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Results demonstrate that the negative charge at amino acid 149 of human lecithin:cholesterol acyltransferase determines the specificity of the enzyme for phosphatidylcholine containing 20-carbon sn-2 fatty acyl chain as shown by mutational and substrate specificity experiments. Data indicate that reverse mutation changes the substrate specificity and 18-carbon sn-2 fatty acyl chains are not optimum for the enzyme activity.

Molecular ions are generated in induced electrospray ionization, and they can be transported to grounded ambient surfaces in the form of charged microdroplets. Efficient amide bonds formation between amines and carboxylic acids were observed inside charged droplets during transfer to the surface. Biomolecules derivatized using this method were self-assembled on a bare gold surface via Au-S bonds under the charged microdroplet environment. Cyclic voltammetric analysis of the self-assembled molecular film showed accelerated protein derivatization with cysteine, which allowed the covalent immobilization of the protein to the gold surface. Cytochrome C-functionalized electrodes prepared using the induced dual nanoelectrospray process showed bioactivity toward aqueous solutions of hydrogen peroxide below 50 μM. In effect, we have developed a method that allows derivatization of biomolecules and their immobilization at ambient surfaces in a single experimental step. [ABSTRACT FROM AUTHOR]

Ambient ion soft landing, a process in which polyatomic ions are deposited from air onto a surface at a specified location under atmospheric pressure, is described. Ions generated by electrospray ionization are passed pneumatically through a heated metal drying tube, their ion polarity is selected using ion deflectors, and the dry selected ions are soft-landed onto a selected surface. Unlike the corresponding vacuum soft-landing experiment, where ions are mass-selected and soft-landed within a mass spectrometer, here the ions to be deposited are selected through the choice of a compound that gives predominantly one ionic species upon ambient ionization; no mass analysis is performed during the soft landing experiment. The desired dry ions, after electrical separation from neutrals and counterions, are deposited on a surface. Characterization of the landed material was achieved by dissolution and analysis using mass spectrometry or spectrofluorimetry. The treated surface was also characterized using fluorescence microscopy, which allowed surfaces patterned with fluorescent compounds to be imaged. The pure dry ions were used as reagents in heterogeneous ion/surface reactions including the reaction of pyrylium cations with d-lysine to form the N-substituted pyridinium cation. The charged microdroplets associated with incompletely dried ions could be selected for soft landing or surface reaction by choice of the temperature of a drying tube inserted between the ion source and the electrical ion deflectors. [ABSTRACT FROM AUTHOR]

The concept of programmable cascade reactions in charged microdroplets is introduced using carbon-carbon (C-C) bond formation via uncatalyzed Michael addition in a three-tier study culminating in programmable Hantzsch multicomponent, multistep reactions. In situ generated reactive oxygen species (ROS) from nonthermal plasma discharge are fused with charged water microdroplets (devoid of ROS) in real time for accelerated chemical reactions. This plasma-microdroplet fusion platform utilizing a coaxial spray configuration enabled product selection while avoiding unwanted side reactions. Hydrogen abstraction via ROS facilitated the formation of enolate anions without strong base use. Reaction enhancement factors >10 3 were calculated for plasma-microdroplet fusion versus microdroplet-only reactions. The platform programmability was showcased through (i) uncatalyzed 1,4-Michael addition of α,β-unsaturated carbonyls, (ii) novel C-C bond formation via the use of pro-electrophilic amine and alcohol substrates─activated through collisions in the microdroplet environment to serve as Michael acceptors, and (iii) selective Hantzsch cascade reaction with cross-coupling products, avoiding side reactions including N-alkylation and self-coupling product formation. Milligram quantity product collection is achieved, showcasing plasma-microdroplet fusion as an effective tool for preparative-scale synthesis. Thus, the controlled generation of ROS via plasma discharge during charged water microdroplet evolution establishes a green synthetic method for uncatalyzed C-C bond formation.

