Quantification of Total Ketone Bodies in Biological Matrices Using Stable Isotope-Labeled Internal Standards and RP-UHPLC-MS/MS.

Acetoacetate (AcAc) and D-beta-hydroxybutyrate (D-ßOHB), the two major ketone bodies found in circulation, are linked to multiple physiological and pathophysiological states. Therefore, analytical methodologies surrounding the quantification of total ketone body (TKB) concentrations in biological matrices are paramount. Traditional methods to quantify TKBs relied on indirect spectrophotometric assays with narrow dynamic ranges, which have been significantly improved upon by modern mass spectrometry (MS)-based approaches. However, the lack of stable isotope-labeled internal standards (ISs) for AcAc and the need to distinguish D-ßOHB from its closely related structural and enantiomeric isomers pose significant obstacles. Here, we provide a protocol to synthesize and quantify a [13C] stable isotope-labeled IS for AcAc, which, in conjunction with a commercially available [2H] stable isotope-labeled IS for ßOHB, allows TKBs to be measured across multiple biological matrices. This rapid (7 min) analysis employs reverse phase ultra-high performance liquid chromatography (RP-UHPLC) coupled to tandem MS (MS/MS) to distinguish ßOHB from three structural isomers using parallel reaction monitoring (PRM), providing excellent specificity and selectivity. Finally, a method is provided that distinguishes D-ßOHB from L-ßOHB using a simple one-step derivatization to produce the corresponding diastereomers, which can be chromatographically resolved using the same rapid RP-UHPLC separation with new PRM transitions. In summary, this method provides a rigorous analytical pipeline for the analysis of TKBs in biological matrices via leveraging two authentic stable isotope-labeled ISs and RP-UHPLC-MS/MS.

NMR-Based Stable Isotope Tracing of Cancer Metabolism.

NMR is widely used for metabolite profiling (metabolomics, metabonomics) particularly of various readily obtainable biofluids such as plasma and urine. It is especially valuable for stable isotope tracer studies to track metabolic pathways under control or perturbed conditions in a wide range of cell models as well as animal models and human subjects. NMR has unique properties for utilizing stable isotopes to edit or simplify otherwise complex spectra acquired in vitro and in vivo, while quantifying the level of enrichment at specific atomic positions in various metabolites (i.e., isotopomer distribution analysis).In this protocol, we give an overview with specific protocols for NMR-based stable isotope-resolved metabolomics, or SIRM, with a workflow from administration of isotope-enriched precursors, via sample preparation through to NMR data collection and reduction. We focus on indirect detection of common NMR-active stable isotopes including 13C, 15N, 31P, and 2H, using a variety of 1H-based two-dimensional experiments. We also include the application and analyses of multiplex tracer experiments.

Detection of Inverse Stable Isotopic Labeling in Untargeted Metabolomic Data.

Stable isotopic labeling is a powerful tool for determining the biosynthetic origin of metabolites and for discovering natural products that incorporate precursors of interest. When isotopically substituted precursors are not available commercially or synthetically, inverse stable isotopic labeling (InverSIL) is a useful alternative. With InverSIL, an organism is grown on an isotopically substituted medium and then fed precursors of natural isotopic abundance which can be tracked by mass spectrometry, thereby bypassing issues with precursor availability. Currently, there is no automated way to identify precursor incorporation in untargeted metabolomic data using InverSIL without specifying an expected change in the mass-to-charge ratio of metabolites that have incorporated the precursor. This makes it difficult to identify unknown natural products that may incorporate portions of precursors of interest using new biochemistry or to rapidly identify incorporation of multiple precursors into different metabolites simultaneously. To address this, we developed a new, robust workflow for the automated identification of inverse labeling in untargeted metabolomic data. We then use this method to identify metabolites that incorporate para-aminobenzoic acid and different portions of l-methionine, including in the same sample, and in the process discover the likely biosynthetic origin for the C-7 and C-9 methyl groups of the pterin portion of dephosphotetrahydromethanopterin, a C1 transfer coenzyme used by methylotrophic bacteria. This workflow can be applied in the future to streamline the use of the versatile InverSIL approach for natural product and metabolism research.

