Webinars Sponsored by CIL

Abstract: Persistent toxic pollutants (PTS) have properties such as high toxicity, bioaccumulation, and long-range transport. PTS is ubiquitous in the global environment. This presentation mainly focuses on new pollutants of global concern, such as hexachlorobutadiene, halogenated carbazoles, and so on. Their sources, emission inventory and environmental behaviours will be discussed, which are helpful to protect human health and promote sustainable development goals.

Abstract: The USEPA Office of Water currently requires monitoring of PCBs in wastewater discharges using techniques such as EPA Method 608, a dual-column GC/ECD procedure for organochlorine pesticides and seven Aroclor mixtures, which was the most practical approach available in the late 1970s when the first wastewater methods were proposed at 40 CFR Part 136. In the 40 years since those regulations were finalized, the environmental monitoring landscape has changed dramatically.

Abstract: Isotopic labeling of methyl-substituted proteinogenic amino acids with ¹³C has transformed applications of solution-based NMR spectroscopy and allowed the study of much larger and more complex proteins than previously possible with ¹⁵N labeling. Procedures are well-established for producing methyl-labeled proteins expressed in bacteria, with efficient incorporation of ¹³C-methyl-labeled metabolic precursors to enable the isotopic labeling of Ile, Val, and Leu methyl groups.Recently, similar methodology has been applied to enable ¹³C-methyl labeling of Ile, Val, and Leu in yeast,extending the approach to proteins that do not readily fold when produced in bacteria. Mammalian or insect cells are nonetheless preferable for production of many human proteins, yet ¹³C-methyl labeling using similar metabolic precursors is not feasible as these cells lack the requisite biosynthetic machinery. Herein, we report versatile and high-yielding synthetic routes to ¹³C methyl-labeled amino acids based on palladium-catalyzed C(sp3)-H functionalization. We demonstrate the efficient incorporation of two of the synthesized amino acids, ¹³C-g2-Ile and ¹³C-g1,g2-Val, into human receptor extracellular domains with multiple disulfides using suspension-cultured HEK293 cells. Production costs are reasonable, even at moderate expression levels of 2–3 mg purified protein per liter of medium, and the method can be extended to label other methyl groups, such as ¹³C-d1-Ile and ¹³C-d1,d2-Leu. In summary, we demonstrate the cost-effective production of methyl-labeled proteins in mammalian cells by incorporation of ¹³C methyl-labeled amino acids generated de novo by a versatile synthetic route.

Abstract: Decades ago, Ackerman et al. demonstrated the ability to measure cerebral blood flow in vivo using deuterium (²H) MRS or imaging combined with exogenous deuterated water (D₂O) as a freely diffusible tracer.¹ In 2011, Mateescu et al. reported the feasibility of measuring deuterium-labeled glucose and metabolically produced deuterated water (HDO) in the mouse head using ²H MRS and uniformly deuterated glucose as an substrate.² In 2014, we presented the first rat brain ²H MRS study at 16.4T that measured the deuterium-labeled glucose (Glc), mixed glutamate and glutamine (Glx), and lactate (Lac) with excellent SNR and temporal resolution after an IV administration of deuterated D-Glucose-6,6-d₂ (d66), and demonstrated the feasibility of simultaneously determining the cerebral metabolic rates of glucose consumption (CMRGlc) and TCA cycle (VTAC) using a kinetic model and the dynamic changes of [Glx] and [Glc] in the rat brain.^3,4 In 2016, we reported the first 3D ²H MRS imaging (DMRSI) study of rat brain tumors, which used the [Lac]/[Glx] ratio as a sensitive marker of the “Warburg effect” associated with cancer biology, showing excellent contrast between brain tumors and normal appearing brain tissues.^5,6 Since then the ²H MRS/DMRSI or DMI technique has been applied to study various tumors in preclinical models or human brains.^7-10 Recently, we have employed the advanced subspace-based denoising and machine learning methods to largely improve the SNR and spatial-temporal resolution of the DMRSI, and made it possible to map intra-tumor heterogeneity.^11,12 Furthermore, we reported for the first time the possibility of mapping three key metabolic rates of CMRGlc, VTAC, and lactate production rate (CMRLac) using high-resolution dynamic DMRSI covering the entire human brain at 7T.¹³ In summary, DMRSI is emerging as an important metabolic imaging modality with significant merits compared to other imaging methods. It is becoming an important tool for studying metabolic reprograming between glycolytic and oxidative metabolism in healthy brain, brain aging and many brain disorders including cancer, stroke and neurodegeneration, and it has a potential for translation.

