures without sample preparation. Significant attention has been given to innovating ionization methods so that multiple options are now available, allowing for ready selection of the best methods for particular analyte classes. These ambient techniques are commonly implemented on benchtop systems, but their potential application with miniature mass spectrometers for in situ measurements is even more powerful. These applications require that attention be paid to tailoring the mass spectrometric methodology for the on-site operation. In this study, combinations of scan modes are employed to efficiently determine what tandem mass spectrometry (MS/MS) operations are most useful for detecting sulfonamides using a miniature ion trap after ionization. First, mixtures of representative sulfonamide antibiotics were interrogated using a 2D MS/MS scan on a benchtop ion trap in order to determine which class-specific fragments (ionic or neutral) are shared between the sulfonamides and thus have diagnostic value. Then, three less-used combination scans were recorded: (i) a simultaneous precursor ion scan was used to detect both analytes and an internal standard in a single ion injection event to optimize quantitative performance; (ii) a simultaneous precursor/neutral loss scan was used to improve detection limits; and finally, (iii) the simultaneous precursor/neutral loss scan was implemented in a miniature mass spectrometer and representative sulfonamides were detected at concentrations as low as 100 ng/mL by nano-electrospray and 0.5 ng absolute by paper spray ionization, although improvements in the stability of the home-built instrumentation are needed to further optimize performance.

Accelerated Reaction Kinetics in Microdroplets: Overview and Recent Developments.

Various organic reactions, including important synthetic reactions involving C-C, C-N, and C-O bond formation as well as reactions of biomolecules, are accelerated when the reagents are present in sprayed or levitated microdroplets or in thin films. The reaction rates increase by orders of magnitude with decreasing droplet size or film thickness. The effect is associated with reactions at the solution-air interface. A key factor is partial solvation of the reagents at the interface, which reduces the critical energy for reaction. This phenomenon is of intrinsic interest and potentially of practical value as a simple, rapid method of performing small-scale synthesis.

Immediate and sensitive detection of sporulated Bacillus subtilis by microwave release and tandem mass spectrometry of dipicolinic acid.

Spore lysis of Bacillus species is achieved by brief (1 min) microwave irradiation while tandem mass spectrometry (MS/MS) allows identification of the characteristic spore marker, dipicolinic acid. This rapid measurement, made on 105-108 spores, has significant implications for biothreat recognition.

Metabolic profiles of human brain parenchyma and glioma for rapid tissue diagnosis by targeted desorption electrospray ionization mass spectrometry.

Desorption electrospray ionization mass spectrometry (DESI-MS) is well suited for intraoperative tissue analysis since it requires little sample preparation and offers rapid and sensitive molecular diagnostics. Currently, intraoperative assessment of the tumor cell percentage of glioma biopsies can be made by measuring a single metabolite, N-acetylaspartate (NAA). The inclusion of additional biomarkers will likely improve the accuracy when distinguishing brain parenchyma from glioma by DESI-MS. To explore this possibility, mass spectra were recorded for extracts from 32 unmodified human brain samples with known pathology. Statistical analysis of data obtained from full-scan and multiple reaction monitoring (MRM) profiles identified discriminatory metabolites, namely gamma-aminobutyric acid (GABA), creatine, glutamic acid, carnitine, and hexane-1,2,3,4,5,6-hexol (abbreviated as hexol), as well as the established biomarker NAA. Brain parenchyma was readily differentiated from glioma based on these metabolites as measured both in full-scan mass spectra and by the intensities of their characteristic MRM transitions. New DESI-MS methods (5 min acquisition using full scans and MS/MS), developed to measure ion abundance ratios among these metabolites, were tested using smears of 29 brain samples. Ion abundance ratios based on signals for GABA, creatine, carnitine, and hexol all had sensitivities > 90%, specificities > 80%, and accuracies > 85%. Prospectively, the implementation of diagnostic ion abundance ratios should strengthen the discriminatory power of individual biomarkers and enhance method robustness against signal fluctuations, resulting in an improved DESI-MS method of glioma diagnosis.

Reaction Acceleration at Solid/Solution Interfaces: Katritzky Reaction Catalyzed by Glass Particles.

The Katritzky reaction in bulk solution at room temperature is accelerated significantly by the surface of a glass container compared to a plastic container. Remarkably, the reaction rate is increased by more than two orders of magnitude upon the addition of glass particles with the rate increasing linearly with increasing amounts of glass. A similar phenomenon is observed when glass particles are added to levitated droplets, where large acceleration factors are seen. Evidence shows that glass acts as a "green" heterogeneous catalyst: it participates as a base in the deprotonation step and is recovered unchanged from the reaction mixture. Reaction acceleration at two separate interfaces is recognized in this study: i) air/solution phase acceleration, as is well known in microdroplets; ii) solid/solution phase, where such acceleration appears to be a new phenomenon.