ChemRxiv
Not a member yet
27047 research outputs found
Sort by
Acoustofluidic Chromatography for Extracellular Vesicle Enrichment from µL Blood Plasma Samples
No full textWe present a novel acoustofluidic chromatography platform for high-throughput nanoparticle trapping and enrichment, focusing on extracellular vesicles (EVs) from blood plasma. The system consists of a packed bed of polystyrene beads within a rectangular glass capillary, acoustically excited by a piezoelectric element. Using fluorescent polystyrene particles (1.9 µm and 0.27 µm) as model nanoparticles, we characterized the device by evaluating its trapping efficiency across a frequency range of 0.45–4 MHz. Our results demonstrate efficient trapping of micro- and nanoscale particles, with increased efficiency at higher acoustic powers and lower flow rates. EV isolation from 4 µL of diluted blood plasma showed that abruptly increasing the flow rate during the release step significantly enhanced particle recovery, likely due to hydrodynamic effects. Nanoparticle tracking analysis confirmed the release of EVs at concentrations of ~2x10⁹ particles/mL, with low protein background suitable for downstream mass spectrometry.
This platform offers a promising approach for nanoparticle trapping and EV enrichment with minimal sample volumes, presenting potential applications in diagnostics and therapeutic development. Future work will focus on optimizing bead materials and sizes for EV subpopulation separation and scaling the system for clinical use
Metabolic Profiling of the EmDia Cohort by a Scalable DIA-LC-MS Workflow
No full textThe sodium glucose cotransporter-2 (SGLT2) inhibitor empagliflozin improves glycemic control in type 2 diabetes mellitus (T2DM) and has been suggested to additionally reduce CVD comorbidities. Clinical studies, such as the EmDia trial, investigate the short-term effects of empagliflozin on left ventricular diastolic function. Over the course of the trial, the metabolic effects of empagliflozin has been monitored by a limited set of clinical assays. To expand on this data, we here established a LC-MS workflow for comprehensive metabolic profiling of EmDIA. The workflow established enables profiling of >170 metabolites in plasma covering a broad range of compound classes such as carboxylic acids, amino acids, sugars, nucleotides, steroids and drugs at a rate of >100 samples per day. The method is based on optimized metabolite separation by pentafluorophenyl chromatography and high-confidence metabolite annotation based on a well curated in-house spectral library of more than 480 reference standards. Applied to EmDIA, our methodology is characterized by high predictive power of several clinical parameters, especially fasting blood glucose (R2 = 0.97) and estimated glomerular filtration rate (R2 = 0.63) as determined by elastic net-regularized linear and logistic regression. Our data further shows that administration of empagliflozin in addition to standard T2DM medication results in reduced plasma levels of urate, which has been previously linked to improved cardiovascular disease outcome, and reduced plasma levels of deoxyhexoses such as 1,5-anhydroglucitol, a short-term biomarker for glycemic control
Protein-protein interaction and conformational change in the alpha-helical membrane transporter BtuCD-F in the native cellular envelope
No full textAlpha-helical membrane proteins perform numerous critical functions essential for the survival of living organisms. Traditionally, these proteins are extracted from membranes using detergent solubilization and reconstitution into liposomes or nanodiscs. However, these processes often obscure the effects of nanoconfinement and the native environment on the structure and conformational heterogeneity of the target protein. We demonstrate that pulsed dipolar electron spin resonance spectroscopy, combined with the Gd3+-nitroxide spin pair, enables the selective observation of the vitamin B12 importer BtuCD-F in its native cellular envelope. Despite the high levels of non-specific labeling in the envelope, this orthogonal approach combined with the long phase-memory time for the Gd3+ spin enables the observation of the target protein complex at a few micromolar concentrations with high resolution. In the native envelope, vitamin B12 induces a distinct conformational shift at the BtuCD-BtuF interface, which is not observed in the micelles. This approach offers a general strategy for investigating protein-protein and protein-ligand/drug interactions and conformational changes of the alpha-helical membrane proteins in their native envelope context