Abstract: The ongoing discoveries of regulatory RNAs with diverse activities in gene expression and regulation have transformed our view of RNA’s functions in cellular physiology and disease. Despite this progress, a critical gap remains in elucidating the mechanisms underlying RNA activities, where these highly dynamic molecules constantly morph between alternative conformations, each triggered by specific cellular signals. These conformational transitions can occur across a wide range of timescales, from picoseconds to seconds and beyond; yet, conventional static structures convey little of this dynamic nature of RNA, which is crucial for orchestrating their cellular activities. Hence, to fully understand RNA biology, we need to reimagine RNA structural biology, where RNAs are viewed as dynamic ensembles characterized by probability distributions of fluctuating conformations, each with a distinct lifetime. In this presentation, I will discuss our recent progress in uncovering such an ensemble perspective of RNA molecules toward a quantitative and predictive framework for understanding how RNAs harness conformational dynamics to drive cellular activities.

Abstract: Over 300 million years spiders have evolved to produce seven different types of silk. The silks are comprised almost entirely of protein and are used for a diverse range of applications including web construction, egg case production and wrapping prey. The silks vary dramatically in their mechanical and physical properties with the major ampullate silk (dragline) exhibiting a strength that exceeds steel by weight and a toughness greater than Kevlar while, flagelliform silk has an elasticity comparable to rubber. Our lab is focused on understanding the molecular structure and dynamics of the proteins that comprise the various spider silk fibers with MAS solid-state NMR. It is the folded structures and hierarchical organization of these proteins that imparts spider silks their impressive yet, diverse mechanical properties. Our research team has been developing and applying SSNMR to probe secondary structure, hydrogen-bonding, side chain dynamics, and oligomeric protein assembly all of which are crucial to understanding spider silk formation and the resulting fiber properties. Recently, we have focused on using solution NMR to understand the protein-rich fluid within the various silk producing glands to investigate the conformational structure and dynamics prior to fiber formation and determine the important biochemical triggers responsible for converting this hydrogel-like protein solution to fibers with unparalleled properties. It is our belief that a better fundamental understanding of spider silk protein structure and assembly process will accelerate the ability to mimic and reproduce similar biologically inspired materials in the lab. These NMR approaches all require isotopic enrichment (¹³C/¹⁵N) that are administered to the spider in their water supply. I will discuss how we do it, the types of NMR experiments it enables and the molecular information gained in the context of silk formation.

Abstract: Several different types of vaccines are regularly examined in the pharmaceutical industry. Vaccines can contain large molecules such as proteins and polysaccharides or larger particles such as attenuated viruses, lipid nanoparticles or virus like particles. All these vaccine types contain a complex mixture of small and macromolecules, therefore several analytical tools often need to be assessed in order to assist in the various stages of vaccine process development. Benchtop NMR is an advancing technology that can be a versatile analytical tool that provides quantitative information about the multiple components, using a single internal standard, that arise in the development of a vaccine. Benchtop NMR often involves minimal sample preparation since macromolecular species are usually not detectable or can be removed by T2 filters. The software for benchtop NMR is easy to use and provides automatic data processing further making benchtop NMR an attractive analytical tool that can be quickly assessed when comparing different analytical approaches. In this presentation the use of benchtop NMR as an alternative to chromatographic methods will be discussed. Its use for real-time monitoring and fast method development will be demonstrated. Examples of using benchtop NMR to quickly determine solvent compositions, to confirm excipients and study alum sedimentation in vaccine-related samples are some of the examples that will be covered, with some comparison to chromatography-based methods. Furthermore, we will discuss how benchtop NMR method development is possible even from those with no NMR experience, which are clear advantages for the implementation of benchtop NMR in the pharmaceutical industry.

Abstract: Pediatric metabolic disease is increasing at an astronomical rate, in line with the obesity epidemic. Understanding the underlying pathology, in particular, tissue specific insulin resistance and substrate metabolism is critical for the development of new therapeutics. Intravenous and oral tracers can be utilized in different settings: fasting, with hyperinsulinemic-euglycemic clamps or oral glucose tolerance tests to understand metabolism under different metabolic states. Combining studies in youth with different methodologies and tracers, and with clinical interventions is leading towards new therapeutic targets.

Abstract: Cell culture media (CCM) optimization is a critical step during the development and scale up of biotherapeutic production. In particular, the emphasis on quality by design has made it necessary to understand how the components of CCM change during production and how these changes relate to product quality. There is a vital need to develop analytical assays that can provide comprehensive, yet accurate, CCM profiling for a wide range of biotherapeutic types produced from, or are themselves, living cells. Herein, we present a robust method that allows commendable retention and separation of an excess of 110 compounds spanning a multitude of metabolic classes. By using the MSK-QReSS (Quantification, Retention, and System Suitability) kit containing isotope-enriched ¹³C and ¹⁵N metabolite mixes, the developed method enabled the level of key metabolites in culture, as well as spent media, to be relatively quantified. Through statistical analysis, metabolite levels are visualized to understand similarities and differences throughout various manufacturing stages and provide clear indication of CCM components behaviors during biotherapeutic production.

