Analytical Biochemistry

IntroductionSensitive protein stability assays used during hit confirmation conventionally require high quantities of purified protein. Here, we describe a novel high-throughput 384-well BRET-based thermostability assay allowing for the ultrasensitive determination of Gi1 protein stability. Using this method we make use of the environmentally sensitive dye, SYPRO Red, which interacts with hydrophobic regions in the protein that become exposed upon denaturation. Assays are functional in crude cell lysates, without any requirement for protein purification. MethodsThe SolThermoBRET method measures resonance energy transfer between a thermally stabilised nano-luciferase (tsNLuc) genetically engineered to the N-terminus of Gi1 protein, and SYPRO Red, a fluorescent dye that binds to lipophilic residues exposed upon protein unfolding in response to thermal denaturation. Samples in 384-well PCR plate were subjected to a temperature gradient (30 - 60 {degrees}C) on a PCR block and incubated for 5 minutes. Isothermal SolThermoBRET at a fixed temperature 45{degrees}C allowed the measurement of IC50 curves to various ligands. Furimazine (10 M) was added to the samples and BRET read on the BMG Labtech PHERAstar FSX at room temperature. Melt curves were fit to a Boltzmann sigmoidal equation to obtain Tm values. ResultsLigand-induced stabilisation of Gi1 protein was demonstrated in non-purified samples. A range of Tm values were measured with increasing stabilisation observed for nucleotides/sides in the following order GDP<GTP<GppNHp<GTP{gamma}S. Correlation was observed between SolThermoBRET derived Tms determined in the presence of high ligand concentrations and IC50 determinations using Isothermal SolThermoBRET. ConclusionsSolThermoBRET represents a sensitive nanoscale method for the detection of changes in soluble protein thermostability. This method is attractive for hit conformation and the identification of novel ligands targeting soluble proteins.

When optimized, sedimentation velocity analytical ultracentrifugation (SV-AUC) provides the most-accurate, broadest-range, and highest-resolution size distribution analysis of any method. Generating simulated data for an adeno-associated virus (AAV) sample consisting of four species differing only in their DNA content and having closely spaced sedimentation coefficients, allows manipulation of the SV-AUC experimental protocol to optimize the size distribution resolution. In developing this high speed SV-AUC (hs-SV-AUC) protocol several experimental challenges must be overcome: 1) the need for rapid data acquisition, 2) avoiding optical artifacts from steep boundaries and 3) overcoming the increased potential for convection. A protocol, hs-SV-AUC, has been developed that uses high rotor speeds, interference detection and low temperatures to overcome these challenges. By confining data analysis to a limited radial-time window and using a very short run time (< 20 min after temperature equilibration), the need to match the sample and reference solvent composition and meniscus positions is relaxed, making interference detection is as simple to employ as absorbance detection. Experimental size distributions from the same AAV sample by hs-SV-AUC at 45K rpm and 10 {degrees}C versus low-speed SV-AUC at 15K rpm, and 10 {degrees}C illustrates the improved size distribution resolution offered by the hs-SV-AUC protocol.

We here present a novel and sensitive methodology for determining the melting point (MP) of Bovine Serum Albumin (BSA) from micromolar to picomolar concentration levels under label free conditions. At 1 pM we could model the melting with a sharp gaussian. However, from the transient state observed during the melting process by using a simple exponential decay model we determined a time constant of 67 seconds. We applied this methodology by studying a 3.3 pM sample of a botulinum toxin A (BoNT-A) (stabilized with 2.8 nanomolar denatured Human Serum Albumin (HSA)). We were able to determine the Tm of BoNT-A in the presence of the approximately 1000-fold more concentrated HSA. Entry for the Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=153 SRC="FIGDIR/small/624557v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@12d93a7org.highwire.dtl.DTLVardef@138d01dorg.highwire.dtl.DTLVardef@e725acorg.highwire.dtl.DTLVardef@15a5f77_HPS_FORMAT_FIGEXP M_FIG C_FIG Protein label-free melting point (MP) determination at ultralow concentrations is a huge problem which concern to the biopharmaceutical industry. Here, we present a novel method to determine the MP of bovine serum albumin (BSA) from 1M to 1pM under label-free conditions. The benefits of this study match the purposes of stability studies in formulations, in which the protein active component is successful at very low concentrations, such as botulinum toxin A (BoNT-A). We used BOCOUTRE, a commercially available pharmaceutical product based on BoNT-A, and Human Serum Albumin (HSA) as a stabilizer. Our method can detect the MP of the stabilizer protein, even if its concentration is markedly different from that of the active component protein (1000-fold) in the case of BOCOUTRE.