Methyleugenol (ME), found in numerous plants and spices, is a rodent carcinogen and is classified as "possibly carcinogenic to humans". The hypothesis of a carcinogenic risk for humans is supported by the observation of ME-derived DNA adducts in almost all human liver and lung samples examined. Therefore, a risk assessment of ME is needed. Unfortunately, biomarkers of exposure for epidemiological studies are not yet available. We hereby present the first detection of N -acetyl-l-cysteine conjugates (mercapturic acids) of ME in human urine samples after consumption of a popular ME-containing meal, pasta with basil pesto. We synthesized mercapturic acid conjugates of ME, identified the major product as N -acetyl- S -[3'-(3,4-dimethoxyphenyl)allyl]-l-cysteine ( E -3'-MEMA), and developed methods for its extraction and LC-MS/MS quantification in human urine. For conducting an exposure study in humans, a basil cultivar with a suitable ME content was grown for the preparation of basil pesto. A defined meal containing 100 g of basil pesto, corresponding to 1.7 mg ME, was served to 12 participants, who collected the complete urine at defined time intervals for 48 h. Using d 6 - E -3'-MEMA as an internal standard for LC-MS/MS quantification, we were able to detect E -3'-MEMA in urine samples of all participants collected after the ME-containing meal. Excretion was maximal between 2 and 6 h after the meal and was completed within about 12 h (concentrations below the limit of detection). Excreted amounts were only between 1 and 85 ppm of the ME intake, indicating that the ultimate genotoxicant, 1'-sulfooxy-ME, is formed to a subordinate extent or is not efficiently detoxified by glutathione conjugation and subsequent conversion to mercapturic acids. Both explanations may apply cumulatively, with the ubiquitous detection of ME DNA adducts in human lung and liver specimens arguing against an extremely low formation of 1'-sulfooxy-ME. Taken together, we hereby present the first noninvasive human biomarker reflecting an internal exposure toward reactive ME species.

Neutral triacylglyceride (TG) lipids are critical in cellular function, signaling, and energy storage. Multiple molecular pathways control TG structure via nonselective routes making them structurally complex and analytically challenging to characterize. The presence of C=C bond positional isomers exacerbates this challenge as complete structural elucidation is not possible by conventional tandem mass spectrometric methods such as collision-induced dissociation (CID), alone. Herein, we report a custom-made coaxial contained-electrospray ionization (ESI) emitter that allows the fusion of plasma discharge with charged microdroplets during electrospray (ES). Etched capillaries were incorporated into this contained-ES emitter, facilitating the generation of reactive oxygen species (ROS) at low (3 kV) ESI voltages and allowing stable ESI ion signal to be achieved at an unprecedented high (7 kV) spray voltage. The analytical utility of inducing plasma discharge during electrospray was investigated using online ionization of neutral TGs, in situ epoxidation of unsaturation sites, and C=C bond localization via conventional CID mass spectrometry. Collisional activation of the lipid epoxide generated during the online plasma-droplet fusion experiment resulted in a novel fragmentation pattern that showed a quadruplet of diagnostic ions for confident assignment of C=C bond positions and subsequent isomer differentiation. This phenomenon enabled the identification of a novel TG lipid, composed of conjugated linoleic acid, that is isomeric with two other TG lipids naturally found in extra virgin olive oil. To validate our findings, we analyzed various standards of TG lipids, including triolein, trilinolein, and trilinolenin, and isomeric mixtures in the positive-ion mode, each of which produced the expected quadruplet diagnostic fragment ions. Further validation was obtained by analyzing standards of free fatty acids expected from the hydrolysis of the TG lipids in the negative-ion mode, together with isomeric mixtures. The chemistry governing the gas-phase fragmentation of the lipid epoxides was carefully elucidated for each TG lipid analyzed. This comprehensive shotgun lipidomic approach has the potential to impact biomedical research since it can be accomplished on readily available mass spectrometers without the need for instrument modification., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

In this work, we have developed a paper-based microfluidic device capable of remote biofluid collection followed by an analysis of the dried clinical samples using a miniature mass spectrometer. We have evaluated a portable mass spectrometer as a possible surveillance platform by analyzing the clinical malaria samples (whole blood) collected from Ghana. We synthesized pH-sensitive ionic probes and coupled them with monoclonal antibodies specific to the Plasmodium falciparum histidine-rich protein 2 ( Pf HRP2) malaria antigen. We then used the antibody-ionic probe conjugates in a paper-based immunoassay to capture Pf HRP2 antigen from untreated whole blood. After the immunoassay, the bound ionic probes were cleaved, and the released mass tags were analyzed through an on-chip paper spray mass spectrometry strategy. During process optimization, we determined the detection limit for Pf HRP2 in untreated human serum to be 0.216 nmol/L when using the miniature mass spectrometer. This sensitivity is comparable to the World Health Organization's suggested threshold of 0.227 nmol/L for Pf HRP2, proving that our method will be applicable to diagnose symptomatic malaria infection (≥200 parasites per μL blood). The paper device can be stored at room temperature for at least 25 days without affecting the clinical outcome, with each stored paper chip offering good repeatability and reproducibility (RSD = 4-12%). The stability and sensitivity of the developed paper-based immunoassay platform will allow miniature mass spectrometers to be used for point-of-care malaria detection as well as in large-scale surveillance screening to aid eradication programs.