Abstract: The dystrophin associated protein complex (DAPC) is an important glycoprotein complex that helps to maintain the membrane stability of muscle fiber sarcolemma. Central to this complex is the protein dystrophin which anchors the DAPC to the actin cytoskeleton of the cell. In Duchenne muscular dystrophy (DMD) this protein is absent, leading to fiber degradation, muscle atrophy, and loss of ambulation by age 12. Current FDA-approved therapies aim to restore dystrophin expression in patient tissue, but the amount and function of restored dystrophins are not well characterized. To address this, we used two different SILAC strategies to improve our current understanding of the interaction between dystrophin and the DAPC. In one study we used a pulse-chase SILAC labeling strategy to study the DAPC protein turnover in mdx mice treated with an exon-skipping therapy to restore dystrophin expression. In a second study we spiked human muscle lysate with SILAC-labeled myotubes in order to quantify the DAPC in patients with a milder form of muscular dystrophy where dystrophin is present in varying decreased amounts. By using these two different SILAC strategies we were able to better characterize the role dystrophin amount plays in the stability of the DAPC. This greater understanding of the complex will help to explain the efficacy of current dystrophin replacement therapies, as well as aid the designing of newer ones.

Abstract: Metabolomics has demonstrated the ability to measure hundreds of metabolites in diverse sample types. Nonetheless, data analysis remains a key bottleneck in targeted and nontargeted approaches. For example, system suitability testing (SST) has been widely adopted as common practice in metabolomics but human intervention is often required to accept or reject system suitability. After experiments have been run, data quality assessment is often made by somewhat arbitrary and manual approaches, resulting in inconsistent results and wasted time. Finally, errors in metabolite/peak assignment require arduous manual curation. Heavily utilizing stable isotope-labeled internal standards (SIL-IS), we have developed a novel Windows application which automates instrument orchestration, SST interrogation, raw data QC, and the quantitative data analysis pipeline for microchip CE-MS metabolomics. This presentation will focus on the use of SIL-IS for migration time indexing, rapid data quality checking, and use in automated correction of common peak-selection errors made by metabolomics software.

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

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

Abstract: Nuclear magnetic resonance (NMR) spectroscopy is a valuable tool used to gather information regarding the structure and dynamics of protein and/or nucleic acids at the atomic level. Determination of the three-dimensional structure of these such macromolecules and their complexes is vital for rational drug design and expanding knowledge within the field of mechanistic biology. Here, we briefly describe the benefits and importance of stable isotopes in NMR-based research, as well as highlight the common reagents and labeling schemes for enriching protein and nucleic acids for most types of NMR investigations. Additionally, we will present some new isotopically labeled offerings in this space.

Abstract: In my contribution, I will discuss key lessons learnt in the context of the global Covid19-NMR project. In the context of this project, we assigned 20 RNAs derived from the untranslated parts of SARS-CoV-2. They range in size from 20 – 80 nucleotides (Wacker, Weigand et al., 2020). In this process, we used multiple samples with various labeling pattern. A general pipeline will be discussed. The assignment forms the basis for screening of fragments towards binding of RNA.

Abstract: I will discuss recent advances in MAS NMR methods for atomic-resolution structural analysis of large biological assemblies. Using examples of distinct systems studied by our lab, I will illustrate the unique information revealed by MAS NMR about atomic-level 3D structures and drug binding, inaccessible by other techniques. I will demonstrate the power of integrating MAS NMR with medium-resolution cryo-EM and data-driven MD simulations, and discuss the challenges and opportunities for magnetic resonance in integrative structural biology. I will talk about the isotope labeling strategies and needs for NMR-based structural biology.

Abstract: Fatty liver disease is commonly associated with disrupted fatty acid oxidation. While experiments measuring hepatic TCA cycle turnover using ¹³C-labeled substrates have produced incongruous results, none of the methods have directly assayed β-oxidation per se. Here we demonstrate that in the perfused mouse liver, a common model of hepatic metabolism, administration of [D₁₅]octanoate results in the production of partially deuterated water (HDO). HDO is produced at multiple steps of β-oxidation and its generation produces a linear correlation with the O₂ consumption of the liver. Using isotopomer network compartmental analysis (INCA), we produce a quantitative model liver metabolism.

Abstract: This presentation will cover essential components for quantitative NMR analysis, and its application to pharmaceutical R&D.

Abstract: The impact of NMR for studies of proteins has skyrocketed from the first recording of a ¹D NMR spectrum of ribonuclease in 1957 to NMR structures of proteins, high resolution multiple resonance experiments at high field and structures of large proteins and protein complexes in solution. A key step was introduction of ¹⁵N and ¹³C labeling by bacterial expression in the late 1980s, and the demonstration of the benefits of perdeuteration on spectral resolution, and the impact of methyl TROSY. Over the recent years we focused on a judicious optimizing expression precursors for more efficient backbone assignments, ¹⁵N detection, aromatic carbon TROSY and ¹⁹F-¹³C TROSYs. Need for suitable isotope-labeled precursors will be presented.