Recent identification of new human muscle glycogen phosphorylation sites has renewed interest in understanding human variations in the regulation of glycogen metabolism and glucose homeostasis. This paper presents a detailed method for the measurement of glycogen phosphorylase (GPh) activity in skeletal muscle. Our approach incorporates modifications to existing radiolabelling assays, optimizing specificity and sensitivity while enabling the assessment of both active and total enzyme activity levels. The utilization of radioisotope tracers and scintillation counting ensures accurate quantification of GPh activity, which we use to validate a previously published reduction in GPh activity in an Actn3 deficient mouse model. Moreover, we introduce a step-by-step guide for data acquisition, highlight the use of appropriate homogenization, discuss the need for allosteric activators/inhibitors and the importance of assay optimization to record a GPh activity assay for skeletal muscle. In conclusion, our refined method not only contributes to a deeper understanding of glycogen metabolism in muscle tissue but also provides a framework for future investigations, underscoring its role in advancing research on glycogen utilization and glucose homeostasis. NEW & NOTEWORTHYThe study optimizes the glycogen phosphorylase radiolabelled activity assay, unveiling nuances in muscle homogenization, sample dilution, and caffeine inclusion. The research introduces standardized conditions, enhancing assay reliability and reproducibility across mouse strains to reveal sex specific variations in GPh activity and underscore novel distinctions in an Actn3 deficient mouse model. These findings advance our understanding of muscle glycogen metabolism, offering a crucial tool for researchers and facilitating meaningful inter-laboratory comparisons.

Creatine plays a fundamental role in cellular energy homeostasis. The current protocol describes an alternative method for creatine quantification in biological tissue samples using capillary electrophoresis, with high separation efficiency that enables rapid analysis with low sample volumes. The protocol involves homogenization of snap-frozen tissue in phosphate buffer, followed by electrophoresis through a bare-fused capillary (75 {micro}m internal diameter) and measurement at 200 nm on the Agilent 7100 CE system. Under the optimised conditions, there was excellent linearity in creatine standards between 6.3 - 100 {micro}M. The overall intra-assay variability for concentrations between 6.3 - 100 {micro}M was 1.5 %, and the inter-assay variability was 6.4 %, with a limit of detection at 6 nmol/mg protein. The protocol was further benchmarked against a commercially available enzyme assay kit using lung samples from lambs that received continuous creatine or saline supplementation. There was good agreement between the two methods (mean difference = 0.42 [-0.26-1.1] nmol/mg protein). Importantly, capillary electrophoresis enables reliable detection of creatine in biological samples from just [~]1.5 mg of wet-weight lung tissue. Capillary electrophoresis enables rapid (<10 minutes) and highly efficient analysis of tissue samples and avoids challenges faced with traditional enzymatic assays. The current protocol was developed and optimised with ovine lung tissue, but it can be easily adapted to analyse various tissue types. For tissues with higher baseline creatine content, such as the skeletal muscles or brain, <1 mg wet weight tissue would be sufficient to detect creatine using capillary electrophoresis.