Exposing the role of pre-association in interfacial proton-coupled electron transfer
No full textInterfacial proton-coupled electron transfer (I-PCET) reactions are typically viewed as single elementary reaction steps even though analogous solution-phase reactivity is known to require pre-association of proton donor and acceptor. Herein, we examine the role of pre-association in I-PCET to a molecularly well-defined graphite-conjugated carboxylic acid (GC-COOH) surface site. We quantify electrolyte proton activity and I PCET kinetics in acidic, acetate buffered, and alkaline electrolytes as a function of NaClO4 concentration, ranging from 1 mole kg−1 to 17 mole kg−1. Upon accounting for the previously measured proton activity dependence of I-PCET kinetics to GC-COOH, we find that rate of I-PCET is systematically attenuated by factors of 4.3 and 4.6 over this range of NaClO4 concentration in acidic and acetate buffered media, respectively. In contrast, the rate of I-PCET remains unchanged within error across NaClO4 concentration in alkaline electrolyte. Based on these observations, we propose a multiple-step model for I-PCET in acidic media that invokes quasi-equilibrated displacement of Na+ from the interface to form hydrogen-bonded pre-association complexes prior to rate-limiting concerted proton-electron transfer. Increased NaClO4 concentration is invoked to increase Na+ activity in the bulk vs the interface, inhibiting pre-association complex formation and the overall I-PCET rate. These studies emphasize the non-innocent role of support electrolyte species and expose the key role of pre-association equilibria in I-PCET mechanisms. The work also suggests that control over pre-association equilibria could be used as an additional handle for tailoring the kinetics of interfacial ion transfer reactions
Atomic View of Photosynthetic Metabolite Permeability Pathways and Confinement in Cyanobacterial Carboxysomes
No full textCarboxysomes are protein microcompartments found in cyanobacteria, whose shell encapsulates rubisco at the heart of carbon fixation in the Calvin-cycle. Carboxysomes are thought to locally concentrate CO2 in the shell interior to improve rubisco efficiency through selective metabolite permeability, creating a concentrated catalytic center. However, permeability coefficients have not previously been determined for these gases, or for Calvin-cycle intermediates such as bicarbonate (HCO3-), 3-phosphoglycerate (3-PGA), or ribulose-1,5-bisphosphate (RuBP). Starting from a high resolution cryo-EM structure of a synthetic beta-carboxysome shell, we perform unbiased all-atom molecular dynamics (MD) to track metabolite permeability across the shell. The synthetic carboxysome shell structure, lacking the BMC trimer proteins and encapsulation peptides, is found to have similar permeability coefficients for multiple metabolites, and is not selectively permeable to HCO3- relative to CO2. To resolve how these comparable permeabilities can be reconciled with the clear role of the carboxysome in the CO2-concentrating mechanism in cyanobacteria, complementary atomic-resolution Brownian Dynamics (ARBD) simulations estimate the mean first passage time for CO2 assimilation in a crowded model carboxysome. Despite a relatively high CO2 permeability of approximately 10^-2 cm/s across the carboxysome shell, the shell proteins reflect enough CO2 back towards rubisco that 2650 CO2 molecules can be fixed by rubisco for every 1 CO2 molecule that escapes under typical conditions. The permeabilities determined from all-atom molecular simulation are key inputs into flux modeling, and the insight gained into carbon fixation can facilitate the engineering of carboxysomes and other bacterial microcompartments for multiple applications
Direct Nitrogen Oxidation via Pd-nanoparticle Electrocatalysis
No full textPalladium nanoparticles with two different ligands were used as electrocatalysts at low temperature in a hermetic, one-pot electrochemical cell with either a LiOH or LiOD electrolyte. During initial, low-pressure experiments involving palladium (Pd) nanoparticle electrocatalysts with Epigallocatechin Gallate (EGCG) ligands an abrupt,
significant pressure drop occurred in the cell headspace air from 1.59 to 0.055 bar. This astounding, repeatable, and unexplained result initiated further experimentation.