Abstract: The rapidly growing area of metabolomics, in which a large number of metabolites in biological mixtures are analyzed in one step, promises immense potential for numerous areas of basic and applied sciences. Because of its unique ability to detect metabolites in intact biological mixtures as well as live systems in real-time, reproducibly and quantitatively, nuclear magnetic resonance (NMR) spectroscopy has emerged as one of the most powerful analytical techniques in metabolomics. NMR spectroscopy combined with isotope labeling of metabolites has dramatically improved our ability to trace important metabolic pathways as well as quantitatively analyze metabolites. Increased sensitivity and selectivity achieved through isotope labeling have paved novel avenues to unravel biological complexity and gain insights into cellular functions in health and disease conditions. In this presentation, some of the results from ex vivo and in vitro studies made using isotope-labeled compounds will be discussed.

Abstract: NMR spectroscopy of large RNAs has traditionally been hindered by unfavorable relaxation properties and severe spectral overlap. I will discuss the use of partial deuterium labeling to facilitate resonance assignments and structure determination of large RNAs.

Abstract: Mass spectrometry (MS)-based methods can return degrees of variability that impacts the rigor and reproducibility of the acquired results. To help monitor this potential issue, quality control (QC) measures must be entrenched in experimental designs and analytical workflows. The type and placement of QC samples in a batch sequence are important considerations to the assessment process. Here, we describe a systematic approach to designing a MS-based QC workflow utilizing different types of QC samples (with and without isotopically labeled QReSS standards) positioned around a metabolomics study application. The guidelines and lessons gleaned will be presented along with insights into the significance of QCs in MS ‘omics studies.

Abstract: In-depth coverage of highly complex protein mixtures such as plasma, cell and tissue proteomes, in discovery studies has become routine because of advances in liquid chromatography and mass spectrometry instrumentation. However, accurate quantitation of proteins requires targeted approaches such multiple or parallel reaction monitoring (MRM/PRM) and the use of heavy isotope labeled versions of peptides/proteins. I will describe how we have used isotopically labeled standards including the SISCAPA (stable isotope standards and capture with anti-peptide antibody) approach in our studies to develop mass spectrometry-based assays for SARS-CoV-2 viral antigens, variant peptides and host response proteins. Finally, I will also discuss development of novel assays involving triggered acquisition based on heavy spiked-in peptides using the SureQuant approach.

Abstract: Targeted analysis methods for chemicals in environmental media are the backbone of monitoring studies. These methods are highly multi-laboratory validated and time-tested approaches for a host of environmental studies. However, there are shortcomings to this approach. The chosen methods only monitor for a select set of analytes. High-resolution mass spectrometry (HRMS) instrumentation (such as quadrupole time of flight and orbital ion trap systems) partially fills this void by allowing the analysis of chemicals without any preconceived notion of what is in the sample being analyzed. This is done through two main approaches: 1) suspect screening—screening for chemicals in a large list, or 2) non-targeted screening—true novel compound discovery and structure elucidation. This approach is not without its own deficiencies as well. The selected extraction techniques and instrument used (such as GC vs LC) bias the possible detected analytes and is not comprehensive. Additional challenges include generally more expensive instrumentation, more laborious data processing and mining techniques, extremely large data sets, and often a lack of chemical standards for compound confirmation and quantitation. An added benefit of HRMS analysis is that data of previously run samples can be mined in future efforts to explore chemicals that were either unknown at the time of analysis or were overlooked. Non-targeted methods should inform analysts about what chemicals should be added to targeted analysis methods, not take the place of them. This talk will present approaches used in the discovery of xenobiotic PFAS chemicals in environmental media using HRMS application.

Abstract: Bile is a liver-produced secretion that is stored in the gallbladder. Bile contains a broad constellation of bile acids (BAs). The liver synthesizes primary BAs that can be further modified by intestinal microbes to form secondary BAs that can exist in non-conjugated, and amino acid conjugated forms. The amphipathic properties of BAs aid in emulsification of ingested food, absorption of fat-soluble vitamins, and digestion of dietary lipids in the gut. BAs also serve as signaling compounds by which host and intestinal microbes communicate along the gut-liver-axis. Due to the physicochemical properties of bile acids/salts (i.e., hydrophobic and ionizable), the application of reverse-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based methods are ideally suited for the measurement of these compounds in a number of biofluids and tissues often surveyed in biomedical research. Recently, the molecular motor Myosin Vb has been implicated in a variety of cholestatic liver disorders that arise from an inability to properly secrete bile acids. In this presentation we will present our findings of altered bile acid concentrations and changes in proteins in the liver and ileum of mice lacking the molecular motor Myosin Vb.

During this webinar, you will learn about:

• Environmental chemistry would not be what it is today without isotopically labeled analogues.
• There is so much focus on LOQ and without isotope dilution, these data would not hold the same confidence.
• There will always be the “next” dioxin or PFAS or emerging contaminant.

