2023 Webinars

Metabolic Flux Analysis Webinar | November 9 | Sponsored by CIL

Use of Stable Isotopes in Metabolic Flux Analysis (MFA) Studies and How the MFA Results Are Benefiting the Biomanufacturing Industry

Daniel Benjamin | Chief Scientific Officer | Metalytics

Abstract: Metabolic flux analysis (MFA) is the science of using stable isotopes to elucidate both the route of metabolism as well as the rate of metabolism. In this presentation we will demonstrate how stable isotopes are used in MFA studies with a description of the data that is collected, processed, and converted into actionable information for the biomanufacturing industry. We will present three different case studies demonstrating what knowledge was gained and used to improve productivity. We will also highlight the ability to use this stable isotope-derived MFA data to train a machine learning model that enables the rapid and accurate prediction of metabolic fluxes with limited experimental data.

  • Stable isotopes are crucial for calculating the precise rate and route of metabolite flow within cells using a technology called metabolic flux analysis (MFA)

  • Metalytics has successfully used this stable isotope-enabled MFA data to improve the productivity of biomanufacturing

  • Metalytics is currently using this MFA data to train a digital twin that can rapidly and accurately predict metabolic flux with limited experimental data

A transcript of this presentation in English is available here.

Isotope Days 2023 | October 5 | Sponsored by CIL

 Environmental Focus 

Exposure to Per- and Polyfluoroalkyl Substances (PFAS) in the Indoor Environment: Measurement of PFAS in House Dust and Silicone Wristbands

Heather Stapleton, PhD | Ronie-Richele Garcia-Johnson Distinguished Professor | Duke University, Nicholas School of the Environment (USA)

Abstract: Per- and polyfluoroalkyl substances (PFAS) are a large and complex group of synthetic chemicals with over 9,000 different compounds that may be found in various everyday products, including paint, personal care products, stain- and water-resistant fabrics and carpets, firefighting foam, and food-packing materials. Due to their widespread use, persistence and toxicity, there are increasing efforts to understand sources of PFAS exposure. While significant attention has focused on drinking water and food as a source of exposure, less attention has focused on exposure in the indoor environment, particularly for short-chain polyfluorinated alkyl acids and per- and polyfluoroalkyl ether acids. As such, our laboratory developed methods to quantify both volatile and non-volatile PFAS in indoor dust and in personal passive samplers, namely silicone wristbands. Silicone wristbands have been a popular exposure tool to assess individual level exposures and they provide several advantages over more traditional exposure approaches such as analysis of blood and urine. We therefore optimized extraction and analytical conditions to measure 46 different PFAS using LC-MS/MS and 13 different PFAS using GC-HRMS in both house dust and silicone wristbands. This presentation will highlight results from our research investigating levels of PFAS in paired samples of house dust and silicone wristbands from a cohort of adults collected in 2021 residing in the US.

On-demand recording and transcript are not available.

Dynamite Comes in Small Packages

Bryan Vining, PhD | Laboratory Director | Enthalpy Analytical Ultratrace (USA)

Abstract: I'll be presenting on our method(s) for the analysis of ultra-short-chain PFAS compounds using isotope dilution techniques. The nature of these compounds is such that they cannot be readily analyzed alongside their longer-chain cousins. How we get there and the unique challenges these compounds present will be discussed.

A transcript of this presentation in English is available here.

Exploring Freshwater Harmful Algal Blooms: Microcystin Toxin Structural Diversity and Other Bioactive Cyanopeptides

Wendy Strangman, PhD | Assistant Professor | University of North Carolina at Wilmington (USA)

Abstract: Freshwater harmful algal blooms (HABs) caused by cyanobacteria pose significant ecological and health risks. Microcystins, a class of over 200 cyclic heptapeptide toxins, exhibit structural variations that impact toxicity and behavior. Analytical techniques like mass spectrometry unveil this diversity, aiding in understanding their effects and guiding management strategies. Cyanobacteria also produce diverse bioactive peptides with applications in pharmaceuticals and biotechnology. Exploring their structures and functions sheds light on ecological roles and potential benefits. This presentation will highlight advances in microcystin and cyanopeptide characterization. Understanding these compounds is vital for effective HAB management and sustainable usage, safeguarding aquatic ecosystems and human health.

