One-carbon metabolism influences gene expression by providing methyl units for DNA, RNA, and histone methylation. Robust methylation requires rapid hydrolysis of the methylation by-product S- adenosylhomocysteine (SAH) by S- adenosylhomocysteinase (Ahcy). Here, we show Ahcy is a redox-sensitive enzyme that is inhibited by oxidation of a conserved cysteine, C195, in vitro and in vivo . Transient oxidation of Ahcy is neuroprotective in a Drosophila light stress model where it results in rapid gene expression changes and protects against retinal degeneration. Thus, redox sensing by the one-carbon metabolic enzyme Ahcy enables rapid changes in gene expression in response to changes in redox homeostasis.

Multiphase reduction of ambient dinitrogen (N2) was observed during the deposition of charged or uncharged aqueous microdroplets containing a lithium salt pre-catalyst onto conducting or inert surfaces to yield mixed lithium nanoparticles. We propose that this method leverages the high electric fields and partial solvation of ions at the gas-water-solid interface to enhance reaction rates and perform normally moisture-sensitive chemistry in aqueous droplets. Ammonia (NH3) is formed and Li+ is regenerated from the hydrolysis of transient lithium nitride Li3N, constituting a complete catalytic cycle. The ammonia is captured in situ using formaldehyde to yield hexamethylenetetramine, a solid nitrogenous fuel. By measuring product formation with mass spectrometry, it was determined that 2.97 ± 0.36 μg h-1 of NH3 was produced by a single spray source when using pure N2 sheath gas and an applied potential, though microdroplets alone appear a sufficient source of electrons in cases where an external potential is not applied; air is also an adequate source of N2 to produce NH3. The mixed lithium metal nanoparticles were characterized with (S)TEM, EDS, and EELS. This ammonia synthesis has implications for the formation of nitrogenous compounds in environmental, prebiotic, and traditional synthetic contexts.

Phosphorylation plays important roles in biology by modulating the structure, reactivity, and biological function of a broad range of molecules. Biocatalytic phosphorylation has attracted attention from synthetic chemists due to its selectivity and mild reaction conditions using ATP as a phosphate donor. Given the potential synthetic utility of kinases with activity on small molecule substrates, we explored the activity of PsiK, the enzyme responsible for selective 4‐O‐phosphorylation of 4‐hydroxytryptamine or psilocin in psylocybin biosynthesis by Psilocybe cubensis. We find that PsiK has good activity on a range of substituted phenols and benzenediols beyond its native substrate, enabling preparative phosphorylation of different substrates, and substantially expands the substrate scope of biocatalytic phosphorylation. We also show that active site mutations can further expand substrate scope and improve site‐selectivity. This engineering effort was greatly expedited using DESI‐MS screening, which enabled analysis of 2,688 reactions in only 40 min. Finally, gram‐scale phosphorylation of a representative substrate was achieved with a turnover number over 10,000. Together, these results highlight the biocatalytic utility of PsiK and derivatives thereof for selective phosphorylation of phenols and benzenediols under mild conditions.