Abstract: Skeletal muscle protein is constantly being synthesized and broken down, with a turnover rate of about 1-2% per day. The rate of skeletal muscle protein synthesis is regulated by two main metabolic stimuli, food intake and physical activity. Using stable isotope-labeled amino acid tracers and/or the use of specifically produced, intrinsically labeled protein we can study post-prandial protein handling and muscle protein synthesis rates in vivo in humans. Ingestion of a single meal-like amount of protein allows ~55% of the protein derived amino acids to become available in the circulation, with approximately 20% of the protein derived plasma amino acids taken up in skeletal muscle tissue during a 5 h post-prandial period, thereby stimulating muscle protein synthesis rates and providing precursors for de novo muscle protein. In conclusion “you are what you just ate.”

Abstract: I will describe our recent discovery of intracellular long-lived proteins using metabolic pulse-chase labeling of rodents and proteomic analysis. First, I will summarize our finding that a subset of the mitochondrial proteome unexpectedly persists for months selectively in tissues enriched with long-lived post-mitotic cells. Second, I will describe how we used a similar strategy to identify synaptic vesicle associated proteins with impaired degradation during the onset of amyloid pathology in mouse models of Alzheimer’s disease and how this has advanced our understanding.

Abstract: Homocystinuria (HCU) results from the inability to convert homocysteine to cystathionine, due to enzymatic or vitamin B12 deficiencies, causing an elevation of total homocysteine (tHcy) in the blood and urine. Methionine is used as a primary-tier marker for HCU, but methionine concentrations physiologically vary in newborn blood which impacts false positive rates. Furthermore, in HCU patients, methionine concentrations may take a few days after birth to be elevated above cutoffs which leads to false negatives (dried blood spot collection takes place 24-48 hours after birth).  Here we present a breakthrough method to selectively derivatize the Hcy thiol function group to successfully multiplex tHcy into primary-tier assays with amino acids, acylcarnitines, succinylacetone, adenosine, deoxyadenosine, creatine, creatinine, and guanidinoacetic acid for analysis by FIA-MS/MS.

Abstract: To improve the process efficiency and to minimize errors in the processing of newborn dried blood spot samples, isotopically labeled compounds are imperative and are procedurally inserted as internal standards. To facilitate targeted screening assays, the Utah newborn screening laboratory contracted a manufacturer to prepare a custom amino acid isotope mix. This dried down mix consists of three disorder-relevant amino acids that can be easily reconstituted in 1 mL of solvent, eliminating the need to weigh and dilute the individual compounds prior to use. The pre-formulated mix helped increase the throughput, accuracy, and reproducibility of internal standard preparation while maintaining the desired assay sensitivity. This presentation will discuss the analytical development and performance of the amino acid isotope mix in the context of newborn screening applications.

Abstract: To be presented is an overview of how CIL’s NSK-A and NSK-B2-UK isotopically labelled mixes are used to screen metabolic conditions (e.g., PKU, MSUD, IVA, GA-1, MCAD, HCU) in the UK by tandem MS/MS. A summary of our complete pipeline will be described stepwise. This process includes ordering, validation, stock and internal standard preparation, analysis, result review, and reporting. An overview of the screening pathway (i.e., initial analysis, retest, follow-up sample analysis, and referrals) will also be discussed along with case examples from our NBS patient applications.

Abstract: Metabolomics is a series of systematic analysis of endogenous metabolites in biological samples. To improve the clinical course of diseases, prevention and precision, clinical diagnostics are desired. In order to achieve this, metabolomics offers new opportunities for biomarker discovery in complex diseases and may provide pathological understanding of diseases beyond traditional clinical diagnostics. It is critical to have reliable and consistent data for product development. This presentation will highlight the opportunities and challenges of metabolomics biomarkers discovery and development in clinical diagnostics.

Abstract: In this seminar I will discuss three topics relevant to our latest efforts to develop mass spectrometric technologies for proteome analysis. First, I will present a multi-protease strategy that provides the deepest coverage of the human proteome to date with detection of over 17,000 gene products. By use of various enzymes and MS/MS dissociation technologies we obtain extremely high sequence coverage allowing for a global mapping of alternative splicing on the proteome level. Second, I will present on the use of an IR laser, coupled with ion parking in a quadrupole linear ion trap, to double the production of TMT reporter ions for quantitative proteomic experiments. This approach boosts the number of quantifiable peptides in a global experiment and overall improves the quantitative accuracy and precision. Finally, I will describe new approaches to soft landing intact protein complexes onto transmission electron microscope grids for structural analysis. This approach offers a direct path to connect the nascent field of native MS to cryoEM.