Synthetic methods for Pd nanoparticle electrocatalysts with EGCG ligands and with polyvinylpyrrolidone (PVP) ligands are described. These Pd nanoparticles are capable
of simultaneously generating ammonium, nitrite, and nitrate ions. Ion chromatography was used for quantification of nitrogen-containing anions and cations in the alkaline
electrolyte. The Pd nanoparticles with PVP ligands and a 5 bar 3:1 H2/N2 gas mixture resulted in a nitrate yield rate of 1.16 µg h−1 mg−1 with current applied for 132 hr. The
Pd nanoparticles with PVP ligands also achieved the highest ammonium and nitrite ion yield rates at 10 bar under similar conditions while the Pd-EGCG nanoparticles
achieved lower yield rates Faradaic efficiencies (FE) values. This study demonstrates that direct generation of nitrite and nitrate salts avoids the need to further process
ammonia into ammonium nitrate
A DFT-driven Analysis of Aggregation-Dependent Stability in Alkylpyrazines: Monomers, Dimers, and Beyond
No full textPrecisely determining the structure and properties of analytes and their ions, such as proton-bound clusters, holds significant importance in both the theoretical understanding and practical applications of mass spectrometry, ion mobility spectrometry, and other related chemical ionization methods. Density functional theory (DFT) calculations were utilized to investigate the confor mational constraints governing the formation of stable proton-bound clusters of alkyl pyrazines, encompassing monomers, dimers, and trimers. Employ ing the B3LYP/6-31+G(d, p) method with D3 dispersion correction, molec ular properties, including electric dipole moment, polarizability, and proton affinity, were presented and compared with results from higher basis sets like Aug-cc-PVTZ, demonstrating the efficiency of the chosen approach. Natural bond orbital (NBO) calculations provided insights into natural charges, charge transfer, and stability of proton-bound dimer and trimer structures, revealing a decrease in stability from monomers to trimers. Notably, protonated trimers exhibited stacked structures instead of expected protonated forms, aligning with experimental observations. The stability of alkyl pyrazine clusters was found to be influenced by various factors, including structure, electric dipole moment, polarizability, charge transfer, and steric hindrance. Additionally, proton affinity calculations indicated a linear relationship between stability and proton affinity in monomers, with constant dissociation energies observed in proton-bound dimers regardless of proton affinity variations. The study provides comprehensive insights into the stability paradigm of alkyl pyrazines, facilitating a deeper understanding of their behavior across different structural configurations and molecular concentrations
Towards data-driven design of visible-light photoswitches using structural features
No full textIn this manuscript we present the strategy for modeling of the photoswitch properties (maximum absorption wavelength and thermal half-life of photoisomers) of visible-light azo-photoswitches using structural data. We compile a comprehensive data set from literature sources and perform a rigorous benchmark to select the best feature type and modeling approach. The fragment counts have demonstrated excellent performance in the benchmark for both properties. We validate the models in cross-validation and on an external set. The predictions for this set are highly accurate, despite the modest size of the data set related to thermal half-life of photoisomers, especially when consensus modeling approach is applied. We also provide the interpretation of the modeling results using ColorAtom approach and the insights into the chemical space covered by the data set
Nanoflow Size Exclusion Chromatography – Native Mass Spectrometry of Intact Proteoforms and Protein Complexes
No full textNative size-exclusion chromatography (SEC) coupled with native mass spectrometry (nMS) enables the characterization of proteins and protein complexes by combining liquid-phase separation (SEC) and mass measurement of the complexes (nMS). This approach allows for an increase in the throughput of native MS experiments, reduces the bias that may be present due to the co-ionization of oligomers, and facilitates online sample buffer exchange. SEC-nMS uses volatile buffers and relatively wide-diameter columns (e.g., ≥ 1 mm), with flow rates in the tens of µL/minute. To ionize sample components under this flow regime, relatively harsh electrospray ionization (ESI) desolvation conditions are needed, which may result in protein dissociation/denaturation. Also, relatively large amounts of samples are required (several µgs). Herein, we describe the development of a nanoflow SEC-nMS method using 200 µm I.D. columns, operated at 500 nL•min-1. This approach allows buffer exchange, oligomer separation, and mild ionization conditions (e.g., without the assistance of heated gas flow or temperature). Compared to microflow (1 mm I.D. column), the nanoflow method achieved a 4-fold increase in MS peak intensity, despite using a sample 20 times less concentrated (0.05 mg mL-1 for nanoflow vs. 1 mg mL-1 for microflow). Furthermore, we evaluated the impact in terms of sensitivity and separation efficiency of three injection approaches: large-volume injection (1μL), nano-volume injection (50nL), and an online mix-bed ion-exchange capillary trap column. The final method, using nano-volume injection, was applied to several model protein and protein complexes to showcase performance and applicability to the study of sample-limited analysis
PFAS destruction and near complete defluorination of undiluted aqueous film-forming foams at ambient conditions by piezoelectric ball milling
No full textThe non-thermal destruction of aqueous film-forming foam (AFFF) stockpiles, one of the major culprits responsible for water and soil contamination by per- and polyfluoroalkyl substances (PFAS), is extremely challenging because of the coexistence of mixed recalcitrant PFAS and complicated organic matrices at extremely high concentrations. To date, the complete defluorination of undiluted AFFF at ambient conditions has not been demonstrated. This study reports a novel piezoelectric ball milling (BM) approach for treating AFFF with a total organic fluorine concentration of 9,080 mg/L and total organic carbon of 234 g/L. Near-complete defluorination (> 95% conversion of organofluorine to fluoride) of undiluted AFFF was achieved by co-milling with boron nitride (BN). By carefully examining the experimental data, we identified AFFF liquid film thickness (Z) at the collision interface as a descriptor of treatment performance. We further validated that effective defluorination proceeded when Z was less than a criteria value of 2.3 μm. In light of this new understanding, the addition of SiO2 as a dispersant and the pre-evaporation solvents to reduce Z have been validated as effective strategies to promote AFFF treatment capacity