Glycogen is the primary storage carbohydrate in mammals and it is synthesized in most tissues. Glycogen contains covalently attached phosphate groups on hydroxyls of glucose units. The addition of phosphate modulates branching pattern, granular size, and crystallinity of a glycogen molecule, which all impact its accessibility to glycogen interacting enzymes during catabolism. As glycogen architecture modulates its role in metabolism, it is essential to accurately evaluate and quantify phosphate content in glycogen. Simultaneous quantitation of glucose and its phosphate esters is challenging and requires an assay with high sensitivity and a robust dynamic range. Currently, this method is lacking in the field. Herein, we describe a highly-sensitive method for the detection of both glycogen-derived glucose and glucose-phosphate esters utilizing gas-chromatography coupled mass spectrometry. Using this method, we observed higher glycogen levels in the liver compared to skeletal muscle, but skeletal muscle contained much more phosphate esters. These results confirm previous findings and establish the validity of the method. Importantly, this method can detect femtomole levels of glucose and glucose phosphate esters within an extremely robust dynamic range with excellent accuracy and reproducibility. The method can also be easily adapted for the quantification of glucose from plant starch, amylopectin or other biopolymers as well as covalently attached phosphate within them.

Hyperpolarized carbon-13 MRI has shown promise for non-invasive assessment of the cerebral metabolism of [1-13C]pyruvate in both healthy volunteers and in patients. Exchange of pyruvate to lactate catalyzed by lactate dehydrogenase (LDH), and pyruvate flux to bicarbonate through pyruvate dehydrogenase (PDH), are the most widely studied reactions in vivo. Here we show the potential of the technique to probe other metabolic reactions in the human brain. Approximately 50 s after intravenous injection of hyperpolarized pyruvate, high flip angle pulses were used to detect cerebral 13C-labelled carbon dioxide (13CO2), in addition to the 13C-bicarbonate (H13CO2-) subsequently formed by carbonic anhydrase. Brain pH weighted towards the extracellular compartment was calculated from the ratio of H13CO3- to 13CO2 in seven volunteers using the Henderson-Hasselbalch equation, demonstrating an average pH {+/-} S.D. of 7.40 {+/-} 0.02, with inter-observer reproducibility of 0.04. In addition, hyperpolarized [1-13C]aspartate was also detected in four of nine volunteers demonstrating irreversible pyruvate carboxylation to oxaloacetate by pyruvate carboxylase (PC), and subsequent transamination by aspartate aminotransferase (AST), with this flux being approximately 6% of that through PDH. Hyperpolarized [1-13C]alanine signal was also detected within the head but this was localized to muscle tissue in keeping with skeletal alanine aminotransferase (ALT) activity. The results demonstrate the potential of hyperpolarized carbon-13 MRI to assess cerebral and extracerebral [1-13C]pyruvate metabolism in addition to LDH and PDH activity. Non-invasive measurements of brain pH could be particularly important in assessing cerebral pathology given the wide range of disease processes that alter acid-base balance.

BackgroundThe clinical translation of molecularly targeted therapeutics and imaging agents represents a cornerstone of precision oncology, with the global theranostics market projected to exceed $25 billion by 2030. However, the development of theragnostic agents or diagnostic companions remains constrained by analytical bottlenecks in quality control, such as target-binding specificity, which are increasingly required by regulatory agencies as product release criteria during the translation process. Current methods, including enzyme-linked immunosorbent assay (ELISA), which require specialized resources or external CROs, or bead-based assays for radiolabeled compounds, which involve complex multi-step protocols; these limitations and others hamper their practical implementation in clinical manufacturing environments. Assay delays can postpone clinical trial initiation, increase development costs, and delay patient access to these agents. ResultsWe have developed and validated a rapid, size-exclusion high-performance liquid chromatography (SE-HPLC) method for the determination of target-binding fractions of labeled biologics. The method separates the unbound biologic from the larger antigen-bound complex, allowing for rapid quantification. We validated the method using a panel of fluorescently labeled antibodies (panitumumab-IRDye800CW, nivolumab-IRDye800CW) and radiolabeled biologics ([18F]GEH200521, [18F]NOTA-ABY-030), assessing linearity, specificity, and concentration independence. The SE-HPLC method achieved excellent separation of bound and unbound species with a resolution (Rs) of 3.2. A strong linear relationship (R2 = 0.999) was observed between the antigen-to-antibody ratio and the measured binding fraction. The method demonstrated high specificity, with no binding detected with non-target antigens. The total assay and analysis time was less than 35 minutes, a significant improvement over traditional methods. ConclusionsSE-HPLC provides a rapid, specific, and cost-effective alternative to traditional binding fraction assessment methods, reducing quality control timelines from weeks/hours to minutes. The methods compatibility with both fluorescent and radiolabeled biologics and integration with existing HPLC infrastructure represents a significant advancement in development workflows.

