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Chemical and isotopic indicators were used to recognize the origin of hydrothermal groundwater, to assess the mineralization processes and groundwater quality, to identify the source of solutes and the likely mixing with cold, and elucidate the fluid geothermometry in the Jérid field of Southern Tunisia. The results show that the geothermal groundwater is neutral to slightly alkaline. They are characterized by SO4-Cl-Na-Ca water type. The dissolution of evaporates and pyrite-bearing rocks is the dominant mineralization process. The groundwater quality index indicates that the majority of samples are very hard and belong to poor to unsuitable for drinking classes. Applications and calculations of hydrogeochemical parameters, including SAR, %Na, PI, Kr, and MAR, showed that the majority of samples are unsuitable for agricultural practices. The human health risk was assessed based on hazard quotient and total hazard index through ingestion and dermal contact with iron-rich groundwater. The consumption of CI groundwaters does not present non-carcinogenic risk to adults and children. The δ18 O and δ2H signatures indicate that the geothermal groundwater was recharged by ocean precipitation during cold and wet paleoclimatic periods. The slight enrichment of oxygen-18 and deuterium contents suggests a limited mixing effect between geothermal water and cold groundwater within the same aquifer. This mixing effect is confirmed by the Na-K-Mg and the chloride-enthalpy diagrams. The K-Mg and SiO2 geothermometers provided fairly reliable reservoir temperature values, ranging between 69.6 and 99 °C. Calculated geothermal potential values, varying between 469 and 16987 kWth, which allow several applications such as domestic and agricultural heating.

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The progesterone receptor (PR) is a steroid-responsive nuclear receptor, expressed as two isoforms: PR-A and PR-B. The isoforms display distinct expression patterns and biological actions in reproductive target tissues and disruption of PR-A:PR-B signaling is associated with breast cancer development potentially by altering interactions with oncogenic co-regulatory protein (CoRs). However, the molecular details of isoform-specific PR-CoR interactions that influence progesterone signaling remain poorly understood. We employed structural mass spectrometry in this study to investigate the sequential binding mechanism of purified full-length PR and full-length CoRs, steroid receptor coactivator 3 (SRC3) and p300, as complexes with target DNA. Our findings reveal selective CoR NR-box binding by PR and novel interaction surfaces between PR, SRC3, and p300, which change during complex assembly. This provides a structural model for a sequential priming mechanism that activates PR. Comparisons of PR bound to progesterone agonist versus antagonist challenges the classical model of nuclear receptor activation and repression. Collectively, we offer a peptide-level perspective on the organization of the PR transcriptional complex and elucidate the mechanisms behind the interactions of these proteins, both in active and inactive conformations.

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Contrast matching by isotopic exchange in cellulose allows visualizing functional groups, biomolecules, polymers and nanoparticles embedded in cellulosic composites. This isotopic exchange varies the scattering length density of cellulose to match its contrast with the background network. Here, contrast matching of microcrystalline-cellulose (MCC) and the functionalized nanocellulose-fiber (CNF) and cellulose nanocrystals (CNC) are elucidated by small angle neutron scattering (SANS). Results show no isotopic exchange occurs for the CNF surface functionalized with carboxyl nor for the CNC-High with a high sulfate groups concentration. Both CNC-Low, with low sulfate groups, and MCC exchange 1H with 1D in D2O. This is due to the high exchange probability of the labile C6 position primary -OH group. The structure of thermo-responsive poly-N-isopropylacrylamide (PNIPAM) chains grafted onto CNF (PNIPAM-grafted-CNF) was extracted by CNF contrast matching near the lower critical solution temperature. Contrast matching eradicates the CNF scattering to retain only the scattering from the grafted-PNIPAM chains. The coil to globule thermo-transition of PNIPAM was revealed by the power law variation from q-1.3 to q-4 in SANS. Isotopic exchange in functionalized cellulosic materials reveals the nano- and micro-scale structure of its individual components. This improved visualization by contrast matching can be extended to carbohydrate polymers to engineer biopharmaceutical and food applications.