A transcript of this presentation in English is available here.

An Isotope Dilution-Based Method for the Analysis of Microcystins and Anatoxin-a for Improved Accuracy and Robustness

Xavier Ortiz Almirall, PhD | Assistant Professor | IQS Barcelona (Spain)

Abstract: The occurrence of cyanobacterial harmful algal blooms in freshwater around the world has been increasing steadily during the last decades due to the global warming and extensive use of fertilizers in agriculture, which contribute to the eutrophication of lakes and rivers. Cyanobacteria can produce different families of toxins which can be harmful or even lethal to living organisms, such as microcystins or anatoxin-a. During this presentation, an automated method for the targeted and non-targeted analysis of microcystins and anatoxin-a is presented, which is based on the use of mass labelled internal standards for an improved method accuracy and robustness.

Quantitative Analysis of Microplastics Using Isotope-Labelled Polystyrene (Styrene-d8) by Pyrolysis-GCxGC-TOFMS

Yukari Ishikawa, PhD | Research Associate | Imperial College London, Environmental Research Group, School of Public Health (UK)

Abstract: Whilst pyrolysis-GC-MS presents a promising technique for the quantitative analysis of micro- and nanoplastics, researchers face issues such calibration curve preparation and what to use as an internal standard. There are several ways to create a calibration curve, including using ASE (accelerated solvent extraction), dissolving in a solvent (although there are limitations), or adjusting the concentration of solid standards by mixing with a non-reactive solid.

For internal standards, there are two approaches: (1) the use of organic compounds that mimic to some extent the pyrolysis behavior of the polymer under investigation, and (2) the use of stable isotope-labelled polymer materials. A mixture of androstane, 9-dodecyl-1,2,3,4,5,6,7,8-octahydro anthracene (DOHA), 9-tetradecyl-1,2,3,4,5,6,7,8-octahydro anthracene (TOHA), d10-anthracene and cholanic acid is an example of approach (1). The most recent example of this approach was published in 2021 with poly-fluoro-styrene (PFS). As for the stable isotope-labelled polymer standard in approach (2), d5-PS, d8-PS, d8-PP, and d6-polybutadiene have been used for pyrolysis-GC-MS analysis. For more accurate analytical data, approach (2), which uses stable isotope-labelled polymer standards, seems to be better than approach (1), but “deuterium-hydrogen exchange” should be carefully monitored. 13C-polymer (e.g. 13C-PE) is also available, but it is significantly more expensive, and there are concerns about handling difficulties due to the unclear particle size and its (lack of) solubility.

If the internal standard is to be used as a clean-up spike, its form must also be taken into consideration. Since the micro-/nanoplastics are present in the sample in solid form, the internal standard should be in solid form with similar size distribution. However, that leaves us with the problem of how to weigh and dilute the ultra-fine amounts of the internal standard. On the other hand, if the internal standard is provided as a solution, it may behave differently from micro-/nanoplastics during the sample preparation process, for example, passing through the filter which usually captures and concentrates microplastic during extract filtration. To determine the best internal standard for polymer analysis with pyrolysis-GC-MS, further study is necessary.

A transcript of this presentation in English is available here.

Isotope Days 2023 | September 28 | Sponsored by CIL

 NMR Focus 

Visualizing RNA Structural Dynamics Using NMR

Hashim Al-Hashimi, PhD | Roy and Diana Vagelos Professor of Biochemistry | Columbia University, Biochemistry and Molecular Biophysics (USA)

Abstract: The talk will describe the development of methods coupling NMR spectroscopy on 13C/15N-labeled RNA samples with computational approaches which are enabling the visualization of RNA structural dynamics at atomic resolution. The focus of the talk will be on HIV-1 TAR RNA and the process of transcriptional activation of the retroviral genome.