We describe a robust, fast and accurate method for the quantification of intra-cellular concentrations of the polyamines, putrescine, spermidine and spermine in cultured cells. Hydrophilic interaction liquid chromatography in combination with Time-of-Flight mass spectrometry was used to obtain high resolution data for the analytes. Assay performance was determined with respect to chromatographic resolution, quantification, analyte recovery and matrix effects. Furthermore, assay variability was determined in a biological context. Based on these variability measurements, minimal detectable effects (MDEs) which would lead to significant differences in a null-hypothesis significance test, were calculated. As such, changes in spermine could be determined with the highest sensitivity with point estimates for the MDEs of 32% between-days and 10% within-days. For spermidine, these values were 38% between-days and 16% within-days. Finally, effects for putrescine were measured least sensitive with 43% between-days and 36% within-days. Finally, we employed the method to analyze the impact of polyamine synthesis pathway inhibition and cell culture conditions which are relevant aspects for the interpretation of the biological role of polyamines.

PurposeTo develop a pipeline for measuring the exchange rates and concentrations of in vivo excgangeable protons, and to demonstrate this for the amide and arginine (Arg) guanidinium (Guan) protons in mobile proteins in the mouse brain. MethodsAn ultra-short echo (UTE) CEST sequence with a continuous wave presaturation (preRadCEST) was applied to acquire Z-spectra with robustness to motion and physiological fluctuations. AmideCEST and Arginine guanCEST (ArgCEST) were extracted and their proton concentrations and exchange rates obtained using a two-step multi-B1 Bloch fitting approach that included the semisolid macromolecular background. To minimize contamination from the amine protons from creatine and phosphocreatine, ArgCEST measurements were performed on the Guanidinoacetate N-methyltransferase deficiency (GAMT-/-) mouse characterized by low creatine and phosphocreatine concentrations in the brain. ResultsFor the amideCEST proton pool, the exchange rate and concentrations were found to be 59.6 {+/-} 9.0 s-1 and 41.7 {+/-} 7.0 mM, respectively, with the maximum signal observed at B1 = 0.8 T. For the ArgCEST proton, the guanidinium exchange these were 70.1 {+/-} 5.5 s-1 and 10.1 {+/-} 1.3 mM, respectively, with the maximum effect observed at B1 = 0.9 T. The current study suggests that the inverse pH dependence in GuanCEST of brain is led by the CrCEST component, not ArgCEST. ConclusionThe current pipeline is expected to have general use for in vivo CEST quantitation and optimization of visible CEST resonances.