Upon malignancy and metastasis to distant organs such as brain, lung and bone marrow, the 5-year survival rate of castration-resistance prostate cancer (CRPC) is only around 30%. Therefore, there is an urgent need for identifying new CRPC drug targets and developing new CRPC treatments. A potential new drug target for CRPC is human sulfotransferase isozyme SULT2B1b. It catalyzes the transfer of the sulfate group from 3’-phosphoadenosine 5’-phosphosulfate (PAPS) to cholesterol to produce cholesterol sulfate (CS). Increased expression of SULT2B1b has been shown to correlate to prostate cancer cell proliferation with decreased level of TNF-mediated apoptosis. Knockdown of SULT2B1b leads to elevation of TNF expression and NF-κB activation. SULT2B1b knockdown also enhances TNF-mediated apoptosis in both TNF-sensitive (LNCaP)and TNF-resistant (C4-2) cells. We demonstrate herein that catalytic knockout mutants, H125A and K70A, of SULT2B1b abolish CS production in cells mimicking genetic knockdown studies. This result suggests that small molecule inhibitors of SULT2B1b activity could serve as potential CRPC therapeutics. Towards this goal, we established a novel high-throughput (50nL, <1s per sample) label-free DESI-MS platform for the screening of small-molecule compounds with excellence Z’ scores ranging from0.73 to 0.86. Using DESI-MS, we first determined Kcat and KM values for catalysis of cholesterol by SULT2B1b to be 0.05 ± 0.002 min-1 and 19.8 ± 2.2 µM respectively. We then screened 4500compounds from three chemical libraries (ChemBridge Kinase Inhibitor, LOPAC 1280 &AnalytiCon MEGx Natural Products) and identified 3 bona fide hit compounds (#099, #170 and#954) that had IC50 values ranging from 1.2 to 1.6 µM. We also showed via DESI-MS that these three compounds, each containing multiple hydroxyl groups, could not be sulfated. NanoDSF was used to show that the hit compounds stabilize the SULT2B1b melting temperature by 1.7°C to3.0°C. Binding interactions of the hit molecules were also examined using microscale thermophoresis (MST) and dissociation constants (KD) of 13.5 µM (#099), 6.9 µM (#170) and 7.2µM (#954) were determined. X-ray structures of SULT2B1b in complex with #099 and #170 were determined to 2.8 Å and 2.9 Å resolution respectively. Both compounds are found bound within the cholesterol substrate binding site. One important structural observation is the N-terminus helix (residues 1 - 28) is displaced due to the binding of the inhibitors. In summary, we used DESI-MS to identify three compounds that potently inhibit SULT2B1b through interactions at the cholesterol binding site. These results should now allow for the structure-based design and development of more potent lead compounds of SULT2B1b which ultimately may be useful forCRPC treatment. Beinan Yang, Nicolás M. Morato, Samadhi Kulathunga, Timothy L. Ratliff, Graham Cooks, Andrew D. Mesecar. Desorption electrospray ionization mass spectrometry (DESI-MS) high-throughput screening of small-molecule inhibitors for human sulfotransferase 2B1b & biophysical and crystallographic characterization of target-hit interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3149.

PLCG2 is signal-transduction protein identified as a potential drug target for the treatment of Alzheimer’s disease (AD). PLCG2 is regulated by stimulation of the TREM2 pathway in microglia, which results in phagocytosis of beta-amyloid. PLCG2 catalyzes the cleavage of PI(4,5)P2 into IP3 and diacylglycerol, resulting in increased cell motility, phagocytosis, and proliferation in microglia. Studies found that a naturally occurring PLCG2 variant, P522R, stimulates PLCG2 activity and is associated with decreased AD risk, making PLCG2 an appealing target as an AD therapeutic. Current drug discovery efforts for this enzyme are limited by no publicly available structures of full-length PLCG2 and limited kinetic characterization studies. In this study, kinetic and structural studies of wild-type and the P522R variant of PLCG2 are explored and presented. PLCG2 was expressed and purified from baculovirus infected Sf9 cells for in-vitro studies. Kinetic characterization of PLCG2 was accomplished using a continuous fluorescence-based assay and a mass spectrometry-based end-point assay called Desorption Ionization Mass Spectrometry (DESI-MS). Structural studies of PLCG2 wild-type and P522R variant were pursued using single-particle cryo-EM and X-ray crystallography. Kinetic parameters were quantified with both the fluorescence and label-free DESI assays and found to be similar between assays. X-ray data on wild-type PLCG2 crystals were collected to 2.8Å. The current X-ray structural model reveals an intact active site with bound calcium atom and first-sphere waters, but a missing nSH2 domain (no observable electron density). Cryo-EM maps of wild-type and P522R are consistent with the X-ray structural model including poor density for the nSH2 domain. Comparison of human PLCG2 structural models to AlphaFold and rat PLCG1 models reveals differences in domain xSH2 domain positions. Human PLCG2 is highly active in solution as determined by kinetic studies using two different biochemical assays. The X-ray and cryo-EM structures of PLCG2 reveal significant conformational flexibility of the nSH2 domain which may be an important function in it binding to other proteins in the plasma membrane. This research was supported by grant 1U54 AG065181 (IUSM-Purdue TREAT-AD Center) from the National Institute of Aging, National Institutes of Health.