Abstract: An estimated 248,530 American men will be diagnosed with prostate cancer in 2021, and roughly 34,000 will die. As with many malignancies, the impact of a urologic cancer on a patient’s life expectancy and quality of life is largely based upon cancer stage. Early-stage prostate cancer (stage I and II) is associated with excellent cancer-specific outcomes, and tremendous emphasis is placed on limiting treatment-related morbidity and preserving quality of life. However, several challenges must be addressed to avoid overtreatment and unnecessary testing. A reliable assessment of early-stage prostate cancer is vital for identifying suitable treatment options, as is a diagnostic test that has biomarkers specific to prostate. One of the failures of biomarker translation to clinical practice is utilizing specimens that do not represent the broad clinical phenotype present in a patient population for biomarker identification and testing. Thus, a true biomarker validation study should include patients with a wide spectrum of urologic conditions so the true accuracy and precision of the biomarker can be assessed. Metabolites represent the closest aspect to phenotype because they are utilized in healthy and disease processes. In fact, many current clinical tests rely on metabolites for health diagnostics (i.e. comprehensive metabolic panel). Our preliminary metabolomics show that urine biomarkers can identify prostate cancer and calibrate Gleason score (severity), while also differentiating prostate cancer from prostatitis, BPH, bladder cancer, and kidney cancer.

Abstract: While the selectivity and specificity of LC-MS/MS methods have become increasingly powerful for feature annotation in untargeted analyses, previous studies have shown that in metabolomics analyses only a small percentage of detected features are actually metabolites occurring from the system and many features result from in-source fragments, multimers, or other artifacts of the MS experiment. This presentation will illustrate two different small molecule analysis pipelines utilized by our group to investigate features from untargeted studies. First, I will demonstrate how combining liquid chromatography, ion mobility spectrometry, and tandem mass spectrometry (LC-IMS-MS/MS) separations with isotopologue workflows enables the detection, identification, and validation of features associated with metabolism. Next, I will showcase how we utilize standards and the LC-IMS-MS/MS measurements to create multidimensional libraries providing additional confidence to our feature annotations. I will then apply our bile acid library containing >200 molecular entries to examine how novel bile acid conjugates change due to specific system perturbations.

Welcome to Isotope Day, a virtual event showcasing cutting-edge applications of stable isotopes in nuclear magnetic resonance (NMR) and mass spectrometry research. This event brings together 12 experts in their fields to share insights and strengthen your understanding of stable isotopes in research. The morning session, moderated by Kevin Millis from Cambridge Isotope Laboratories (CIL), focuses on NMR applications. Speakers include Bob Griffin, Jan Marchant, Hari Arthanari, Lewis Kay, and Christoffer Laustsen. After a live trivia break with prizes, the afternoon session turns to mass spectrometry-based techniques. Moderated by Andrew Percy from CIL, this session features talks by Matthew Vander Heiden, David Muddiman, Lingjun Li, John Yates, and Gary Patti. Throughout the day, you’ll have opportunities to engage with speakers, participate in Q&A sessions, and explore how stable isotopes can advance your research. Whether you’re a seasoned user or new to isotopic applications, this event aims to educate, engage, and inspire. Cambridge Isotope Laboratories is the world’s leading stable isotope company, dedicated to supporting researchers with a vast product range and unparalleled expertise in stable isotope chemistry. Learn more about CIL’s capabilities and how they can partner with you in your research journey. This video includes closed captions for accessibility. Abstracts, speaker bios, and recorded Q&A sessions are available via the link provided in the video description. Thank you for joining Isotope Day. We hope you find the presentations informative and applicable to your work. Please reach out to CIL to explore how stable isotopes can empower your research.

Abstract: Stable Isotopes in Biomolecular Nuclear Magnetic Resonance Research: A Critical Technology. Most communications that pertain to NMR-based research on biopolymers, such as protein or nucleic acids, will devote no more than a few words, if any at all, to describe the isotope labeling scheme or methods used in producing the molecules under study. Although it is true that enriching biopolymers with stable isotopes, such as ¹³C, ¹⁵N, and ²H, is not always needed, as in the case for short-stranded peptides and nucleic acids, it is more often the case than not that samples do in fact require enrichment with at least a subset of these three stable isotopes. Enrichment allows for correlations to be measured, which are crucial for the determination of inter-atomic distances, torsion angles, and relative orientations of domains within the molecule. Enrichment also can be critical to obtain an isolated spin system for which dynamics can be investigated, for an assessment of protein folding, for detection of signals in large proteins and supramolecular complexes, and for ¹⁵N-based chemical shift perturbation experiments. This short talk will highlight the common reagents and labeling schemes for enriching protein and nucleic acids for most types of NMR investigations.