This study investigates the efficient development and production of single-chain variable fragments (scFvs) and antibody fragments (Fabs) using an E. coli-based cell-free protein synthesis system. Validation of the methodology was performed using a fluorescence correlation spectroscopy (FCS)-based assay to determine binding equilibrium constants (KD) between antibodies and the receptor binding domain (RBD) of SARS-CoV-2 Spike protein. An initial assessment employed two conventionally cell-produced anti-RBD antibodies. To optimize cell-free production, folding strategies were developed to enhance the solubility and yields of scFvs, including a two-stage refolding protocol that successfully recovered active proteins from misfolded precipitates. Fab fragments were also produced and characterized, with their binding properties analyzed to assess functionality. This study highlights the potential of cell-free systems for the rapid and efficient production of functional antibody fragments. The integration of advanced techniques, such as FCS-based kinetic measurements, underscores the versatility and applicability of cell-free platforms for antibody development and high-throughput screening. These findings offer a promising avenue for accelerating therapeutic antibody research and production. SIGNIFICANCE of WORKThis study highlights the transformative potential of integrating cell-free protein synthesis (CFPS) with fluorescence correlation spectroscopy (FCS) for the rapid and scalable production and characterization of functional antibody fragments. By leveraging an E. coli-based CFPS platform, we successfully designed and optimized the production of single-chain variable fragments (scFvs) compared to Fab fragments, addressing some common challenges of low solubility and yield. The development of a cost-efficient two-stage refolding strategy further enhanced the scalability and functionality of scFvs, enabling higher recovery of active proteins from the unfolded state. Additionally, FCS provided a sensitive, rapid method for accurately quantifying antigen-antibody binding kinetics across a range of affinities. This work establishes CFPS and FCS as versatile and complementary tools, offering a robust framework for accelerating antibody fragment development, particularly in time-sensitive scenarios like infectious disease outbreaks.

BackgroundDNA polymerase activity assays are required for enzyme quality control in biotechnology and diagnostics, but standard methods rely on specialist reagents, radioactivity and other hazardous materials, or real-time PCR instruments that are not widely accessible in resource-limited settings. This constrains local production of high quality, validated reagents and increases dependence on imported enzymes. MethodsBased on experiences derived from partnerships with scientists in several low and middle-income countries (LMICs) and stakeholder consultations, we adapted a commercial EvaGreen-based fluorometric DNA polymerase activity assay for isothermal operation using minimal equipment. Assay conditions were optimized using Design of Experiments (DOE) methodology, varying temperature, reaction volume, and MgCl2 concentration. To address reagent cost and supply-chain constraints, we developed detailed protocols for in-house synthesis of the off-patent AOAO-12 DNA dye (sold commercially as EvaGreen) and generation of single-stranded DNA templates via asymmetric PCR. ResultsOptimized isothermal assay conditions (40{degrees}C, 7.75 mM MgCl2) reliably quantified activity across multiple DNA polymerase families. In-house synthesized AOAO-12 dye exhibited comparable DNA-binding performance to commercial alternatives (R{superscript 2} = 0.95), reducing costs by more than an order of magnitude when normalized to working concentrations, enabling assay costs of approximately {pound}0.001 per reaction. The assay is effective across multiple polymerases (Bst-LF, OpenVent, Taq, Q5) and is compatible with both plate readers and qByte, a low-cost, open-source fluorometric device. ConclusionsThis stakeholder-informed assay provides an accessible, cost-effective solution for DNA polymerase quality control in resource-limited settings. The combination of optimized commercial protocols and in-house reagent synthesis offers flexibility for different resource contexts, potentially improving access to molecular biology tools globally.

The use of quality control samples in metabolomics ensures data quality, reproducibility and comparability between studies, analytical platforms and laboratories. Long-term, stable and sustainable reference materials (RMs) are a critical component of the QA/QC system, however, the limited selection of currently available matrix matched RMs reduce their applicability for widespread use. To produce a RM in any context, for any matrix that is robust to changes over the course of time we developed IBAT (Iterative Batch Averaging meThod). To illustrate this method, we generated 11 independently grown E. coli batches and made a RM over the course of 10 IBAT iterations. We measured the variance of these materials by NMR and showed that IBAT produces a stable and sustainable RM over time. This E. coli RM was then used as food source to produce a C. elegans RM for a metabolomics experiment. The metabolite extraction of this material alongside 41 independently grown individual C. elegans samples of the same genotype, allowed to estimate the proportion of sample variation in pre-analytical steps. From the NMR data, we found that 40% of the metabolite variance is due to the metabolite extraction process and analysis and 60% is due to sample-to-sample variance. The availability of RMs in untargeted metabolomics is one of the predominant needs of the metabolomics community that reach beyond quality control practices. IBAT addresses this need by facilitating the production of biologically relevant RMs and increasing their widespread use.