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Arginyltransferase 1 (ATE1) catalyzes arginylation, an important post-translational modification (PTM) in eukaryotes that plays a critical role in cellular homeostasis. The disruption of ATE1 function is implicated in mammalian neurodegenerative disorders and cardiovascular maldevelopment, while post-translational arginylation has also been linked to the activities of several important human viruses such as SARS-CoV-2 and HIV. Despite the known significance of ATE1 in mammalian cellular function, past biophysical studies of this enzyme have mainly focused on yeast ATE1, leaving the mechanism of arginylation in mammalian cells unclear. In this study, we sought to structurally and biophysically characterize mouse (Mus musculus) ATE1. Using size-exclusion chromatography (SEC), small angle X-ray scattering (SAXS), and hydrogen deuterium exchange mass spectrometry (HDX-MS), assisted by AlphaFold modeling, we found that mouse ATE1 is structurally more complex than yeast ATE1. Importantly, our data indicate the existence of an intrinsically disordered region (IDR) in all mouse ATE1 splice variants. However, comparative HDX-MS analyses show that yeast ATE1 does not have such an IDR, consistent with prior X-ray, cryo-EM, and SAXS analyses. Furthermore, bioinformatics approaches reveal that mammalian ATE1 sequences, as well as in a large majority of other eukaryotes, contain an IDR-like sequence positioned in proximity to the ATE1 GNAT active-site fold. Computational analysis suggests that the IDR likely facilitates the formation of the complex between ATE1 and tRNAArg, adding a new complexity to ATE1 structure and providing new insights for future studies of ATE1 functions.

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As hydrogen is touted as a key player in the decarbonization of modern society, it is critical to enable quantitative hydrogen (H) analysis at high spatial resolution and, if possible, at the atomic scale. H has a known deleterious impact on the mechanical properties (strength, ductility, toughness) of most materials that can hinder their use as part of the infrastructure of a hydrogen-based economy. Enabling H mapping including local hydrogen concentration analyses at specific microstructural features is essential for understanding the multiple ways that H affect the properties of materials including embrittlement mechanisms and their synergies. In addition, spatial mapping and quantification of hydrogen isotopes is essential to accurately predict tritium inventory of future fusion power plants thus ensuring their safe and efficient operation. Atom probe tomography (APT) has the intrinsic capability to detect H and deuterium (D), and in principle the capacity for performing quantitative mapping of H within a material's microstructure. Yet, the accuracy and precision of H analysis by APT remain affected by complex field evaporation behavior and the influence of residual hydrogen from the ultrahigh vacuum chamber that can obscure the signal of H from within the material. The present article reports a summary of discussions at a focused workshop held at the Max-Planck Institute for Sustainable Materials in April 2024. The workshop was organized to pave the way to establishing best practices in reporting APT data for the analysis of H. We first summarize the key aspects of the intricacies of H analysis by APT and then propose a path for better reporting of the relevant data to support interpretation of APT-based H analysis in materials.

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Metabolic dysfunction-associated steatotic liver disease (MASLD) prevalence is increasing annually and affects over a third of US adults. MASLD can progress to metabolic dysfunction-associated steatohepatitis (MASH), characterized by severe hepatocyte injury, inflammation, and eventual advanced fibrosis or cirrhosis. MASH is predicted to become the primary cause of liver transplant by 2030. Although the etiology of MASLD/MASH is incompletely understood, dysregulated fatty acid oxidation is implicated in disease pathogenesis. Here, we develop a method for estimating hepatic ß-oxidation from the metabolism of [D15]octanoate to deuterated water and detection with deuterium magnetic resonance methods. Perfused livers from a mouse model of MASLD reveal dysregulated hepatic ß-oxidation, findings that corroborate in vivo imaging. The high-fat-diet-induced MASLD mouse studies indicate that decreased ß-oxidative efficiency in the fatty liver could serve as an indicator of MASLD progression. Furthermore, our method provides a clinically translatable imaging approach for determining hepatic ß-oxidation efficiency.

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The use of deuterated compounds is an interesting opportunity to expand the capabilities of Raman spectroscopy to study metabolism in living cells. Different biological objects have different tolerances to different deuterated compounds, and their metabolic chains may differ. Here, we explore the potential of this approach to probe metabolism in early mouse embryos. We investigated the Raman spectra of mouse embryos at different developmental stages cultured with deuterated amino acids, phenylalanine-d8 and leucine-d10, glucose-d7, and D2O. Embryos after in vitro culture with 20 % v/v D2O demonstrate Raman peak at 2186 cm-1 corresponding to newly synthesized proteins. Deuterated amino acids can slow down the development rate in 4-8 cell stage embryos, and deuterated glucose can be used at 2 mM concentration. For blastocyst, it was possible to achieve 75 % fraction of deuterated phenylalanine, when cultured with glucose, the maximal intensity ratio between CD and CH bands was 13.7 %. To demonstrate the capabilities of Raman spectroscopy reinforced by deuterium labeling, we investigated the short-term effect of cryopreservation and revealed that cryopreservation decreases the amount of saccharides in embryos and does not affect the activity of protein de novo synthesis.