Quelling the Geometry Factor Effect in Quantum Chemical Calculations of 13C NMR Chemical Shifts with the Aid of the pecG-n (n = 1, 2) Basis Sets.

A root factor for the accuracy of all quantum chemical calculations of nuclear magnetic resonance (NMR) chemical shifts is the quality of the molecular equilibrium geometry used. In turn, this quality depends largely on the basis set employed at the geometry optimization stage. This parameter represents the main subject of the present study, which is a continuation of our recent work, where new pecG-n (n = 1, 2) basis sets for the geometry optimization were introduced. A goal of this study was to compare the performance of our geometry-oriented pecG-n (n = 1, 2) basis sets against the other basis sets in massive calculations of 13C NMR shielding constants/chemical shifts in terms of their efficacy in reducing geometry factor errors. The testing was carried out with both large-sized biologically active natural products and medium-sized compounds with complicated electronic structures. The former were treated using the computation protocol based on the density functional theory (DFT) and considered in the theoretical benchmarking, while the latter were treated using the computational scheme based on the upper-hierarchy coupled cluster (CC) methods and were used in the practical benchmarking involving the comparison with experimental NMR data. Both the theoretical and practical analyses showed that the pecG-1 and pecG-2 basis sets resulted in substantially reduced geometry factor errors in the calculated 13C NMR chemical shifts/shielding constants compared to their commensurate analogs, with the pecG-2 basis set being the best of all the considered basis sets.

Parahydrogen-enhanced pH measurements using [1-13C]bicarbonate derived from non-enzymatic decarboxylation of [1-13C]pyruvate-d3.

Alterations in pH are a hallmark in several pathologies including cancer, ischemia, and inflammation. Non-invasive magnetic resonance methods to measure pH offer a new approach for early diagnosis of diseases characterized by acid-base imbalances. The hyperpolarization with parahydrogen-induced polarization (PHIP) enhances inherently low signals in magnetic resonance experiments by several orders of magnitude and offers a suitable platform to obtain biocompatible markers in less than one minute. Here, we present an optimized preparation of an hyperpolarized H13CO3-/13CO2 pH sensor via non-enzymatic decarboxylation with H2O2 of [1-13C]pyruvate-d3 obtained by PHIP at 7 T. An improved 13C polarization of purified [1-13C]pyruvate-d3 in water with 36.65 ± 0.06% polarization was obtained starting from 50 mM precursor. Subsequent decarboxylation, H13CO3-/13CO2 exhibited 12.46 ± 0.01% of polarization at physiological pH, 45 seconds after the reaction start. Considering the dilution factor that [1-13C]pyruvate-d3 exhibits in vivo, we optimized our methodology to test the accuracy of the pH sensor at single digit millimolar concentration. In vitro pH estimations on phantoms and cell culture media demonstrated accurate pH calculations with uncertainties of less than 0.08 units. These promising results highlight the efficiency of a pH sensor generated via PHIP in less than one minute, with remarkable polarization, and biocompatibility suitable for future in vivo studies.

High-temperature liquid chromatography-isotope ratio mass spectrometry methodology for carbon isotope ratio determination of anabolic steroids in urine.