Here we report an adapted protocol using the Promega NAD/NADH-Glo Assay kit. The assay normally allows quantification of trace amounts of both oxidized and reduced forms of nicotinamide adenine dinucleotide (NAD) by enzymatic cycling, but we now show that the NAD analog 3- acetylpyridine adenine dinucleotide (AcPyrAD) also acts as a substrate. In fact, AcPyrAD generates amplification signals of larger amplitude than those obtained with NAD. We exploited this finding to devise and validate a novel method for assaying the base exchange activity of SARM1 in reactions containing NAD and an excess of the free base 3-acetylpyridine (AcPyr), where AcPyrAD is the product. We also propose an application of this method based on competition between AcPyr and other free bases to rank their preference for SARM1. This has significant advantages over traditional methods for assaying SARM1 base exchange as it is rapid, sensitive, cost-effective, and easily scalable. This could represent a useful tool given current interest in the role of SARM1 base exchange in programmed axon death and related human disorders. It may also be applicable to other multifunctional NAD glycohydrolases (EC 3.2.2.6) that possess similar base exchange activity.

The SLAPTAG is a novel molecular TAG derived from a protein domain present in the sequence of Lactobacillus acidophilus SlpA (SlpA284-444). Proteins from different biological sources, with different molecular weights or biochemical functions, can be fused in frame to the SLAPTAG and efficiently purified by the specific binding to a bacterial-derived chromatographic matrix named here Bio-Matrix (BM). Different binding and elution conditions were evaluated to set an optimized protocol for the SLAPTAG-based affinity chromatography (SAC). The binding equilibrium between SLAPTAG and BM was reached after a few minutes at 4{degrees}C, being the apparent dissociation constant (KD) of 4.3 {micro}M, a value which is similar to different Kd determined for other S-layer proteins and their respective bacterial cell walls. A reporter protein was generated (H6-GFP-SLAPTAG) to compare the efficiency of the SAC against a commercial system based on a Ni2+-charged agarose matrix, observing no differences in the H6-GFP-SLAPTAG purification performance. The stability and reusability of the BM were evaluated, and it was determined that the matrix was stable for more than a year, being possible to reuse it five times without a significant loss in the efficiency for protein purification. Alternatively, we explored the recovery of bound SLAP-tagged proteins by proteolysis using the SLAPASE (a SLAP-tagged version of the HRV-3c protease) that released a tag-less GFP (SLAPTAG-less). Additionally, iron nanoparticles were linked to the BM and the resulting BMmag was successfully adapted for a magnetic SAC, a technique that can be potentially applied for high-throughput-out protein production and purification.

Urine collection is painless and offers the potential for kidney liquid-biopsy(1), which appears particularly appealing with regard to the diagnosis of kidney disease (2) and patient follow-up after renal transplantation (3). From a nephrological point of view, urinary sediment and the soluble and exosome fractions of urine constitute different biological entities. We here describe a method that allows deep profiling of the protein content of the above-mentioned three fractions of urine by quantitative data-independent label-free proteomics. The method was evaluated using 19 urine samples from the Nephrology outpatient clinic at Vienna General Hospital, comprising a diverse set of chronic kidney disease (CKD) as well as patients after kidney transplantation (NTX). Peptide separation was accomplished through 60 min active gradients. A timsTOF Pro2 mass spectrometer was operated in DIA mode. The total analysis time per urine sample (three fractions) was around four hours. We demonstrate adequate technical and experimental reproducibility. Our data suggest that the protein information content of these three fractions is diverse, strengthening the importance of separate analysis. The depth of our quantitative proteomics approach permitted a detection of proteins characteristic for different parts of the nephron, such as Podocin, CD2-AP and Podocalyxin (Podocytes), SLC22A8 and SLC22A13 (proximal tubule) and Aquaporin-2 (collecting duct), suggesting that our method is sensitive enough to detect and quantify biologically relevant proteins that might serve as potential biomarkers. To the best of our knowledge, the ability to quantify up to 4000 protein groups per urine sample and more than 6000 protein groups in total makes our strategy the deepest proteome profiling study of urine to date. In conclusion, we established a method with promising figures of merit that we consider broadly applicable and useful for future clinical biomarker research studies in urine.