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Hydrogen-Deuterium exchange mass spectrometry's (HDX-MS) utility in identifying and characterizing protein-small molecule interaction sites has been established. The regions that are seen to be protected from exchange upon ligand binding indicate regions that may be interacting with the ligand, giving a qualitative understanding of the ligand binding pocket. However, quantitatively deriving an accurate high-resolution structure of the protein-ligand complex from the HDX-MS data remains a challenge, often limiting its use in applications such as small molecule drug design. Recent efforts have focused on the development of methods to quantitatively model Hydrogen-Deuterium exchange (HDX) data from computationally modeled structures to garner atomic level insights from peptide-level resolution HDX-MS. One such method, HDX ensemble reweighting (HDXer), employs maximum entropy reweighting of simulated HDX data to experimental HDX-MS to model structural ensembles. In this study, we implement and validate a workflow which quantitatively leverages HDX-MS data to accurately model protein-small molecule ligand interactions. To that end, we employ a strategy combining computational protein-ligand docking, molecular dynamics simulations, HDXer, and dimensional reduction and clustering approaches to extract high-resolution drug binding poses that most accurately conform with HDX-MS data. We apply this workflow to model the interaction of ERK2 and FosA with small molecule compounds and inhibitors they are known to bind. In five out of six of the protein-ligand pairs tested, the HDX derived protein-ligand complexes result in a ligand root-mean-square deviation (RMSD) within 2.5 Å of the known crystal structure ligand.

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Since its discovery by Harold Urey in 1932, deuterium has attracted increased amounts of attention from the scientific community, with many previous works aimed to uncover its biological effects on living organisms. Existing studies indicate that deuterium, as a relatively rare isotope, is indispensable for maintaining normal cellular function, while its enrichment and depletion can affect living systems at multiple levels, including but not limited to molecules, organelles, cells, organs, and organisms. As an important compound of deuterium, deuterium-depleted water (DDW) possess various special effects, including but not limited to altering cellular metabolism and potentially inhibiting the growth of cancer cells, demonstrating anxiolytic-like behavior, enhancing long-term memory in rats, reducing free radical oxidation, regulating lipid metabolism, harmonizing indices related to diabetes and metabolic syndrome, and alleviating toxic effects caused by cadmium, manganese, and other harmful substances, implying its tremendous potential in anticancer, neuroprotective, antiaging, antioxidant, obesity alleviation, diabetes and metabolic syndrome treatment, anti-inflammatory, and detoxification, thereby drawing extensive attention from researchers. This review comprehensively summarizes the latest progress in deuterium acting on living organisms. We start by providing a snapshot of the distribution of deuterium in nature and the tolerance of various organisms to it. Then, we discussed the impact of deuterium excess and deprivation, in the form of deuterium-enriched water (DEW) and deuterium-depleted water (DDW), on living organisms at different levels. Finally, we focused on the potential of DDW as an adjuvant therapeutic agent for various diseases and disorders.

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Rates of microbial growth are fundamental to understanding environmental geochemistry and ecology. However, measuring the heterogeneity of microbial activity at the single-cell level, especially within complex populations and environmental matrices, remains a forefront challenge. Stable isotope probing (SIP) is a method for assessing microbial growth and involves measuring the incorporation of an isotopic label into microbial biomass. Here, we assess Raman microspectroscopy as a SIP technique, specifically focusing on the measurement of deuterium (2H), a tracer of microbial biomass production. We correlatively measured cells grown in varying concentrations of deuterated water with both Raman spectroscopy and nanoscale secondary ion mass spectrometry (nanoSIMS), generating isotopic calibrations of microbial 2H. Relative to Raman, we find that nanoSIMS measurements of 2H are subject to substantial dilution due to rapid exchange of H during sample washing. We apply our Raman-derived calibration to a numerical model of microbial growth, explicitly parameterizing the factors controlling growth rate quantification and demonstrating that Raman-SIP can sensitively measure the growth of microorganisms with doubling times ranging from hours to years. The measurement of single-cell growth with Raman spectroscopy, a rapid, nondestructive technique, represents an important step toward application of single-cell analysis into complex sample matrices or cellular assemblages.