BACKGROUND: Gas Chromatography Isotope Ratio Mass Spectrometry (GC-C-IRMS) has long been used in routine laboratories to determine the δ13C values of anabolic steroids in urine, differentiating between, e.g., endogenous and synthetic testosterone (T) in sports doping control. Until now, liquid chromatography (LC-IRMS) has not been used. The LC-IRMS setup doesn't allow organic solvents or modifiers in the mobile phase for δ13C determinations. Mid-to non-polar analytes such as steroids can be analysed in water heated to High Temperatures (HT, up to 200 °C) because at 200 °C has a similar polarity as 80/20 methanol/water at ambient temperature. In this work, we developed a method for steroids in urine, extending the application of the LC-IRMS to non-polar analytes in complex matrices. RESULT: An HT-LC-IRMS method capable of determining the δ13C values of four steroids (i.e., testosterone (T), 5α-androstane-3α,17ß-diol (ααß), 5ß-androstane-3α,17ß-diol (ßαß) and pregnanetriol (PT)) in urine was developed and validated. Accuracy ranged from 0.23 ‰ (ααß and ßαß) to 0.49 ‰ (T), and the detection limit was set at 10 ng mL-1 (T, ααß+ßαß). The validation data and a comparison of authentic urine samples analysed with HT-LC-IRMS and GC-C-IRMS indicated a comparable performance between HT-LC-IRMS and GC-C-IRMS. SIGNIFICANCE: HT-LC-IRMS can be used to determine δ13C values of anabolic steroids, extending the applicability of both HT-LC and LC-IRMS to non-polar substances determined in a complex matrix in routine laboratory practice.

Automated Assignment of 15N And 13C Enrichment Levels in Doubly-Labeled Proteins.

Uniform enrichment of 15N and 13C in proteins is commonly employed for 2D heteronuclear NMR measurements of the three-dimensional protein structure. Achieving a high degree of enrichment of both elements is important for obtaining high quality data. Uniform labeling of proteins and glycoproteins expressed in higher organisms (yeast or mammalian cell lines) is more challenging than expression in Escherichia coli, a prokaryote that grows on simple, chemically defined media but does not provide appropriate eukaryotic modifications. It is difficult to achieve complete incorporation of both heavy isotopes, and quality control measures are important for quantitating the level of their enrichment. Mass spectrometry measurements of the isotopic distribution of the intact protein or its proteolytic fragments provide the means to assess the enrichment level. A mass accuracy of 1 ppm or better is shown to be required to distinguish the correct combination of 13C and 15N enrichment due to subtle shifts in peak centroids with differences in the underlying, but unresolved, isotopic fine structure. A simple computer program was developed to optimize the fitting of experimental isotope patterns to statistically derived distributions. This method can determine the isotopic abundance from isotope patterns and isotopologue masses in conventional MS data for peptides, intact proteins, and glycans. For this purpose, MATLAB's isotope simulator, isotopicdist, has been modified to permit the variation of 15N and 13C enrichment levels and to perform a two-dimensional grid search of enrichment levels of both isotopes. We also incorporated an alternate isotope simulator, js-emass, into MATLAB for use in the same fitting program. Herein we benchmark this technique on natural abundance ubiquitin and uniformly [15N,13C]-labeled ubiquitin at both the intact and peptide level, outline considerations for data quality and mass accuracy, and report several improvements we have made to the previously reported analysis of the [15N,13C]-enriched human IgG Fc domain, a glycoprotein that has been expressed in Saccharomyces cerevisiae.

Two-dimensional high performance liquid chromatography purification of underivatized urinary prednisone and prednisolone for compound-specific stable carbon isotope analysis.

The gas chromatography-combustion isotope ratio mass spectrometry (GC/C/IRMS) confirmation procedure for prednisone (PS) and prednisolone (PSL) is still a great challenge for the doping control laboratory due to the many structurally similar steroids present in urinary matrices. This study aims to establish an innovative online two-dimensional high performance liquid chromatography (2D-HPLC) purification method for measuring the carbon isotope ratios (CIRs) and achieving the identification of the synthetic forms of these two endogenous anabolic androgenic steroids (EAASs). Initially, the one-dimensional chromatographic column was used to separate and purify endogenous reference compounds (ERCs), and the co-elution fluids containing PS and PSL were switched to a two-dimensional chromatographic column for further purification through an online transfer system. Then the purified compounds were analyzed using GC/C/IRMS after sample pretreatments. The results showed that the minimum detection concentration of PS and PSL reached 30 ng mL-1, and no isotope fractionation occurred during the entire collection and preparation process. This method has been validated with the WADA technical document and showed good sensitivity and selectivity, demonstrating its practical applicability for urine samples in doping control laboratories.