The recent alleged use of A-series chemical warfare agents (CWAs) highlights the urgent need to better understand their inhibition of cholinesterase enzymes and the reported shortcomings of traditional oxime countermeasures. Here, using high-throughput (HT) mass spectrometry (MS) technologies, we characterized the largely unknown inhibition kinetics of A-series CWAs on human acetylcholinesterase (hAChE) and its reactivation by oximes, achieving label-free quantitation at rates of up to 7,000 reactions per hour. Our findings indicate i) A-series agents exhibit inhibitory potencies similar to traditional CWAs like sarin and VX, and ii) bipyridinium-based oximes can reactivate A-series-adducted hAChE in vitro, challenging prior reports on oxime efficacy. These results underscore the need for continued exploration of countermeasure candidates against A-series CWAs and demonstrate the potential of HT-MS for rapidly and safely characterizing emerging toxic chemicals.

Automation of chemical synthesis and high-throughput (HT) screening are important for speeding up drug discovery. Here, we describe an automated HT picomole scale synthesis system which uses desorption electrospray ionization (DESI) to create microdroplets of reaction mixtures at individual positions from a two-dimensional reactant array and transfer them to a corresponding position in an array of collected reaction products. On-the-fly chemical transformations are facilitated by the reaction acceleration phenomenon in microdroplets and high reaction conversions are achieved during the milliseconds droplet flight time from the reactant to the product array. Successful functionalization of bioactive molecules is demonstrated through the generation of 172 analogs (64% success rate) using multiple reaction types. Synthesis throughput is ~45 seconds/reaction including droplet formation, reaction, and collection steps, all of which occur in an integrated fashion, generating product amounts sufficient for subsequent bioactivity screening (low ng to low µg). Quantitative performance was validated using LC/MS. This system bridges the demonstrated capabilities of HT-DESI for reaction screening and label-free bioassays, allowing consolidation of the key early drug discovery steps around a single synthetic-analytical technology.

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.

The bile salt export pump (BSEP) assay is widely used to evaluate the potential for drug-induced liver injury (DILI) early in the drug discovery process. While traditional liquid chromatography-mass spectrometry (LC-MS)-based approaches have been utilized for BSEP activity testing, they have intrinsic limitations in either throughput or the requirement for sample preparation and are difficult to scale up in order to screen drug candidates. Here we demonstrate the use of two different high-throughput MS methods based on solid-phase extraction (SPE) and desorption electrospray ionization (DESI) for high-throughput BSEP activity assessment in a label-free manner, with minimal needs for sample workup, at sampling rates of ∼11 and ∼5.5 s/sample, respectively. Both approaches were validated, compared, and successfully applied to the evaluation of 96 drug candidates for the inhibition of taurocholic acid (TCA) transport using BSEP vesicles.