Enzyme-catalyzed hydrolysis of lipids was monitored directly in immiscible microdroplet environments using contained-electrospray mass spectrometry. Aqueous solution of the lipase enzyme from Pseudomonas cepacia and the chloroform solution of the lipids were sprayed from separate capillaries, and the resultant droplets were merged within a reaction cavity that is included at the outlet of the contained-electrospray ionization source. By varying the length of the reaction cavity, the interaction time between the enzyme and its substrate was altered, enabling the quantification of reaction product as a function of time. Consequently, enhancement factors were estimated by comparing rate constants derived from the droplet experiment to rate constants calculated from solution-phase conditions. These experiments showed enhancement factors greater than 100 in favor of the droplet experiment. By using various lipid types, two possible mechanisms were identified to account for lipase reactivity in aerosols: in-droplet reactions for relatively highly soluble lipids and a droplet coalescence mechanism that allows interfacial reactions for the two immiscible systems.

Fennel and other herbs contain the secondary plant metabolites estragole and trans -anethole, of which estragole is carcinogenic in rodents. It is metabolically activated by cytochrome P450-catalyzed conversion to 1'-hydroxyestragole and subsequent sulfo conjugation to the genotoxic 1'-sulfoxyestragole. The current study followed the hypothesis that the reactive sulfate ester may be detoxified by glutathione conjugation, leading to the urinary excretion of a resultant mercapturic acid. We identified the assumed downstream metabolite N -acetyl- S -[3'-(4-methoxyphenyl)allyl]-l-Cys (AMPAC) in human urine samples after consumption of fennel tea. An isotope-dilution technique for its quantification by ultraperformance liquid chromatography-tandem mass spectrometry and [ 13 C 3 , 15 N]AMPAC in urine samples was developed. The method was applied to determine the AMPAC concentration in urine samples following uptake of 500 mL of fennel tea containing 2.2 mg of estragole by 12 healthy participants (six females and six males). Before drinking the tea, the urinary AMPAC concentration was below the limit of detection. In most of the participants, the highest amounts of urinary AMPAC were found in the first-hour urine after exposure. The excretion by first-order kinetics (range of t 1/2 = 0.78-1.54 h; mean ± SD: 1.13 ± 0.21 h) led to a nearly complete clearance within 8 h in all participants. The total AMPAC excreted was in the range of 93-1076 ng, reflecting pronounced interindividual variations of enzymes taking part in estragole metabolism. Importantly, AMPAC was also formed in one volunteer following oral uptake of a single dose of isolated trans -anethole, albeit to a much smaller extent compared to estragole. AMPAC may be of future use as a human biomarker for the internal exposure to the carbocation formed from either 1'-sulfoxyestragole or 3'-sulfoxyisoestragole, the reactive sulfate ester metabolites of estragole and trans -anethole, respectively.

A droplet imbibition experiment is proposed to study interfacial effects, which appears to be the main factor influencing reaction acceleration in charged microdroplets produced by electrospray ionization (ESI). One reagent is deposited onto the surface of rapidly moving microdroplets containing the second reagent to be reacted. In this manner, reactions are hindered from reaching equilibrium and monitored in real time by mass spectrometry. We demonstrated this phenomenon using Katritzky chemistry, which is known to proceed either by the solvent-stabilized 2 H -pyran intermediate or via the surface-active pseudobase intermediate. Comparisons with reactions performed using ESI show obvious surface effects in favor of the droplet imbibition experiment. By keeping reactant mole ratio constant, it was demonstrated that similar interfacial effects observed in the droplet imbibition experiment can be reached by allowing ESI microdroplets containing premixed reagents to traverse a distance >16 mm. At such spray distance, molecular diffusion and droplet lifetime become comparable allowing reactants to be enriched at droplet surface. Reactions were also conducted in rapid mixing, theta capillary-based droplets, which showed markedly reduced yields compared with the interfacial droplet imbibition experiment.

We have developed a new spray-based method for characterizing surface energies of planar, porous substrates. Distinct spray modes (electrospray versus electrostatic spray), from the porous substrates, occur in the presence of an applied DC potential after wetting with solvents of different surface tension. The ion current resulting from the spray process is maximized when the surface energy of the porous substrate approaches the surface tension of the wetting solvent. By monitoring the selected ion current (e.g., benzoylecgonine, m / z 290 → 168) with a mass spectrometer or the total ion current with an ammeter, we determined the solvent surface tension yielding the maximum ion current to indicate the surface energy of the solid. Detailed evaluations using polymeric substrates of known surface energies enabled effective calibration of the approach that resulted in the correct estimation of the surface energy of hydrophobic paper substrates prepared by gas-phase silanization. A three-parameter empirical model suggests that the experimentally observed ion current profile is governed by differential partitioning of analyte controlled by the interfacial forces between the wetting solvent and the porous substrate.