Background and PurposeIn-vivo magnetic resonance spectroscopy (MRS) of 2-hydroxyglutarate (2HG) may provide diagnostic and monitoring biomarkers in isocitrate dehydrogenase (IDH)-mutated glioma. A previous meta-analysis has shown good diagnostic accuracy of TE-optimized PRESS for IDH-mutated glioma, but most studies feature IDH-wildtype glioma as a comparison. However, when considering newly identified brain lesions that may mimic glioma, full characterization of its diagnostic utility should also consider the accuracy of 2HG measurement in non-tumor tissue. Therefore, we tested how well TE-optimized 2HG levels distinguish between IDH-mutated glioma and non-tumor tissue, in this case, normal-appearing brain. We further examined the impact of different spectral modeling strategies (baseline stiffness, macromolecule inclusion, and basis set composition). Materials and Methods48 patients with diagnosed/suspected IDH-mutated glioma were enrolled. 3T MRS data were acquired from tumor and contralateral non-tumor tissue with PRESS localization (TE = 97 ms, optimized for 2HG detection) and analyzed with LCModel software. Receiver operating characteristic analysis evaluated 2HG estimates ability to distinguish IDH-mutated glioma from non-tumor brain tissue. Modeling interactions between 2HG and other metabolites were evaluated to identify reasons for potential false-positive 2HG detection. ResultsTE-optimized PRESS distinguished IDH-mutated glioma from non-tumor tissue with lower sensitivity (range 0.76-0.62) and specificity (0.85-0.78) than literature suggests for IDH-mutated vs. IDH-wildtype glioma. Strong negative correlations between gamma-aminobutyric acid (GABA) and 2HG persisted across all modeling strategies and may lead to false-positive 2HG detection in non-tumor tissue. We further present a cautionary example from a patient on a ketogenic diet, showing that the ketone body acetone can interfere with 2HG detection. ConclusionsSpectral overlap with GABA and acetone can lead to false-positive 2HG detection in non-tumor tissue. Clinicians need to be mindful of these pitfalls when interpreting 2HG estimates.

Over the last decade chemical exchange saturation transfer (CEST) NMR methods have emerged as powerful tools to characterize biomolecular conformational dynamics occurring between a visible major state and invisible minor states. The ability of the CEST experiment to detect these minor states, and provide precise exchange parameters, hinges on using appropriate B1 field strengths during the saturation period. Typically, a pair of B1 fields with{omega} 1 (= 2{pi}B1) values around the exchange rate kex are chosen. Here we show that the transverse relaxation rate of the minor state resonance (R2,B) also plays a crucial role in determining the B1 fields that lead to the most informative datasets. Using [Formula], to guide the choice of B1, instead of kex, leads to data wherefrom substantially more accurate exchange parameters can be derived. The need for higher B1 fields, guided by K, is demonstrated by studying the conformational exchange in two mutants of the 71 residue FF domain with kex [~]11 s-1 and [~]72 s-1, respectively. In both cases analysis of CEST datasets recorded using B1 field values guided by kex lead to imprecise exchange parameters, whereas using B1 values guided by K resulted in precise site-specific exchange parameters. The conclusions presented here will be valuable while using CEST to study slow processes at sites with large intrinsic relaxation rates, including carbonyl sites in small to medium sized proteins, amide 15N sites in large proteins and when the minor state dips are broadened due to exchange among the minor states.