Linking biosynthetic genes to natural products using inverse stable isotopic labeling (InverSIL).

The sequencing of microbial genomes has far outpaced their functional annotation. Stable isotopic labeling can be used to link biosynthetic genes with their natural products; however, the availability of the required isotopically substituted precursors can limit the accessibility of this approach. Here, we describe a method for using inverse stable isotopic labeling (InverSIL) to link biosynthetic genes with their natural products. With InverSIL, a microbe is grown on an isotopically substituted medium to create a fully substituted culture, and subsequently, the incorporation of precursors of natural isotopic abundance can be tracked by mass spectrometry. This eliminates issues with isotopically substituted precursor availability. We demonstrate the utility of this approach by linking a luxI-type acyl-homoserine lactone synthase gene in a bacterium that grows on methanol with its quorum sensing signal products. In the future, InverSIL can also be used to link biosynthetic gene clusters hypothesized to produce siderophores with their natural products.

Redox Transformations of the OX063 Radical in Biological Media: Oxidative Decay of Initial Trityl with Further Formation of Structurally-Modified TAM.

Being a low-toxic and hydrophilic representative of TAM, OX063 has shown its suitability for in-vivo and in-cell EPR experiments and design of spin labels. Using 13C labeling, we investigated the course of oxidative degradation of OX063 into quinone-methide (QM) under the influence of superoxide as well as further thiol-promoted reduction of QM into TAM radical, which formally corresponds to substitution of a carboxyl function by a hydroxyl group. We found these transformations being quantitative in model reactions mimicking specific features of biological media and confirmed the presence of these reactions in the blood and liver homogenate of mice in vitro. The emergence of the trityl with the hydroxyl group can be masked by an initial TAM in EPR spectra and may introduce distortions into EPR-derived oximetry data if they have been obtained for objects under hypoxia. 13C labeling allows one to detect its presence, considering its different hyperfine splitting constant on 13C1 (2.04 mT) as compared to OX063 (2.30 mT). The potential involvement of these reactions should be considered when using TAM in spin-labeling of biopolymers intended for subsequent EPR experiments, as well as in the successful application of TAM in experiments in vivo and in cell.

Magic-angle spinning NMR spectral editing of polysaccharides in whole cells using the DREAM scheme.

Most bacterial, plant and fungal cells possess at their surface a protective layer called the cell wall, conferring strength, plasticity and rigidity to withstand the osmotic pressure. This molecular barrier is crucial for pathogenic microorganisms, as it protects the cell from the local environment and often constitutes the first structural component encountered in the host-pathogen interaction. In pathogenic molds and yeasts, the cell wall constitutes the main target for the development of clinically-relevant antifungal drugs. In the past decade, solid-state NMR has emerged as a powerful analytical technique to investigate the molecular organization of microbial cell walls in the context of intact cells. 13C NMR chemical shift is an exquisite source of information to identify the polysaccharides present in the cell wall, and two-dimensional 13C-13C correlation experiments provide an efficient tool to rapidly access the polysaccharide composition in whole cells. Here we investigate the use of the adiabatic DREAM (for dipolar recoupling enhancement through amplitude modulation) recoupling scheme to improve solid-state NMR analysis of polysaccharides in intact cells. We demonstrate the advantages of two-dimensional 13C-13C experiments using the DREAM recoupling scheme. We report the spectral editing of polysaccharide signals by varying the radio-frequency carrier position. We provide practical considerations for the implementation of DREAM experiments to characterize polysaccharides in whole cells. We demonstrate the approach on intact fungal cells of Neurospora crassa and Aspergillus fumigatus, a model and a pathogenic filamentous fungus, respectively. The approach could be envisioned to efficiently reduce the spectral crowding of more complex cell surfaces, such as cell wall and peptidoglycan in bacteria.