A transcript of this presentation in English is available here.

Binding Site of Hexamethylene Amiloride in the SARS-CoV-2 Envelope Protein from Solid-state NMR

Mei Hong, PhD | Professor of Chemistry | Massachusetts Institute of Technology (MIT) (USA)

Abstract: The SARS-CoV-2 envelope (E) protein forms a five-helix bundle in lipid bilayers whose cation-conducting activity is associated with the inflammatory response and respiratory distress symptoms of COVID-19. E channel activity is inhibited by the drug 5-(N,N-hexamethylene) amiloride (HMA). However, the binding site of HMA in E has not been determined. Here we use solid-state NMR to measure distances between HMA and the E transmembrane domain (ETM) in lipid bilayers. 13C, 15N-labeled HMA is combined with fluorinated or 13C-labeled ETM. Conversely, fluorinated HMA is combined with 13C, 15N-labeled ETM. These orthogonal isotopic labeling patterns allow us to conduct dipolar recoupling NMR experiments to determine the HMA binding stoichiometry to ETM as well as HMA-protein distances. We find that HMA binds ETM with a stoichiometry of one drug per pentamer. Unexpectedly, the bound HMA is not centrally located within the channel pore, but lies on the lipid-facing surface in the middle of the TM domain. This result suggests that HMA may inhibit the E channel activity by interfering with the gating function of an aromatic network. These distance data are obtained under much lower drug concentrations than in previous chemical shift data, which showed the largest perturbation for N-terminal residues. This difference suggests that HMA has higher affinity for the protein-lipid interface than the channel pore, which gives insight into the inhibition mechanism of HMA for SARS-CoV-2 E.

A transcript of this presentation in English is available here.

Imaging Tumor Metabolism – From Mouse to Man

Kevin Brindle, PhD | Professor of Biomedical Magnetic Resonance | University of Cambridge, Cancer Research (UK)

Abstract: Molecular imaging is likely to play an increasingly important role in predicting and detecting tumor responses to treatment and thus in guiding treatment in individual patients. We have been using MRI-based metabolic imaging techniques to detect tumor treatment response, to monitor disease progression and to investigate the tumor microenvironment. Initially this was using hyperpolarized 13C-labelled substrates. Nuclear spin hyperpolarization increases sensitivity in the 13C magnetic resonance experiment by >10,000x, which allows imaging of injected hyperpolarized 13C-labelled cell substrates in vivo and, more importantly, the kinetics of their metabolic conversion into other cell metabolites. More recently we have been using 2H-labelled substrates; the relatively low sensitivity of detection is compensated by the very short T1s displayed by this quadrupolar nucleus, which enables extensive signal averaging in the absence of signal saturation. Both imaging techniques have translated to the clinic. In this talk I will describe recent studies in which we have used these techniques to detect the early responses of tumors to treatment.

On-demand recording and transcript are not available.

Protein-based NMR for Generating Small Molecule Hits and Drug Leads

Andrew Namanja, PhD | Principal Research Scientist | AbbVie Inc. (Discovery) (USA)

Abstract: Protein-based nuclear magnetic resonance (NMR) has emerged as a powerful tool for early-stage drug discovery. The ability of NMR to provide atomic-resolution insights of protein-ligand interactions in solution can greatly facilitate unambiguous confirmation of screening hits and drug leads spanning a wide affinity range (mM to nM). Drug discovery programs enabled by protein-detected 2D NMR are less prone to false positives that can stem from biochemical assays and this, in turn, can mitigate an unnecessary waste of resources and time. In this talk, we will discuss our incorporation of the protein-based NMR workflow in our early discovery pipeline involving various screening modalities such as DNA-encoded libraries (DEL), high-throughput screening (HTS), virtual library screening (VLS), and fragment-based drug design (FBDD).

On-demand recording and transcript are not available.

