Thank you for joining us for Isotope Days 2023 – a three-part series of webinars, all featuring science facilitated by stable isotopes. During this series, you will experience a variety of presentations from top-ranked scientists on the wide-ranging applications of stable isotopes in mass spectrometry (MS), nuclear magnetic resonance (NMR), and environmental analysis. 

Isotope Days 2023 – Mass Spectrometry

On Demand Recording Now Available
Event Date: September 21, 2023 

Presenters* include:

Dave Muddiman, PhD | Professor
North Carolina State University (Department of Chemistry) (USA)
The Critical Role of Stable Isotopes to Unravel Complex Biological Questions

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Sonia Fargue, PhD | Assistant Professor
University of Alabama (Urology Department) (USA)
Using Isotopes to Probe the Metabolism of Oxalate

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Ye Yang, PhD | Research Fellow
NIH (Urologic Oncology Branch) (USA)
Metabolic Characterization of Birt-Hogg-Dubé Syndrome Renal Tumor Cells and Tissues Using Stable Isotope-Resolved Metabolomics

On demand recording and transcript not available

Jurre Kamphorst, PhD | VP of MS Technology & Biomarkers
Olaris Therapeutics (USA)
Highly Standardized Metabolomic Analysis of Clinical Samples Using Triple Quadruple Mass Spectrometry

 transcript 

Junmin Peng, PhD | Member (Director)
St. Jude Children’s Research (Center for Proteomics and Metabolomics, Structural Biology and Developmental Neurology Departments) (USA)
Exploring Proteome Turnover in a Murine Alzheimer′s Disease Model Using Stable Isotope Labeling

transcript 

Moderated by
Andrew Percy, PhD | Senior Applications Chemist, Mass Spectrometry and MS 'Omics Product Manager
Cambridge Isotope Laboratories, Inc. (USA)

* Presenter biographies and presentation abstracts can be found below.

Isotope Days 2023 – Nuclear Magnetic Resonance

On Demand Recording Now Available
Event Date: September 28, 2023 

Presenters* include:

Hashim Al-Hashimi, PhD | Roy and Diana Vagelos Professor of Biochemistry
Columbia University (Biochemistry and Molecular Biophysics) (USA)
Visualizing RNA Structural Dynamics Using NMR

transcript

Mei Hong, PhD | Professor of Chemistry
Massachusetts Institute of Technology (USA)
Binding Site of Hexamethylene Amiloride in the SARS-CoV-2 Envelope Protein from Solid-state NMR

transcript

Kevin Brindle, PhD | Professor of Biomedical Magnetic Resonance
University of Cambridge (Cancer Research) (UK)
Imaging Tumor Metabolism – From Mouse to Man 

On demand recording and transcript not available

Andrew Namanja, PhD | Principal Research Scientist
AbbVie Inc. (USA)
Protein-based NMR for Generating Small Molecule Hits and Drug Leads

On demand recording and transcript not available

Rafal Augustyniak, PhD | Assistant Professor
University of Warsaw (Faculty of Chemistry) (Poland)
Histidine Labelling of Enzymes for Solution and Solid-state NMR

On demand recording and transcript not available

Moderated by
Kelly Andrade | Assistant Product Manager
BioNMR, NMR Solvents, Metabolomics
Cambridge Isotope Laboratories, Inc. (USA)

* Presenter biographies and presentation abstracts can be found below.

Isotope Days 2023 – Environmental Analysis

On Demand Recording Now Available
Event Date: October 5, 2023 

Presenters* include:

Heather Stapleton, PhD | Ronie-Richele Garcia-Johnson Distinguished Professor
Duke University | Nicholas School of the Environment (USA)
Exposure to Per- and Polyfluoroalkyl Substances (PFAS) in the Indoor Environment: Measurement of PFAS in House Dust and Silicone Wristbands

On demand recording and transcript not available

Bryan Vining, PhD | Laboratory Director
Enthalpy Analytical Ultratrace (USA)
Dynamite Comes in Small Packages

transcript

Wendy Strangman, PhD | Assistant Professor
University of North Carolina at Wilmington (USA)
Exploring Freshwater Harmful Algal Blooms: Microcystin Toxin Structural Diversity and Other Bioactive Cyanopeptides

transcript

Xavier Ortiz Almirall, PhD | Assistant Professor
IQS Barcelona (Spain)
An Isotope Dilution-Based Method for the Analysis of Microcystins and Anatoxin-a for Improved Accuracy and Robustness

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Yukari Ishikawa, PhD | Research Associate
Imperial College London | Environmental Research Group, School of Public Health (UK)
Quantitative Analysis of Microplastics Using Isotope Labelled Polystyrene (Styrene-D8) by Pyrolysis-GCxGC-TOFMS

transcript

Moderated by
Ben Priest | Business Development Manager
Environmental Products
Cambridge Isotope Laboratories, Inc. (USA)

*Presenter biographies and presentation abstracts can be found below.