Chemical reactions in micrometer-sized droplets can be accelerated by up to six orders of magnitude. However, this acceleration factor (ratio of rate constants relative to bulk) drops to less than 10 for millimeter-sized droplets due to the reduction in surface/volume ratio. To enhance the acceleration in millimeter-sized droplets, we use a new synthesis platform that directly doses reagent vapor onto the reaction droplet surface from a second levitated droplet. Using Katritzky transamination as a model reaction, we made quantitative measurements on size-controlled vapor-dosed droplets, revealing a 31-fold increase in reaction rate constants when examining the entire droplet contents. This enhancement is attributed to a greater reaction rate constant in the droplet surface region (estimated as 105 times greater than that for the bulk). The capability for substantial reaction acceleration in large droplets highlights the potential for rapid synthesis of important chemicals at useful scales. For example, we successfully prepared 23 pyridinium salts within minutes. This efficiency positions droplets as an exceptional platform for rapid, in situ catalyst synthesis. This is illustrated by the preparation of pyridinium salts as photocatalysts and their subsequent use in mediation of amine oxidation both within the same droplet.

The biologically important thiols (cysteine, homocysteine, N-acetyl cysteine, and glutathione) are key species in redox homeostasis, and there is a clinical need to measure them rapidly, accurately, and simultaneously at low levels in complex biofluids. The solution to the challenge presented here is based on a new derivatizing reagent that combines a thiol-selective unit to optimize the chemical transformation and a precharged pyridinium unit chosen to maximize sensitivity in mass spectrometry. Derivatization is performed simultaneously with ionization ("reactive ionization"), and mass spectrometry is used to record and characterize the thiol reaction products. The method is applicable over the concentration range from 1 μM to 10 mM and is demonstrated for 25 blood serum, 1 plasma, and 3 types of tissue samples. The experiment is characterized by limited sample preparation (<4 min) and short analysis time (<1 min). High precision and accuracy (both better than 8%) are validated using independent HPLC-MS analysis. Cystine-cysteine redox homeostasis can be monitored by introducing an additional reduction step, and the accuracy and precision of these results are also validated by HPLC-MS.

This brief cautionary note reports a failure in a common and useful assumption, namely, that the isotopes of the elements occur in their natural abundance ratios in commercially sourced organic compounds. Some commercial sources of tris(pentafluorophenyl)borane, B(C6F5)3, show severely depleted 10B, while materials from other suppliers display natural isotopic abundances. The depletion varies from lot-to-lot, and it was confirmed by inductively coupled plasma (ICP) mass spectrometry. The isotope 10B is used in the nuclear power industry, as a neutron absorber in the power control rods. It is speculated that the residual 11B generated when preparing 10B-enriched boron carbide for control rod use, provides 11B-rich raw material that is then used for commercial B(C6F5)3 synthesis.

Complex mixtures are typically analyzed by chromatographic separation followed by mass spectrometry. Two-dimensional tandem mass spectrometry (2D MS/MS) may eliminate the need for separation and therefore significantly reduce analysis time while expanding the information space by recording the intensities of all fragment ions generated from all trapped precursor ions. Additional chemical specificity can be provided by using chemical derivatization. In this study phenolic compounds are identified after triazole click derivatization by the observation of two diagnostic features in the 2D MS/MS data domain, a mass shift of 175 Da on the precursor ion axis and a neutral loss (NL) of 119 Da. Additional NL transitions were highly correlated with particular substituents including carbonyl, ester, amino, and alkyl/phenyl groups. Substituent position information was also easily diagnosed. The derivatization reactions are accelerated in the charged electrosprayed microdroplets. Using this method, members of this single class of compounds can be identified within 1-2 minutes while structural information on the individual compounds is acquired simultaneously. Accelerated microdroplet derivatization reactions coupled with 2D MS/MS provide a rapid and convenient approach for the analysis of complex mixtures. This method selectively targets the desired compound classes within the mixture, while simultaneously providing detailed structural information for each component through 2D MS/MS.image

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.