Metal macrocycles are among the most important catalytic systems in electrocatalysis and biocatalysis owing to their rich redox chemistry. Precise understanding of the redox behavior of metal macrocycles in operando is essential for fundamental studies and practical applications of this catalytic system. Here we present electrochemical data for the representative iron phthalocyanine (FePc) in both aqueous and nonaqueous media coupled with in situ Raman and X-ray absorption analyses to challenge the traditional notion of the redox transition of FePc at the low potential end in aqueous media by showing that it arises from the redox transition of the ring. Our data unequivocally demonstrate that the electron is shuttled to the Pc ring via the Fe(II)/Fe(I) redox center. The Fe(II)/Fe(I) redox transition of FePc in aqueous media is indiscernible by normal spectroscopic methods owing to the lack of a suitable axial ligand to stabilize the Fe(I) state.

Current analytical methods, either point-of-care or centralized detection, are not able to meet recent demands of patient-friendly testing and increased reliability of results. Here, we describe a two-point separation on-demand diagnostic strategy based on a paper-based mass spectrometry immunoassay platform that adopts stable and cleavable ionic probes as mass reporter; these probes make possible sensitive, interruptible, storable, and restorable on-demand detection. In addition, a new touch paper spray method was developed for on-chip, sensitive, and cost-effective analyte detection. This concept is successfully demonstrated via (i) the detection of Plasmodium falciparum histidine-rich protein 2 antigen and (ii) multiplexed and simultaneous detection of cancer antigen 125 and carcinoembryonic antigen.

Molecular ions are generated in induced electrospray ionization, and they can be transported to grounded ambient surfaces in the form of charged microdroplets. Efficient amide bonds formation between amines and carboxylic acids were observed inside charged droplets during transfer to the surface. Biomolecules derivatized using this method were self-assembled on a bare gold surface via Au-S bonds under the charged microdroplet environment. Cyclic voltammetric analysis of the self-assembled molecular film showed accelerated protein derivatization with cysteine, which allowed the covalent immobilization of the protein to the gold surface. Cytochrome C-functionalized electrodes prepared using the induced dual nanoelectrospray process showed bioactivity toward aqueous solutions of hydrogen peroxide below 50 μM. In effect, we have developed a method that allows derivatization of biomolecules and their immobilization at ambient surfaces in a single experimental step.

Energy densities of Li ion batteries, limited by the capacities of cathode materials, must increase by a factor of 2 or more to give all-electric automobiles a 300 mile driving range on a single charge. Battery chemical couples with very low equivalent weights have to be sought to produce such batteries. Advanced Li ion batteries may not be able to meet this challenge in the near term. The state-of-the-art of Li ion batteries is discussed, and the challenges of developing ultrahigh energy density rechargeable batteries are identified. Examples of ultrahigh energy density battery chemical couples include Li/O2, Li/S, Li/metal halide, and Li/metal oxide systems. Future efforts are also expected to involve all-solid-state batteries with performance similar to their liquid electrolyte counterparts, biodegradable batteries to address environmental challenges, and low-cost long cycle-life batteries for large-scale energy storage. Ultimately, energy densities of electrochemical energy storage systems are limited by chemistry constraints.

Coumarin is a flavoring which can cause hepatotoxicity in experimental animals and in a proportion of the human population. The tolerable daily intake (TDI) may be exceeded in consumers with high intake of cinnamon containing high levels of coumarin. The objective of this study was to determine these levels in cinnamon samples and to identify possible factors influencing them. A HPLC method to quantify coumarin and related constituents (cinnamaldehyde, cinnamic acid, cinnamyl alcohol, eugenol) in a single run was used. Results found in 47 cinnamon powder samples obtained from the German retail market confirmed high levels of coumarin in cassia cinnamon. A huge variation was observed in stick samples from two packages (range from below the limit of detection to about 10000 mg/kg). Cassia bark samples of five trees received directly from Indonesia were analyzed additionally. Interestingly, a high variation was observed in one of the trees, whereas no coumarin was detected in the samples of two other trees. In conclusion, coumarin levels in cassia cinnamon can vary widely even within a single tree.