 Biographies and Abstracts – Mass Spectrometry

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

David C. Muddiman is the Jacob and Betty Belin Distinguished Professor of Chemistry and was the Founding Director of the Molecular Education, Technology and Research Innovation Center (METRIC) at North Carolina State University in Raleigh, NC (2017-2023). Prior to moving his research group to North Carolina State University in 2005, David was a professor of biochemistry and molecular biology and Founder and Director of the Proteomics Research Center at the Mayo Clinic College of Medicine in Rochester, MN. Prior to this appointment, David was an associate professor of chemistry at Virginia Commonwealth University. It was there that he began his professional career as an assistant professor with an adjunct appointment in the Department of Biochemistry and Molecular Biophysics and as a member of the Massey Cancer Center in 1997. These academic appointments were preceded by a postdoctoral fellowship at Pacific Northwest National Laboratory in the Environmental Molecular Sciences Laboratory under Richard D. Smith from 1995-1997.

David was born in Long Beach, CA, in 1967 but spent most of his formative years in a small town in Pennsylvania. David received his BS in chemistry from Gannon University (Erie, PA) in 1990 and his PhD in analytical chemistry from the University of Pittsburgh in 1995 under the auspices of David M. Hercules.

Dr. Muddiman was editor of Analytical and Biological Chemistry (2015-2020) and he currently is the coordinating editor of Journal of Mass Spectrometry (2022-present), serves on the Editorial Advisory Board of Molecular and Cellular Proteomics, Rapid Communications in Mass Spectrometry, and the Journal of Chromatography B. He also serves as the chair of the advisory board of the NIH Funded Yale/NIDA Neuroproteomics Center, Yale University. Dr. Muddiman has served as a member of the ASMS Board of Directors (2013-2015) and treasurer (2013-2015) and president (2015-2017) of the United States Human Proteome Organization. His group has presented over 700 invited lectures and presentations at national and international meetings including 31 plenary/keynote lectures. His group has published over 300 peer-reviewed papers and has received six US patents. He was awarded the 2023 Donald F. Hunt Distinguished Contribution in Proteomics Award (US HUPO), the 2016 Graduate School Outstanding Graduate Faculty Mentor Award in the Mathematical, Physical Sciences, and Engineering, 2015 ACS Award in Chemical Instrumentation, 2010 Biemann Medal (American Society for Mass Spectrometry), 2009 NCSU Alumni Outstanding Research Award, the 2004 ACS Arthur F. Findeis Award, the 1999 American Society for Mass Spectrometry Research Award, and the 1990-1991 Safford Award for Excellence in Teaching (University of Pittsburgh).

Dr. Muddiman’s research is at the intersection of innovative mass spectrometry platform technologies, systems biology, environmental science, and model organisms to understand human disease and is largely funded by the National Institutes of Health. His personal interests include playing ice hockey, cycling, hiking, restoring sports cars, and spending time with family.

The Critical Role of Stable Isotopes to Unravel Complex Biological Questions

Since its first demonstration in the 1960’s, 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.

During this talk you will learn about:

• Functional mass spectrometry imaging using stable isotopes
• Learning the role of diet on pancreatic health
• Building a MSI community-based QC standard: Get involved

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

Sonia Fargue received her PhD in Cell Biology at the University College London, United Kingdom, and her MD at the Universite Claude Bernard Lyon 1, France. She is an assistant professor in the Department of Urology at the University of Alabama at Birmingham. Dr. Fargue’s research over the past 18 years has focused on the metabolism of oxalate in the rare inherited disease primary hyperoxaluria and in idiopathic calcium oxalate kidney stone disease with the aim to contribute to the development of therapies for patients affected with these disorders. A large component of Dr. Fargue’s current research relies on the use of isotope tracers in human studies.

Using Isotopes to Probe the Metabolism of Oxalate

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.

During this talk you will learn about:

• How to leverage the peculiarities of a metabolic pathway to further investigate it using isotope tracers
• What is oxalate and its role in kidney stone disease
• How isotope tracer studies have help shape the therapeutic landscape for the rare disease primary hyperoxaluria

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

Dr. Ye Yang is currently serving as a Research Fellow at the Urologic Oncology Branch and Clinical Cancer Metabolism Facility at NCI, NIH, where she focuses on mass spectrometry-based stable isotope resolved metabolomics in human renal cancers. Her academic journey started with a bachelor of science degree in chemistry from China Agricultural University in 2008. Seeking to advance her research career, she pursued further studies in the United States and joined the University of Louisville, KY, in 2009. There, she joined the laboratory of Dr. Richard M. Higashi, Dr. Teresa W.M. Fan, and Dr. Andrew N. Lane, earning a master's degree in chemistry in 2013. Throughout her academic pursuit, Ye Yang's fascination with cancer metabolism intensified, particularly in the application of stable isotope labeling. As a result, she decided to continue her journey in this field in Dr. Higashi's lab at the University of Kentucky, where she obtained her PhD in toxicology and cancer biology. In 2017, driven by a passion for advancing cancer research, Dr. Yang joined Dr. Marston Linehan's group at the Urologic Oncology Branch, NCI. Here, she actively engages in groundbreaking research focused on the metabolic characterization of kidney cancers using stable isotope labeling and contributing valuable insights to the field.