Abstract: Many peptides and proteins form amyloid fibrils whose detailed molecular structure is of considerable functional and/or pathological importance. For example, amyloid is closely associated with the neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases. In this presentation we review the macroscopic structural properties of fibrils and outline approaches to determining the microscopic structure of these systems to atomic resolution using magic angle spinning (MAS) NMR in combination with cryoEM. In particular, we discuss a series of 2D and 3D heteronuclear and homonuclear dipole recoupling experiments involving spectral assignments and distance and torsion angle measurements aimed at accomplishing this goal. Key to obtaining high resolution is the ability to measure a sufficient number of NMR structural constraints (¹³C-¹³C and ¹³C-¹⁵N distances and torsion angles per residue). We discuss the structures of different systems determined using these approaches but focusing on (1) fibrils formed by Aβ1-42, the toxic species in Alzheimer’ discases, using a set of >500 distance constraints; and (2) a structure of fibrils forned by β2-microglobulin, the 99 amino acid protein associated with dialysis related amylosis, using ~1200 constraints. The spectra also provide information on the arrangement of the monomers in the strands that form sheets, and the sheets that ultimately form the fibrils. Contrary to conventional wisdom, the spectral data indicate that the molecules in the fibril are microscopically well ordered.

Abstract: The application of NMR spectroscopy to the study of large, biologically relevant RNAs is complicated by a number of factors, including limited chemical shift dispersion, undesirable relaxation parameters and a relative lack of long-range distance constraints. This talk will describe a number of NMR approaches we use to mitigate these difficulties, with a focus on nucleotide-specific deuterium labeling schemes and multiple bond heteronuclear couplings.

Abstract: Solution NMR has the unique capacity to determine structures of proteins and characterize their dynamic states at an atomic resolution. With the maturity of X-ray crystallography and the emergence of cryo-EM, the power of NMR lies in complementing structural information with dynamics and characterizing transient interactions. Such studies have profound implications for protein function and therapeutic drug design. The starting point for most NMR protein investigations is “sequence specific resonance assignment” – matching each observed resonance peak to a particular nucleus in the protein. Currently, resonance assignment of large proteins (> 25 kDa) demands long hours of expert analysis. Studies of proteins with molecular weights above 50 kDa are not routine given two predominant challenges: i) Larger proteins produce more peaks, leading to overlap and degeneracy ii) Large proteins suffer rapid relaxation, which broadens the peaks and diminishes both sensitivity and resolution – especially for experiments with multiple lengthy delays for transfer of magnetization. The HNCA is the most sensitive triple resonance experiment that provides sequential connectivity for resonance assignment. Degeneracy of Cα chemical shifts impedes the complete assignment of large proteins. However, HNCACB and HNCACO, which are typically used to resolve the ambiguities, have poor sensitivity for large systems. We use a mixed pyruvate labeling strategy to modulate the isotope-environment of different amino acids, producing unique signature peak shapes. We design tailored ¹³C-homonuclear decoupling pulses to generate fingerprint patterns of CO and Cβ resonances directly in the observed Cα peak patterns, suitable for pyruvate or traditional isotope labeled samples. Cβ and CO information will then be collected using the superior resolution and sensitivity of the HNCA.

Abstract: Over the past four decades solution NMR spectroscopy has made huge advances both in terms of the biochemical problems that can be explored as well as the quantitative nature of investigations that can be performed. The use of stable isotopes has been absolutely critical in this process, certainly as important as advances in spectrometer hardware and software, and improvements to NMR experiments that continue to evolve. I will present an overview of methyl labeling as applied to both proteins and DNA, focusing on the nucleosome core particle, the 220 kDa building block of chromatin. Examples from NMR spin relaxation studies of invisible protein states will also be presented, showing that different applications are best performed with different labeling strategies.

Abstract: In this presentation I will introduce hyperpolarized carbon ¹³C MRI and DMI and discuss the translation and consideration that goes into a good metabolic MRI biomarker.

Abstract: Stable isotope standards provide valuable experimental and informatic solutions to improving the validity of qualitative / quantitative determinations in analytical and diagnostic science. To help enable routine implementation, CIL offers a broad and diverse collection of stable isotope-labeled standards in their individual and mixture forms. Applications of these are innumerable, ranging from exploratory ‘omics research to modern biomedicine. This presentation will provide an overview into example applications of isotopically labeled standards (and their mixtures) in MS-based measurements.

Abstract: Complex regulatory mechanisms enable cell metabolism to match physiological state. The major pathways cells use to turn nutrients into energy and to synthesize macromolecules have been elucidated; however, there remain many unanswered questions regarding how metabolism supports cancer cell proliferation and thus how best to target metabolism for cancer treatment. By tracing the fate of isotope labeled nutrients in different cancer contexts, we are working to identify how different cancers use metabolism differently to grow. This includes tracing nutrient use into stable biomass to assess differential pathway use by different cancer cells in tumors, as well as isotope tracing to uncover whether cells use synthesis or salvage pathways to acquire nucleotides.

Abstract: Mass spectrometry offers a versatile and robust platform to discover and subsequently quantify new diagnostic, prognostic, and therapeutic biomarkers for disease as well as understand the role of the environment (exposure) on human health. This presentation will cover two main topics: 1) a CE-MS approach related to human exposure to environmental neurotoxins as well as new discoveries, followed by translation of those findings to a new even more rapid MS-platform; and 2) novel methods for the quantification of per- and polyfluoroalkyl substances (PFAS) in human serum.