Enhanced Solubility and Polarization of 13C-Fumarate with Meglumine Allows for In Vivo Detection of Gluconeogenic Metabolism in Kidneys.

Hyperpolarized 13C-labeled fumarate probes tissue necrosis via the production of 13C-malate. Despite its promises in detecting tumor necrosis and kidney injuries, its clinical translation has been limited, primarily due to the low solubility in conventional glassing solvents. In this study, we introduce a new formulation of fumarate for dissolution dynamic nuclear polarization (DNP) by using meglumine as a counterion, a nonmetabolizable derivative of sorbitol. We have found that meglumine fumarate vitrifies by itself with enhanced water solubility (4.8 M), which is expected to overcome the solubility-restricted maximum concentration of hyperpolarized fumarate after dissolution. The achievable liquid-state polarization level of meglumine-fumarate is more than doubled (29.4 ± 1.3%) as compared to conventional dimethyl sulfoxide (DMSO)-mixed fumarate (13.5 ± 2.4%). In vivo comparison of DMSO- and meglumine-prepared 50-mM hyperpolarized [1,4-13C2]fumarate shows that the signal sensitivity in rat kidneys increases by 10-fold. As a result, [1,4-13C2]aspartate and [13C]bicarbonate in addition to [1,4-13C2]malate can be detected in healthy rat kidneys in vivo using hyperpolarized meglumine [1,4-13C2]fumarate. In particular, the appearance of [13C]bicarbonate indicates that hyperpolarized meglumine [1,4-13C2]fumarate can be used to investigate phosphoenolpyruvate carboxykinase, a key regulatory enzyme in gluconeogenesis.

Isotope dilution with isotopically labeled biomass: An effective alternative for quantitative metabolomics.

BACKGROUND: State-of-the-art quantitative metabolomics relies on isotope dilution using internal standards (IS) derived from fully 13C labeled biomass. By spiking samples and external standards with known amounts of IS, the spike characterization demands are kept to a minimum. In fact, it is sufficient to experimentally assess the isotopic enrichment of the IS. This study develops the yeast derived IS toolbox further, (1) by characterizing the concentration levels of hydrophilic metabolites in a yeast fermentation batch and (2) by exploring the analytical figures of merit of one-point IS versus multipoint external calibration using IS, the established gold-standard for quantitative metabolomics. RESULTS: Independent reverse isotope dilution experiments using different chromatographic methods over a period of several months, delivered a list of 83 13C-labeled metabolites with fully characterized concentration and their uncertainty, covering 5 orders of magnitude, from the nanomolar to the low millimolar range. The 13C-labeled yeast-derived IS showed excellent intermediate stability with 92 % of molecules showing inter-method RSDs ≤30 % (75 % of molecules showed RSDs ≤15 %) over a timeframe of five months. One-point internal standardization with the characterized labeled biomass achieved figures of merit equivalent to multipoint calibrations for the majority of metabolites. SIGNIFICANCE: The proposed calibration workflow rationalizes time and standard expenditure and is particularly beneficial for laboratories dealing with wide-target assays and small analysis batches. The present assessment serves as a seminal study for further developments of the concept towards absolute quantification from archive high-resolution MS data of U13C-biomass-spiked samples and the implementation of quick biomass recalibration with each experiment, promising seamless transition between internal standards derived from different fermentation batches.

Protocol for producing hyperpolarized 13C-bicarbonate for clinical MRI of extracellular pH in aggressive tumors.

Tumor acidosis is one of the hallmarks indicating the initiation and progression of various cancers. Here, we present a protocol for preparing a hyperpolarized (HP) 13C-bicarbonate tissue pH MRI imaging contrast agent to detect aggressive tumors. We describe the steps for the formulation and polarization of a precursor molecule 13C-glycerol carbonate (13C-GLC), the post-dissolution reaction, and converting HP 13C-GLC to an injectable HP 13C-bicarbonate solution. We then detail procedures for MRI data acquisition to generate tumor pH maps for assessing tumor aggressiveness. For complete details on the use and execution of this protocol, please refer to Mu et al.1.