Plant glutamate decarboxylase (GAD) is a Ca2+-calmodulin activated cytosolic enzyme that produces {gamma}-aminobutyrate (GABA) as the first committed step of the GABA shunt. This pathway circumvents the 2-oxoglutarate to succinate reactions of the mitochondrial tricarboxylic acid cycle. Our prior research established that in vivo phosphorylation of the root-specific AtGAD1 isozyme (AT5G17330) occurs at multiple N-terminal serine residues, following Pi resupply to Pi-starved cell cultures of the model plant Arabidopsis thaliana. The aim of the current investigation was to purify recombinant AtGAD1 following its expression in Escherichia coli to facilitate studies of the impact of site-specific phosphorylation on its kinetic properties. However, in vitro proteolytic truncation of a 5 kDa polypeptide from the C-terminus of 59 kDa AtGAD1 subunits occurred during its purification. Immunoblotting demonstrated that most protease inhibitors or cocktails that we tested were ineffective in suppressing partial AtGAD1 proteolysis during incubation of clarified extracts at 23 {degrees}C. Although the thiol modifiers N-ethylmaleimide or 2,2-dipyridyl disulfide negated AtGAD1 proteolysis, they also abolished its GAD activity. This indicates that an essential -SH group is needed for catalytic activity, and that AtGAD1 is susceptible to partial degradation either by an E. coli cysteine endopeptidase, or possibly via autoproteolytic activity. The inclusion of exogenous Ca2+/calmodulin in extraction and chromatography buffers facilitated the purification of non-proteolyzed AtGAD1 to a specific activity of 27 ({micro}mol GABA produced/mg) at optimal pH 5.8, while exhibiting an approximate 3-fold activation by Ca2+/CaM at pH 7.3. By contrast, the purified partially proteolyzed His6-AtGAD1 was >40% less active at both pH values, and only activated 2-fold by Ca2+/CaM at pH 7.3. These results emphasize the need to diagnose and prevent unwanted proteolysis before conducting kinetic studies of purified regulatory enzymes.

Lysine lactoylation is a recently described protein post-translational modification (PTM). However, the biochemical pathways responsible for this acylation remain unclear. Two metabolite-dependent mechanisms have been proposed: enzymatic histone lysine lactoylation derived from lactoyl-coenzyme A (lactoyl-CoA, also termed lactyl-CoA), and non-enzymatic lysine lactoylation resulting from acyl-transfer via lactoyl-glutathione. While the former has precedent in the form of enzyme-catalyzed lysine acylation, the lactoyl-CoA metabolite has not been previously quantified in mammalian systems. Here we use liquid chromatography-high resolution mass spectrometry (LC-HRMS) together with a synthetic standard to detect and validate the presence of lactoyl-CoA in cell and tissue samples. Conducting a retrospective analysis of data from previously analyzed samples revealed the presence of lactoyl-CoA in diverse cell and tissue contexts. In addition, we describe a biosynthetic route to generate 13C3 15N1 -isotopically-labeled lactoyl-CoA, providing a co-eluting internal standard for analysis of this metabolite. We estimate lactoyl-CoA concentrations of 1.14x10-8 pmol/cell in cell culture and 0.0172 pmol/mg tissue wet weight in mouse heart. These levels are similar to crotonyl-CoA, but between 20-350 times lower than predominant acyl-CoAs such as acetyl-, propionyl-, and succinyl-CoA. Overall our studies provide the first quantitative measurements of lactoyl-CoA and provide a methodological foundation for the interrogation of this novel metabolite in biology and disease. Highlights- Detection of lactoyl-CoA at picomole concentrations across tissues and cells - Lactoyl-CoA was detected at concentrations similar to crontonyl-CoA within HepG2 cells - Isotopically labeled 13C315N1-lactoyl-CoA can be prepared by SILEC