Abstract: Recent advances in mass spectrometry (MS) have made MS-based omics a central technology for biomedical research. Quantification of proteins, peptides and metabolites present in complex biological systems is often key to understanding dynamic changes of many essential physiological and pathological processes. Chemical labeling with multiplex isobaric tags offers an effective strategy for parallel comparative analyses of many samples during liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. In this presentation, I will present our recent progress on the design and development of several novel chemical tags, including dimethylated leucine (DiLeu) isobaric tagging reagents, which offer cost-effective implementations that enable higher orders of multiplexing. The utilities of these novel chemical tags are further demonstrated through their application in the study of targeted proteomic and glycoproteomic changes in Alzheimer’s disease. Additionally, we report on a multiplexed quantification method for simultaneous proteomics and amine metabolomics analyses via nanoflow reversed phase LC-MS/MS, exploiting mass defect-based DiLeu (mdDiLeu) labeling. Paralleled proteomics and amine metabolomics analyses using mdDiLeu will be presented for application to pancreatic cancer cells. Collectively, we present a versatile chemical tagging toolbox enabled by high solution FTMS platform for system-wide omics studies.

Abstract: Identifying molecular changes associated with disease is a major challenge and to do so at the earliest time point prior to pathology is desired. At early time points, however, molecular changes may be small and difficult to identify hidden by the overwhelming static proteome. The second method is to measure protein degradation in tissues. Traditional methods of using stable isotope labeled amino acids is complicated by a decreasing signal in a high background. One solution we are exploring is to pulse in azidohomoalanine (AHA) into an animal model and then use click chemistry to enrich labeled peptides at various timepoints to plot degradation curves. Azidohomoalanine (AHA) is a modified methionine that is accepted by the endogenous methionine tRNA and inserted into proteins in vivo. AHA can be covalently linked to a biotin alkyne through click chemistry. Thus, AHA proteins or peptides can be enriched and efficiently separated from the whole proteome through avidin bead enrichment. Newly synthesized proteins (NSP) within a discrete time period in conjunction with the development of disease can be identified using this method. We’ve also developed sophisticated software tools to analyze the data.

Abstract: Stable isotopes are a cornerstone of metabolic research, with applications ranging from quantitation to flux analysis. This presentation will outline three different use cases of stable isotopes in mass spectrometry-based metabolomics. First, an experimental approach called credentialing will be discussed as a strategy for data reduction. Given that only peaks derived from biological compounds can become isotopically labeled, credentialing enables the annotation of signals in metabolomics data that correspond to contaminants and artifacts. Second, an application of stable isotopes to mammalian cell culture will be described. Rapidly dividing cells will be compared to quiescent cells to demonstrate metabolic fluxes that change in support of proliferation. Finally, third, experimental strategies for performing isotope-tracer analysis in animals will be reviewed. An example of metabolic crosstalk, where molecules are exchanged between different tissues, will be highlighted.

Join us for Isotope Day 2021, a virtual conference featuring renowned researchers discussing the latest advances in stable isotope applications. From preclinical disease models to real-world implications, our expert speakers delve into the breadth and depth of isotope usage in heart failure, Alzheimer’s, ALS, cancer, and beyond. Presented by Cambridge Isotope Laboratories (CIL), this conference aims to share new insights, lessons, and techniques to enhance your research. Learn how CIL supports the evolving demands of researchers across various fields. Conference Highlights: -Explore cutting-edge isotope applications in preclinical and clinical scenarios -Gain insights into disease models, including heart failure, Alzheimer’s, ALS, and cancer -Discover how isotopes are used in real-world research -Learn about CIL’s commitment to supporting researchers A Special Thank You: To our participants, speakers, moderators, and the entire CIL team for making this event possible.

Participants will:

• Understand necessary components of a successful MS-based metablomics experiment
• Learn technologies that improve throughput of identifications, as well as characterize the pathway, function, and localization of metabolites
• Discover a new benchmarking approach, called credentialing, for removal of artifactual features

Sponsored by Thermo Scientific

Dr. Percy will discuss:

• Method development
– Overview
– Target selection
– Significance of standards
– Workflow and examples
• Quantitative proteomic kits
– Products: PeptiQuant™ and ProteusQC™
– Utility
– Applications

Sponsored by Agilent

Dr. Borchers will discuss:

• Applications of multiple-reaction monitoring (MRM) mass spectrometry
• Systematic development of MRM assays
• Key advantages of MRM assays

Sponsored by CIL

Dr. Muddiman will discuss:

• Background on Glycans
• The Isomer Barrier
• Ionization Methods and Mass Analyzers
• Chemical Tagging
• Separation Modes
• and Future Directions

Sponsored by CIL