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We show that in plasmas generated in deuterium in the presence of sputtered W surfaces, various molecular tungsten species are formed, whose chemical composition depends on the presence of gaseous impurities, namely, nitrogen, oxygen, and hydrogen. A magnetron discharge was used for plasma sustaining, and the species were investigated by mass spectrometry and optical emission spectroscopy. The identified tungsten-containing molecules are described by the chemical formula WOxNyDzHt, where x = 0-4, y = 0-3, z = 0-3, t = 0-5. Presumptively, even higher mass tungsten molecular species are present in plasma, which were not detected because of the limitation of the spectrometer measurement range to 300 amu. The presence of these molecules will likely impact the W particle balance and dust formation mechanisms in fusion plasmas.

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Metabolic imaging in clinical practice has long relied on PET with fluorodeoxyglucose (FDG), a radioactive tracer. However, this conventional method presents inherent limitations such as exposure to ionizing radiation and potential diagnostic uncertainties, particularly in organs with heightened glucose uptake like the brain. This review underscores the transformative potential of traditional deuterium MR spectroscopy (MRS) when integrated with gradient techniques, culminating in an advanced metabolic imaging modality known as deuterium MRI (DMRI). While recent advancements in hyperpolarized MRS hold promise for metabolic analysis, their widespread clinical usage is hindered by cost constraints and the availability of hyperpolarizer devices or facilities. DMRI, also denoted as deuterium metabolic imaging (DMI), represents a pioneering, single-shot, and noninvasive paradigm that fuses conventional MRS with nonradioactive deuterium-labelled substrates. Extensively tested in animal models and patient cohorts, particularly in cases of brain tumours, DMI's standout feature lies in its seamless integration into standard clinical MRI scanners, necessitating only minor adjustments such as radiofrequency coil tuning to the deuterium frequency. DMRI emerges as a versatile tool for quantifying crucial metabolites in clinical oncology, including glucose, lactate, glutamate, glutamine, and characterizing IDH mutations. Its potential applications in this domain are broad, spanning diagnostic profiling, treatment response monitoring, and the identification of novel therapeutic targets across diverse cancer subtypes.

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Deuterium-depleted water (DDW) is used in the treatment of many diseases, including cancer and diabetes. To detect the effect of DDW on gene expression and activation of the insulin-responsive transporter GLUT4 as a mechanism for improving the pathology of diabetes, we investigated the GLUT4 expression and glucose uptake at various concentrations of DDW using the myoblast cell line C2C12 differentiated into myotubes. GLUT4 gene expression significantly increased under deuterium depletion, reaching a maximum value at a deuterium concentration of approximately 50 ppm, which was approximately nine times that of natural water with a deuterium concentration of 150 ppm. GLUT4 protein also showed an increase at similar DDW concentrations. The membrane translocation of GLUT4 by insulin stimulation reached a maximum value at a deuterium concentration of approximately 50-75 ppm, which was approximately 2.2 times that in natural water. Accordingly, glucose uptake also increased by up to 2.2 times at a deuterium concentration of approximately 50 ppm. Drug-induced insulin resistance was attenuated, and the glucose uptake was four times higher in the presence of 10 ng/mL TNF-α and three times higher in the presence of 1 µg/mL resistin at a deuterium concentration of approximately 50 ppm relative to natural water. These results suggest that DDW promotes GLUT4 expression and insulin-stimulated activation in muscle cells and reduces insulin resistance, making it an effective treatment for diabetes.

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Labeling with deuterium oxide (D2O) has emerged as one of the preferred approaches for measuring the synthesis of individual proteins in vivo. In these experiments, the synthesis rates of proteins are determined by modeling mass shifts in peptides during the labeling period. This modeling depends on a theoretical maximum enrichment determined by the number of labeling sites (NEH) of each amino acid in the peptide sequence. Currently, NEH is determined from one set of published values. However, it has been demonstrated that NEH can differ between species and potentially tissues. The goal of this work was to determine the number of NEH for each amino acid within a given experiment to capture the conditions unique to that experiment. We used four methods to compute the NEH values. To test these approaches, we used two publicly available data sets. In a de novo approach, we compute NEH values and the label enrichment from the abundances of three mass isotopomers. The other three methods use the complete isotope profiles and body water enrichment in deuterium as an input parameter. They determine the NEH values by (1) minimizing the residual sum of squares, (2) from the mole percent excess of labeling, and (3) the time course profile of the depletion of the relative isotope abundance of monoisotope. In the test samples, the method using residual sum of squares performed the best. The methods are implemented in a tool for determining the NEH for each amino acid within a given experiment to use in the determination of protein synthesis rates using D2O.