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

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.

During this talk you will learn about:

• ¹³C₆-glucose tracer experiments showed BHD tumor tissues exhibited enhanced pyruvate dehydrogenase activity.
• Metformin inhibited cell growth and pyruvate dehydrogenase activity in BHD tumor cell lines, revealed by ¹³C₆-glucose tracer experiments.
• 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.

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

Jurre Kamphorst is the VP of Mass Spectrometry Technology & Biomarkers at Olaris, Inc., a company enabling precision diagnostics by combining comprehensive metabolomics with AI. Jurre has over 15 years of experience in MS-based metabolomics and lipidomics and their application in the life sciences. His primary interest is in translating omics-driven research into clinical biomarkers and precision diagnostics.

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

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.

During this talk you will learn about:

• Triple quadrupole MS-based methods hold great promise in clinical metabolomics
• The quality of metabolite measurements, rather than the quantity of metabolites measured, will dictate the success of clinical metabolomics
• How ¹³C-standards improve the accuracy and reproducibility of metabolite measurements in clinical samples

Junmin Peng, PhD | Member (Director)

St. Jude Children’s Research | Center for Proteomics and Metabolomics Structural Biology and Developmental Neurology Departments) (USA)

Dr. Junmin Peng is an accomplished researcher and expert in the fields of proteomics and neuroscience at St. Jude Children's Research Hospital in Memphis. With a PhD from the University of Iowa, postdoctoral training at Harvard Medical School, and over 20 years of independent research at Emory University and St. Jude, Dr. Peng has published over 240 scientific papers and has been invited to present his work at numerous conferences and academic institutions worldwide. As an academic leader, Dr. Peng has trained over 90 graduate students, postdocs, and staff scientists. He has made significant contributions to the development of high-throughput mass spectrometry-based proteomics, using these approaches to study complex biological systems, including ubiquitin and protein turnover, Alzheimer's disease, cancer, and immunity. Dr. Peng's work has led to the identification of novel disease pathways and potential therapeutic interventions, contributing to the advancement of precision medicine.

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

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.

During this talk you will learn about:

• Biological significance of studying protein turnover
• Developing pSILAC labeling and JUMPt software for accurately analyzing proteome turnover
• A proteome turnover case study in a human disease model

Biographies and Abstracts – Nuclear Magnetic Resonance

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

Al-Hashimi was born in Beirut, Lebanon, and grew up in Greece, Italy, Jordan, and the UK. As a graduate student at Yale, Al-Hashimi helped develop residual dipolar coupling methodology, which revolutionized the study of protein structure and dynamics by NMR. As a postdoctoral fellow at the Memorial Sloan Kettering Cancer Center, Al-Hashimi expanded the domain of applicability of these methods to nucleic acids. As a principal investigator, Al-Hashimi and his trainees discovered many of the ubiquitous motional modes underlying the biological activities of nucleic acids, with important implications for drug discovery and for understanding the mechanisms that cause genome instability and cancer. These motions include transitions between Watson-Crick and Hoogsteen base pairs, which shape the DNA protein recognition and damage landscapes; transitions between mismatch and tautomeric/anionic Watson-Crick base pairs, which are responsible for errors during replication, transcription, and translation; motions that determine proper folding of RNA; and transient changes in RNA secondary structure that underlie gene regulation and viral genomic replication by non-coding RNAs. His group developed methods harnessing the predictive power of RNA dynamic ensembles to identify small molecule inhibitors of HIV-1 replication. In 2009, Al-Hashimi co-founded Nymirum Inc. to enable RNA-targeted drug discovery using RNA dynamics.

Visualizing RNA Structural Dynamics Using NMR

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.