Real-Time Metabolic Magnetic Resonance Spectroscopy of Pancreatic and Colon Cancer Tumor-Xenografts with Parahydrogen Hyperpolarized 1-13C Pyruvate-d3.

Parahydrogen-induced polarization (PHIP) is an emerging technique to enhance the signal of stable isotope metabolic contrast agents for Magnetic Resonance (MR). The objective of this study is to continue establishing 1-13C-pyruvate-d3, signal-enhanced via PHIP, as a hyperpolarized contrast agent, obtained in seconds, to monitor metabolism in human cancer. Our focus was on human pancreatic and colon tumor xenografts. 1-13C-vinylpyruvate-d6 was hydrogenated using parahydrogen. Thereafter, the polarization of the protons was transferred to 13C. Following a workup procedure, the free hyperpolarized 1-13C-pyruvate-d3 was obtained in clean aqueous solution. After injection into animals bearing either pancreatic or colon cancer xenografts, slice-selective MR spectra were acquired and analyzed to determine rate constants of metabolic conversion into lactate and alanine. 1-13C-pyruvate-d3 proved to follow the increased metabolic rate to lactate and alanine in the tumor xenografts.

In vivo Metabolic Sensing of Hyperpolarized [1-13C]Pyruvate in Mice Using a Recyclable Perfluorinated Iridium Signal Amplification by Reversible Exchange Catalyst.

Real-time visualization of metabolic processes in vivo provides crucial insights into conditions like cancer and metabolic disorders. Metabolic magnetic resonance imaging (MRI), by amplifying the signal of pyruvate molecules through hyperpolarization, enables non-invasive monitoring of metabolic fluxes, aiding in understanding disease progression and treatment response. Signal Amplification By Reversible Exchange (SABRE) presents a simpler, cost-effective alternative to dissolution dynamic nuclear polarization, eliminating the need for expensive equipment and complex procedures. We present the first in vivo demonstration of metabolic sensing in a human pancreatic cancer xenograft model compared to healthy mice. A novel perfluorinated Iridium SABRE catalyst in a fluorinated solvent and methanol blend facilitated this breakthrough with a 1.2-fold increase in [1-13C]pyruvate SABRE hyperpolarization. The perfluorinated moiety allowed easy separation of the heavy-metal-containing catalyst from the hyperpolarized [1-13C]pyruvate target. The perfluorinated catalyst exhibited recyclability, maintaining SABRE-SHEATH activity through subsequent hyperpolarization cycles with minimal activity loss after the initial two cycles. Remarkably, the catalyst retained activity for at least 10 cycles, with a 3.3-fold decrease in hyperpolarization potency. This proof-of-concept study encourages wider adoption of SABRE hyperpolarized [1-13C]pyruvate MR for studying in vivo metabolism, aiding in diagnosing stages and monitoring treatment responses in cancer and other diseases.

Hyperpolarized [2-13C, 3-2H3]Pyruvate Detects Hepatic Gluconeogenesis In Vivo.