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In-depth research into the precise evaluation of enzymatic digestion efficiency and the selection of a suitable deuterium-labelled internal standard remains a gap in the accurate determination of ß2-agonists in animal-derived food by isotope dilution-liquid chromatography/tandem mass spectrometry (ID-LC-MS/MS). In this study, the enzymatic digestion conditions were optimized by monitoring the presence of ß2-agonist conjugates in positive samples, which proved to be reliable for ensuring complete enzymatic digestion. Comparative analysis of deuterium-labelled internal standards for salbutamol (SAL), ractopamine (RAC), and clenbuterol (CLB) revealed that CLB-D6 and SAL-D9 were less effective in compensating for matrix effects due to hydrogen­deuterium exchange during MS fragment formation. Consequently, SAL-D3, RAC-D3 and CLB-D9 were chosen for the implementation of ID-LC-MS/MS. The developed method demonstrates high accuracy and precision, with the average recoveries ranging from 93.8% to 107.3% with RSD <6.1%, which can provide higher-order measurement results for ß2-agonists in pork.

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The latest iteration of modular, open-source rolled ion mobility spectrometers was characterized and tailored for heated ion chemistry experiments. Because the nature of ion-neutral interactions is innately linked to the temperature of the drift cell, heated IMS experiments explicitly probe the fundamental characteristics of these collisions. While classic mobility experiments examine ions through inert buffer gases, doping the drift cell with reactive vapor enables desolvated chemical reactions to be studied. By using materials with minimal outgassing and ensuring the isolation of the drift tube from the surrounding ambient conditions, an open-source drift cell outfitted with heating components enables investigations of chemical reactions as a function of temperature. We show here that elevated temperatures facilitate an increase in deuterium incorporation and allow for hydrogen/deuterium exchanges otherwise unattainable under ambient conditions. While the initial fast exchanges get faster as temperature is increased, the slow rate which rises from the kinetic nonlinearity though to be attributed to ion-neutral clustering, remains constant with no change in mobility shifts. Additionally, we show the analytical merit of multiplexing mobility data by comparing the performance of traditional signal-averaging and FT-IMS modes.

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This study aimed to synthesise and interpret stable isotopic data (δ2H and δ18O) from various sources to understand the isotope hydrology around coal mine operations in Elk Valley, B.C., Canada. The data, including precipitation, groundwaters, seeps, and mine rock drains, were used to construct a local meteoric water line (LMWL) for the Elk Valley, evaluate the spatiotemporal isotopic composition of its groundwater, and assess mine seepage and mine rock drain discharge. The study revealed a robust LMWL relation (δ2H = 7.4 ± 0.2 · δ18O - 4.3 ± 4.1). The groundwater and seep data indicated a winter season bias and a north-south latitudinal gradient, suggesting rapid near-surface groundwater flow without significant post-precipitation evaporation. Porewater isotope samples from unsaturated mine rock piles (MRPs) showed site-specific evaporation patterns, potentially due to convective air flows or exothermic sulphide oxidation. This research revealed the influence of groundwater and meltwater on rock drain discharge. Based on evaporative mass balance calculations, MRPs seasonally contributed ca. 5 %(December base flow) and 22 % (snowmelt) to drain discharge. The findings underscore the value of stable isotope data collections in the Elk Valley to help better define and quantify the hydrology-hydrogeology, including a better understanding of evaporative conditions in MRPs.