Histidine Labelling of Enzymes for Solution and Solid-state NMR

Rafal Augustyniak, PhD | Researcher | University of Warsaw (Poland)

Abstract: Histidine plays an important role in enzyme catalysis and protein stability. As it is one of very few amino acids that change the protonation state within a physiological pH range, histidine imidazole side chain is often involved in proton transfer during chemical reactions in biological systems. Needless to say, full understanding of the protonation state, tautomerism and elucidation of the interaction network is required to get a complete picture of protein molecules containing histidine residues in active centers. NMR spectroscopy offers a wide range of experiments to visualize histidine side chains but to date such studies were limited to rather small proteins. Here, we show how selective labelling of histidines in conjunction with uniform deuteration can expand the applicability of these tools to larger objects. As an example we use a 70 kDa viral protease that is amenable by both solution and solid state NMR – techniques providing complementary data.

On-demand recording and transcript are not available.

Isotope Days 2023 | September 21 | Sponsored by CIL

 Mass Spectrometry Focus 

The Critical Role of Stable Isotopes to Unravel Complex Biological Questions

Dave Muddiman, PhD | Professor | North Carolina State University, Department of Chemistry (USA)

Abstract: Since its first demonstration in the 1960s, the field of mass spectrometry imaging (MSI) has emerged as a fruitful area of scientific research with significant impacts to human health. To date, SIMS, MALDI, and DESI have been the primary ionization methods utilized in the field and these approaches have resulted in key new findings for a diverse range of scientific questions. However, other emerging ionization methods have great potential to impact the field of MSI. We invented matrix-assisted laser desorption electrospray ionization (MALDESI) in 2005 and over the past 18 years, we have made tremendous progress in the fundamentals, source development, and demonstrated the principal advantages of this ionization technique. Mass spectrometry imaging offers a versatile and robust platform to discover and characterize new diagnostic, prognostic, and therapeutic biomarkers for disease, elucidate and understand pathways including protein-protein interactions, visualize endogenous and exogenous compound distributions in tissues via mass spectrometry imaging, and characterize post-translational modifications. Moreover, a Multi-OMIC approach will allow the underlying biology to be defined, enabling modeling of pathways and identify potential drug targets. This presentation will cover two biological questions which are understanding xenobiotic metabolism (stable isotope-labeled glycerate) and cancer (stable isotope-labeled cysteine). Moreover, derivative approaches will be presented to enable diverse MSI platforms to be qualified prior to their application. The biological and QC approaches are made possible by stable-isotope labeled compounds made at high purity. The fundamentals of these strategies will be integrated throughout the presentation.

A transcript of this presentation in English is available here.

Using Isotopes to Probe the Metabolism of Oxalate

Sonia Fargue | Assistant Professor | University of Alabama, Urology Department (USA)

Abstract: Using Isotopes to probe the metabolism of oxalate urinary oxalate excretion is a well-known risk factor for calcium oxalate kidney stone formation. Oxalate is derived from both gut absorption of dietary oxalate and from endogenous synthesis of oxalate. The metabolic pathways leading to endogenous oxalate synthesis are still incompletely characterized in humans and in animals, in health and in disease. Isotope tracers have been a major source of knowledge in the field of oxalate metabolism for decades and current research still heavily relies on these techniques, alongside technical innovations. We describe old and current work using 13C-oxalate and 13C-oxalate precursors in humans to determine the relative influence of different precursors, enzymes and their deficiencies and how this has helped the development of new therapeutic strategies for the rare genetic disease primary hyperoxalurias.

A transcript of this presentation in English is available here.

Metabolic Characterization of Birt-Hogg-Dubé Syndrome Renal Tumor Cells and Tissues Using Stable Isotope-Resolved Metabolomics

Ye Yang, PhD | Postdoc Fellow | NIH, Urologic Oncology Branch, (USA)