We previously described a point mutation in human LCAT (E to A at residue 149; hE149A) that demonstrated greater activity with phosphatidylcholine (PC) substrate containing 20:4 in the sn-2 position compared with the wild-type enzyme [hLCAT; Wang et al. (1997) J. Biol. Chem. 272, 280-286], resulting in a human enzyme with the substrate specificity similar to that of rat LCAT. The purpose of the present study was to explore the molecular basis for the role of amino acid 149 in determining fatty acyl substrate specificity. In the first experiment, the reverse mutation in rat LCAT (rA149E) converted substrate specificity of rat LCAT toward that of the human enzyme, demonstrating that the mutation was context independent and reversible. In the second experiment, we found that hE149A compared with hLCAT demonstrated higher activity with PC species containing 20-carbon, but not 18-carbon, sn-2 fatty acyl chains. The increased activity of hE149A was due to an increase in apparent V(max) but not to apparent K(m) or LCAT binding to the PC surface. Substitution of different amino acids in the 149 position of hLCAT showed that activation of the enzyme with sn-2 20:4 containing PC substrate was only observed when the negative charge at residue 149 was removed. We conclude that the negative charge at amino acid 149 of LCAT is a critical determinant for the specificity of the enzyme for PC containing 18- vs 20-carbon sn-2 fatty acyl chains.

We have found that 8-azidoadenosine 5'-triphosphate (8-azido-ATP) and its photolyzed product are competitive inhibitors of terminal deoxynucleotidyltransferase with respect to substrate deoxynucleoside triphosphates. A detailed characterization of the inhibitory effect of 8-azido-ATP revealed that its mechanism of inhibition is identical with that reported for ATP [Modak, M. J. (1978) Biochemistry 17, 3116-3120]. Photoactivation of the azido-ATP-enzyme complex results in the covalent binding of azido-ATP to terminal deoxynucleotidyltransferase. No significant incorporation of prephotolyzed azido-ATP or unsubstituted ATP into enzyme protein is noted when complexes of these nucleotides with enzyme were exposed to identical photoactivation conditions. The majority of incorporated analogue was associated with the 26 000-dalton subunit of terminal deoxynucleotidyltransferase. Incorporation of azido-ATP was further found to be absolutely dependent on the presence of a divalent cation. All four deoxyribonucleoside triphosphates as well as ATP and guanosine 5'-triphosphate were able to compete with azido-ATP during the incorporation experiment as judged by the competitive reduction in the cross-linking of the photoaffinity analogue to terminal deoxynucleotidyltransferase (TDT). In addition, substrate binding site directed inhibitors, pyrophosphate and pyridoxal 5'-phosphate, effectively blocked the incorporation of azido-ATP into enzyme protein, while several other inhibitors of TDT catalysis, namely, ethylenediaminetetraacetic acid, alpha, alpha'-dipyridyl, 1,10-phenanthroline, p-(chloromercuri)benzoate, Rose Bengal, and the presence of 0.5 M KCl, influenced the cross-linking reaction to varying degrees. A tryptic peptide analysis of the azido-ATP-labeled 26K subunit of TDT revealed that the majority of the incorporated photoaffinity analogue was present in two peptides.

To identify the DNA binding site(s) in Escherichia coli DNA polymerase I (pol I) (Klenow fragment), we have used an active-site-directed reagent, phenylglyoxal (PG), which specifically reacts with arginine residues. Preincubation of DNA pol I with PG resulted in the loss of polymerase, 3'-5'-exonuclease, and DNA binding functions. Furthermore, the presence of DNA but not deoxynucleoside triphosphates protected the enzyme from inactivation. Labeling studies with [7-14C]PG indicated that two arginine residues were modified per mole of enzyme. In order to locate the site of PG modification, we digested the PG-treated enzyme with trypsin and V-8 protease. The resulting peptides from each digest were then resolved on reverse-phase hydrophobic columns. An appearance of a new peptide peak was observed in both tryptic and V-8 protease digests. Since inclusion of template-primer during PG modification of enzyme blocks the appearance of these peaks, these peptides were concluded to represent the template-primer binding domain of pol I. Indeed, the extent of inactivation of enzyme by PG treatment correlated very well with the quantitative increase in the new tryptic peptide peak. Amino acid composition analysis of both tryptic peptide and V-8 peptide revealed that the two peptides were derived from the same general region; tryptic peptide spanned between residues 837 and 857 while V-8 peptide spanned between residues 841 and 870 in the primary sequence of pol I. Sequence analysis of tryptic peptide further identified arginine-841 as the site of PG modification, which implicates this residue in the DNA binding function of pol I.