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BACKGROUND: Deuterium metabolic imaging (DMI) is an innovative, noninvasive metabolic MR imaging method conducted after administration of 2H-labeled substrates. DMI after [6,6'-2H2]glucose consumption has been used to investigate brain metabolic processes, but the impact of different [6,6'-2H2]glucose doses on DMI brain data is not well known. PURPOSE: To investigate three different [6,6'-2H2]glucose doses for DMI in the human brain at 7 T. STUDY TYPE: Prospective. POPULATION: Six healthy participants (age: 28 ± 8 years, male/female: 3/3). FIELD STRENGTH/SEQUENCE: 7 T, 3D 2H free-induction-decay (FID)-magnetic resonance spectroscopic imaging (MRSI) sequence. ASSESSMENT: Three subjects received two different doses (0.25 g/kg, 0.50 g/kg or 0.75 g/kg body weight) of [6,6'-2H2]glucose on two occasions and underwent consecutive 2H-MRSI scans for 120 minutes. Blood was sampled every 10 minutes during the scan, to determine plasma glucose levels and plasma 2H-Glucose atom percent excess (APE) (part-1). Three subjects underwent the same protocol once after receiving 0.50 g/kg [6,6'-2H2]glucose (part-2). STATISTICAL TEST: Mean plasma 2H-Glucose APE and glucose plasma concentrations were compared using one-way ANOVA. Brain 2H-Glc and brain 2H-Glx (part-1) were analyzed with a two-level Linear Mixed Model. In part-2, a General Linear Model was used to compare brain metabolite signals. Statistical significance was set at P < 0.05. RESULTS: Between 60 and 100 minutes after ingesting [6,6'-2H2]glucose, plasma 2H-Glc APE did not differ between 0.50 g/kg and 0.75 g/kg doses (P = 0.961), but was significantly lower for 0.25 g/kg. Time and doses significantly affected brain 2H-Glucose levels (estimate ± standard error [SE]: 0.89 ± 0.01, 1.09 ± 0.01, and 1.27 ± 0.01, for 0.25 g/kg, 0.50 g/kg, and 0.75 g/kg, respectively) and brain 2H-Glutamate/Glutamine levels (estimate ± SE: 1.91 ± 0.03, 2.27 ± 0.03, and 2.46 ± 0.03, for 0.25 g/kg, 0.50 g/kg, and 0.75 g/kg, respectively). Plasma 2H-Glc APE, brain 2H-Glc, and brain 2H-Glx levels were comparable among subjects receiving 0.50 g/kg [6,6'-2H2]glucose. DATA CONCLUSION: Brain 2H-Glucose and brain 2H-Glutamate/Glutamine showed to be [6,6'-2H2]glucose dose dependent. A dose of 0.50 g/kg demonstrated comparable, and well-detectable, 2H-Glucose and 2H-Glutamate/Glutamine signals in the brain. EVIDENCE LEVEL: 1 TECHNICAL EFFICACY: Stage 2.

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Objective: To identify factors contributing to sex-differences in OA risk by evaluating the short-term effect of high-fat (HF) diet on sex-specific changes in cartilage cell proliferation, ribosomal biogenesis, and targeted extra-cellular and cellular protein abundance. Materials and methods: Knee cartilage was harvested to the subchondral bone from 20-week-old female and male C57BL/6J mice fed a low-fat or HF diet for 4 weeks and labeled with deuterium oxide for 1, 3, 5, 7, 15, or 21 days. Deuterium enrichment was quantified in isolated DNA and RNA to measure cell proliferation and ribosomal biogenesis, respectively. Protein concentration was measured using targeted high resolution accurate mass spectrometry. Results: HF diet increased the maximal deuterium incorporation into DNA from approximately 40 to 50%, albeit at a slower rate. These findings, which were magnified in female versus male mice, indicate a greater number of proliferating cells with longer half-lives under HF diet conditions. HF diet caused distinct sex-dependent effects on deuterium incorporation into RNA, increasing the fraction of ribosomes undergoing biogenesis in male mice and doubling the rate of ribosome biogenesis in female mice. HF diet altered cartilage protein abundance similarly in both sexes, except for matrilin-3, which was more abundant in HF versus LF conditions in female mice only. Overall, HF diet treatment had a stronger effect than sex on cartilage protein abundance, with most changes involving extracellular matrix and matrix-associated proteins. Conclusions: Short-term HF diet broadly altered cartilage matrix protein abundance, while sex-dependent effects primarily involved differences in cell proliferation and ribosomal biogenesis.

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Therapeutic antibodies play an important role in the public healthcare system to treat patients with a variety of diseases. Protein characterization using an array of analytical tools provides in-depth information for drug quality, safety, efficacy, and the further understanding of the molecule. A therapeutic antibody candidate MAB1 exhibits unique binding properties to both cation and anion exchange columns at neutral pH. This uniqueness disrupts standard purification processes and necessitates adjustments in manufacturing. This study identifies that the charge heterogeneity of MAB1 is primarily due to the N-terminal cyclization of glutamine to pyroglutamine and, to a lesser extent, succinimide intermediate, deamidation, and C-terminal lysine. Using three approaches, i.e., deferential chemical labeling, H/D exchange, and molecular modeling, the binding to anion exchange resins is attributed to negatively charged patches on the antibody's surface, involving specific carboxylic acid residues. The methodologies shown here can be extended to study protein binding orientation in column chromatography.