The feasibility of hyperpolarized [2-13C, 3-2H3]pyruvate for probing gluconeogenesis in vivo was investigated in this study. Whereas hyperpolarized [1-13C]pyruvate has clear access to metabolic pathways that convert pyruvate to lactate, alanine, and bicarbonate, its utility for assessing pyruvate carboxylation and gluconeogenesis has been limited by technical challenges, including spectral overlap and an obscure enzymatic step that decarboxylates the labeled carbon. To achieve unambiguous detection of gluconeogenic products, the carbonyl carbon in pyruvate was labeled with 13C. To prolong the T1 relaxation time, [2-13C, 3-2H3]pyruvate was synthesized and dissolved with D2O after dynamic nuclear polarization. The T1 of [2-13C, 3-2H3]pyruvate in D2O could be improved by 76.9% (79.6 s at 1 T and 74.5 s at 3 T) as compared to [2-13C]pyruvate in water. Hyperpolarized [2-13C, 3-2H3]pyruvate with D2O dissolution was applied to rat livers in vivo under normal feeding and fasting conditions. A gluconeogenic product, [2-13C]phosphoenolpyruvate, was observed at 149.9 ppm from fasted rats only, highlighting the utility of [2-13C, 3-2H3]pyruvate in detecting key gluconeogenic enzyme activities such as pyruvate carboxylase and phosphoenolpyruvate carboxykinase in vivo.

Fabrication of Isotope-Enriched Nanostructures Using Ultrafast Laser Pulses under Ambient Conditions for Biomolecular Sensing.

Recent advances in the use of stable isotopes necessitate novel synthesis techniques for isotope separation and enrichment that are scalable and offer high throughput. Stable-isotope-enriched nanostructures can offer unique advantages as nanomedicines, safe tracers, and labels and are critical for applications in various industrial processes, metabolic research, and medicine. So far, there exists no method to synthesize miniature isotope-enriched materials at the nanoscale. In this study, an ultrafast Laser-induced isotope enrichment at nanoscale (LIIEN) is put forward to synthesize isotope-enriched nanostructures, eliminating the need for large equipment and expenses, thereby demonstrating a lab-scale isotope enrichment process. A significant isotope enrichment for Carbon nanostructures is observed. The isotope enrichment can be attributed to the redistribution of isotope ions in the plasma plume explained by the plasma centrifuge model. The LIIEN synthesized structures exhibit excellent Surface-Enhanced Raman Scattering (SERS) signal enhancement and reproducibility, making them potential candidates for SERS-based biomolecule sensing. This technique is an efficient method to fabricate nanosized isotope-enriched structures of characteristic properties by carefully tuning laser parameters at ambient conditions.

Solution-state methyl NMR spectroscopy of large non-deuterated proteins enabled by deep neural networks.

Methyl-TROSY nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for characterising large biomolecules in solution. However, preparing samples for these experiments is demanding and entails deuteration, limiting its use. Here we demonstrate that NMR spectra recorded on protonated, uniformly 13C labelled samples can be processed using deep neural networks to yield spectra that are of similar quality to typical deuterated methyl-TROSY spectra, potentially providing information for proteins that cannot be produced in bacterial systems. We validate the methodology experimentally on three proteins with molecular weights in the range 42-360 kDa. We further demonstrate the applicability of our methodology to 3D NOESY spectra of Escherichia coli Malate Synthase G (81 kDa), where observed NOE cross-peaks are in good agreement with the available structure. The method represents an advance in the field of using deep learning to analyse complex magnetic resonance data and could have an impact on the study of large biomolecules in years to come.

Combining Gas Exchange and Rapid Quenching of Leaf Tissue for Mass Spectrometry Analysis Directly in Gas Exchange Cuvette.

Isotopically nonstationary metabolic flux analysis (INST-MFA) is a powerful technique for studying plant central metabolism, which involves introducing a 13CO2 tracer to plant leaves and sampling the labeled metabolic intermediates during the transient period before reaching an isotopic steady state. The metabolic intermediates involved in the C3 cycle have exceptionally fast turnover rates, with some intermediates turning over many times a second. As a result, it is necessary to rapidly introduce the label and then rapidly quench the plant tissue to determine concentrations in the light or capture the labeling kinetics of these intermediates at early labeling time points. Here, we describe a rapid quenching (0.1-0.5 s) system for 13CO2 labeling experiments in plant leaves to minimize metabolic changes during labeling and quenching experiments. This system is integrated into a commercially available gas exchange analyzer to measure initial rates of gas exchange, precisely control ambient conditions, and monitor the conversion from 12CO2 to 13CO2.