2022 Webinars

 NMR Focus 

CIL Products for Biomolecular NMR

Kelly Andrade | Assistant Product Manager | Cambridge Isotope Laboratories, Inc. (USA)

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.

A transcript of this presentation in English is available here.

Rapid RNA NMR Assignment Process

Harald Schwalbe, PhD | Professor | Jaohann Wolfgang Goethe-Universität, Institut für Organische Chemie und Chemische Biologie (Germany)

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.

A transcript of this presentation in English is available here.

Integrative Structural Biology of Protein Assemblies: Challenges and Opportunities for Magnetic Resonance

Tatyana Polenova, PhD | Professor | University of Delaware (USA)

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.

Measuring β-Oxidation in the Perfused Liver with [D₁₅]Octanoate

Matthew Merritt, PhD | Associate Professor | University of Florida (USA)

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.

A transcript of this presentation in English is available here.

Examples of Quantitative NMR Analyses in Pharmaceutical Industry

Qiuwei Xu, PhD | Senior Principal Scientist | Merck (USA)

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

A transcript of this presentation in English is available here.

The Power of Isotope Labeling NMR of Proteins

Gerhard Wagner, PhD | Professor | Harvard Medical School (USA)

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.

A transcript of this presentation in English is available here.

Isotope Labeling in NMR Spectroscopy-Based Metabolomics

G.A. Nagana Gowda, PhD | Research Associate Professor | University of Washington (USA)

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.

A transcript of this presentation in English is available here.

Structure Determination of Large RNAs by NMR

Sarah Keane, PhD | Assistant Professor | University of Michigan (USA)

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.

 Mass Spectrometry Focus 

Guidelines, Considerations, and Benefits of Incorporating QCs into MS ‘Omics Experimental Practices

Andrew Percy, PhD | MS 'Omics Product Manager | Cambridge Isotope Laboratories, Inc. (USA)

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.

Heavy Isotopes for Discovery and Clinical Assay Development

Akhilesh Pandey, MD, PhD | Professor | Mayo Clinic (USA)

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.

The Discovery of Novel PFAS in Environmental Samples Using High Resolution Mass Spectrometry

Mark Strynar, PhD | Physical Scientist | Office of Research and Development of the U.S. Environmental Protection Agency (USA)

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.

A transcript of this presentation in English is available here.

A Targeted Metabolomics Approach for Quantifying a Broad Constellation of Bile Acids in Biomedical Research

Amy Engevik, PhD | Assistant Professor | The Medical University of South Carolina (USA)

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.

Use of ¹³C- Labeled Internal Standards for Accurate Determination of 6PPD Quinone

Andrew Patterson | Technical Director | Eurofins Environment Testing America (USA)

During this webinar, you will learn about:

A transcript of this presentation in English is available here.

Post-prandial Protein Handling: ‘You Are What You Eat’

Luc van Loon, PhD | Professor | Maastricht University (The Netherlands)

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.”

The Role of Long-Lived Proteins in Health and Neurodegeneration

Jeffery Savas, PhD | Assistant Professor | Northwestern University, Feinberg School of Medicine (USA)

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.

A transcript of this presentation in English is available here.