Abstract: Background Birt-Hogg-Dubé syndrome (BHD) is caused by germline mutations in the FLCN gene, and patients are at risk of developing bilateral, multifocal renal tumors. In this study we utilized stable isotopes to track various metabolic pathways in BHD renal tumor cells and tissues. Ultra-high-resolution mass spectrometry, as well as nuclear magnetic resonance (NMR), were used to analyze the polar/non-polar metabolites extracted from BHD renal tumor cells and tissues. Methods FLCN-deficient renal tumor cell line UOK257 was derived from patient with BHD. The BHD renal tumor slices were obtained intra-operatively from patients undergoing surgery at the NIH Clinical Center. Cells and tissue slices were cultured in medium containing either 13C6-glucose or 13C5, 15N2-glutamine, with or without metformin, to probe the central metabolic pathways. After 24h, cells and tissues were harvested and extracted. IC-UHR-MS was applied as the main tool to analyze the polar extract. NMR was also applied as complementary tool for polar and lipid extract analysis.

Results and Conclusions: Our data revealed that the BHD tumor tissues exhibit enhanced glucose oxidation and reduced glutamine uptake relative to renal cortex tissues. Using 13C6-glucose as the tracer, we found increased citrate (m+2)/pyruvate (m+3) in BHD tumor tissues, which suggested enhanced pyruvate dehydrogenase (PDH) activity relative to renal cortex tissues. This was consistent with the gene expression analysis. Moreover, western blot analysis demonstrated that the respiratory chain was also upregulated in the BHD tumors. Treatment of UOK257 cells with respiratory chain inhibitor metformin inhibited cell growth. 13C6-glucose tracer experiments demonstrated that the oxidation of glucose through PDH pathway was inhibited. Whereas 13C5, 15N2-glutamine tracer experiments showed that while the oxidative glutamine metabolism was inhibited, the reductive carboxylation of glutamine was stimulated with metformin treatment of UOK257 cells. Metformin also decreased the incorporation of glucose derived 13C into lipid acyl chain in UOK257 cells. These findings provide a potential foundation for the development of therapeutic approaches for treatment and/or prevention of BHD renal cancer.

On-demand recording and transcript are not available.

Highly Standardized Metabolomic Analysis of Clinical Samples Using Triple Quadruple Mass Spectrometry

Jurre Kamphorst, PhD | Vice President of MS Technology & Biomarkers | Olaris Therapeutics (USA)

Abstract: High-resolution mass spectrometers are often the preferred choice for discovery metabolomics research efforts, particularly for their ability to perform untargeted metabolite profiling and detect unknowns. Triple quadrupole (QqQ) instruments have been less popular. However, the high accuracy, sensitivity, and dynamic range of these instruments, as well as the relative ease of data processing, remain attractive. Additionally, improvements in cycle time in newer generation instruments make it feasible to profile hundreds of metabolites in a single analysis. As such, the use of QqQ instruments holds great promise for clinical metabolomics, where the emphasis is on the quality of the measurements. We developed a HILIC-QqQ MS method for the accurate analysis of 300+ endogenous metabolites. We combine this with the use of 13C-yeast metabolite extract for comprehensive internal standard coverage. In this talk we will demonstrate how this method enables us to perform clinical metabolomics with high accuracy and reproducibility at scale, across time course studies and multiple batches.

A transcript of this presentation in English is available here.

Exploring Proteome Turnover in a Murine Alzheimer′s Disease Model Using Stable Isotope Labeling

Junmeng Peng, PhD | Member (Director)
 | St. Jude Children’s Research, Center for Proteomics and Metabolomics Structural Biology and Developmental Neurology Departments (USA)

Abstract: We introduced JUMPt, a software utilizing an ordinary differential equation-based mathematical model to determine reliable protein degradation rates. JUMPt considers amino acid recycling and simultaneously fits labeling kinetics and the whole proteome to derive protein half-lives. We applied JUMPt to analyze protein turnover in pSILAC-labeled brain and liver tissues. Notably, we observed enrichment of long-lived proteins in brain compartments. Additionally, JUMPt facilitated the investigation of proteome turnover in an Alzheimer’s disease mouse model, revealing delayed turnover of Abeta peptides and associated proteins due to amyloid plaque formation. Thus, JUMPt enhances protein turnover analysis in complex systems, offering insights into disease-related protein dynamics and potential therapeutic strategies.

A transcript of this presentation in English is available here.