Charged microdroplets accelerate mineral disintegration

This study leverages accelerated reactions at the solution/air interface of microdroplets generated by desorption electrospray ionization (DESI) to explore the chemical space. DESI is utilized to synthesize drug analogs at an overall rate of 1 reaction mixture per second, working on the low-nanogram scale. Transformations of multiple drug molecules at specific functionalities (phenol, hydroxyl, amino, carbonyl, phenyl, thiophenyl, and alkenyl) are achieved using electrophilic/nucleophilic, redox, C-H functionalization, and coupling reactions. These transformations occur under ambient conditions on the millisecond time scale with direct detection of products being successful in all but three of the reaction types studied. The large scope (22 bioactive compounds, >20 chemical transformations, and >300 functionalization reagents) and high speed (>3000 reactions/hour) provide access to a wide array of drug analogs that can be used for bioactivity testing. A total of ∼6800 unique reactions were examined through a data-driven workflow, and more than 3000 unique derivatives (∼44%) were identified tentatively by the m/z value and signal-to-control ratio in single-stage mass spectrometry (MS) analysis, with over 1000 being further characterized by tandem MS. The speed of the DESI-MS reaction screen provides potential advantages for emerging machine learning-based predictions of organic synthesis, and it sets the stage for future online DESI-MS bioassays and scaled-up microdroplet synthesis before formal characterization of hit compounds is sought using traditional methods of drug discovery.

Desorption electrospray ionization (DESI) tandem mass spectrometry (MS) is used to assess mutation status of isocitrate dehydrogenase (IDH) in human gliomas. Due to the diffuse nature of gliomas, total gross resection is not normally achieved during surgery, leading to tumor recurrence. The mutation status of IDH has clinical significance due to better prognosis in IDH-mutant patients. The mutant IDH converts alpha-ketoglutaric acid (alpha-KG) into 2-hydroxyglutarate (2HG), which accumulates abnormally in cells. Immunohistochemical staining (IHC) and genetic testing, the gold standards, are incompatible with intraoperative applications but DESI tandem mass spectrometry (MS/MS) can be used to assess the mutation status of IDH enzyme from tissue intraoperatively. Here, on off-line evaluation is made of the performance of two different types of mass spectrometers in characterization of IDH mutation status. The intensity of 2HG is measured against glutamate (Glu), an intrinsic reference molecule, in both tandem MS measurements. In both cases using DESI clear separation between IDH-mutant (mut) and IDH-wildtype (wt) samples (p < 0.0001) is observed, despite the short analysis time. Due to the higher detection sensitivity, multiple reaction monitoring experiments using a triple quadrupole show slightly better performance compared to product ion MS/MS performed on a simple linear ion trap. Both DESI-MS platforms are capable of providing information on IDH mutation status, which might in future be used at the time of surgery to support decision-making on resection regions, especially at tumor margins.

The current opioid epidemic has incentivized the discovery of new non-addictive analgesics, a process that requires the screening of opioid receptor binding, traditionally performed using radiometric assays. Here we describe a label-free alternative based on high-throughput (1 Hz) ambient mass spectrometry for screening the receptor binding of new opioid analogues.

Biopolymer analysis, including proteomics and glycomics, relies heavily on the use of mass spectrometry for structural elucidation, including sequence determination. Novel methods to improve sample workup, instrument performance, and data analysis continue to be developed to address shortcomings associated with sample preparation, analysis time, data quality, and data interpretation. Here, we present a new method that couples in-source collision-induced dissociation (IS-CID) with two-dimensional tandem mass spectrometry (2D MS/MS) as a way to simplify proteomics and glycomics workflows while also providing additional insight into analyte structures over traditional MS/MS experiments. Specifically, IS-CID is employed as a gas-phase digestion method, i.e., to break down intact full-length polysaccharide or peptide ions prior to mass analysis. The resulting mixtures of oligomeric ions are analyzed by 2D-MS/MS, a technique that allows association of product ions with their precursor ions without isolation of the latter. A novel data analysis strategy is introduced to leverage the second dimension of 2D MS/MS spectra, in which stairstep patterns, representing outputs of a molecule's MSn scans, are extracted for structural interconnectivity information on the oligomer. The results demonstrate the potential applicability of 2D MS/MS strategies to the modern omics workflow and structural analysis of various classes of biopolymers.

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.