During this talk you will learn about:

• RNA structures are highly dynamic
• Knowledge of the dynamics is key for understanding and interfering with cellular function
• NMR spectroscopy on ¹³C/¹⁵N labeled RNA samples provides a powerful approach for visualizing these dynamics

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

Prof. Mei Hong obtained her BA degree in chemistry with summa cum laude from Mount Holyoke College in 1992 and her PhD from the University of California, Berkeley in 1996 (with Professor Alex Pines). After a one-year postdoctoral stint at MIT, she began her independent career at the University of Massachusetts Amherst before moving to Iowa State University in 1999. She became a full professor in 2005, held the first John D. Corbett Professorship in 2007-2010, and returned to MIT as a full professor in 2014. She has received numerous awards for the creative development and application of solid-state NMR spectroscopy to elucidate the structure, dynamics and mechanism of action of membrane proteins, amyloid protein and other biological macromolecules such as plant cell walls.

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

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.

During this talk you will learn about:

• Versatile isotopic labeling is highly beneficial for determining drug binding sites in proteins.
• HMA binds the lipid-facing surface of the E protein.
• Solid-state NMR spectroscopy can yield important insight into small molecule binding mechanisms to membrane proteins.

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

Kevin Brindle is Emeritus Professor of Biomedical Magnetic Resonance in the Department of  Biochemistry at the University of Cambridge and a Senior Group Leader in the Cancer Research UK Cambridge Institute. He got his BA (Biochemistry,1978) and D. Phil (1982) in Oxford, before becoming a Royal Society University Research Fellow in 1986. He moved to a lectureship in Manchester in 1990 and in 1993 to a lectureship in Cambridge, where he became Professor in 2005. The current focus of his work is to develop novel imaging methods to detect cancer, disease progression, and to monitor early tumour responses to treatment. He was elected a Fellow of the Academy of Medical Sciences in 2012, a Fellow of the European Academy of Cancer Sciences in 2014, a Fellow of the International Society of Magnetic Resonance in 2020 and a Fellow of the Royal Society in 2020. He was President of the European Society for Molecular Imaging in 2018 and was awarded the European Society of Molecular Imaging Award in 2013 and the Gold Medal of the World Molecular Imaging Society in 2014.

Imaging Tumor Metabolism – From Mouse to Man 

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.

During this talk you will learn about:

• Hyperpolarized ¹³C-labelled and ²H-labelled substrates have enabled new approaches to metabolic imaging in the clinic
• Metabolic imaging with hyperpolarized [1-¹³C]pyruvate and [6,6-²H₂] glucose can be used to detect early tumor responses to treatment
• Metabolic imaging with [2,3-²H₂] fumarate can provide a sensitive indicator of tumor cell death post-treatment

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

Dr. Andrew Namanja is a principal research scientist at AbbVie. Since 2015, he has been a member of the integrated small-molecule drug discovery team that delivers drug leads for various therapeutic areas, including virology, oncology, neuroscience, immunology, and targeted protein degradation platform. In addition to leading early-stage drug discovery research projects, his research also focuses on applying and developing NMR methods, as well as AI & ML tools to advance the fragment-based drug discovery (FBDD) and structure-based drug design (SBDD) technologies. Prior to joining AbbVie, he was an assistant research professor at City of Hope Beckman Research Institute and a principal investigator on a federal grant. He received his BS degree in chemistry from Indiana University in 2002, his PhD in biochemistry from the laboratory of Professor Jeffrey Peng at the University of Notre Dame in 2009, and postdoctoral training at City of Hope in 2012.

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

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).

During this talk you will learn about:.

• Protein-detected NMR enables rapid go/no-go decisions for hit follow-up
• NMR workflow for hit confirmation, mode of action (MoA) determination, structure elucidation, and driving structure-activity relationships (SAR)
• Rationale for protein isotopic incorporation and expression systems

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

Rafal Augustyniak is an assistant professor at the University of Warsaw (Poland). He obtained his PhD from the Ecole Normale Superieure and Universite Piere et Marie Curie (2007-2012, Paris, France) working with Geoffrey Bodenhausen and Olivier Lequin. After a post-doctoral position at the University of Toronto with Lewis E. Kay (2012-2018), he returned to Poland after 11 years to establish a new biologically oriented protein NMR research group with Polish National Science Centre grant. 

Histidine Labelling of Enzymes for Solution and Solid-state NMR

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.

During this talk you will learn about:.

• Labelling of His residues in larger proteins to get insight into the imidazole protonation and tautomeric states
• How solution and solid state NMR can provide complementary information about histidines in enzymes
• How histidine protonation affects enzyme catalysis. 

 Biographies and Abstracts – Environmental

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

Professor Heather Stapleton is an environmental chemist and exposure scientist in the Nicholas School of the Environment at Duke University. Her research interests focus on identification of halogenated and organophosphate chemicals in building materials, furnishings and consumer products, and estimation of human exposure, particularly in vulnerable populations such as pregnant women and children. Her laboratory specializes in analysis of environmental and biological tissues for organic contaminants to support environmental health research. Currently she serves as the Director for the Duke Superfund Research Center, and Director of the Duke Environmental Analysis Laboratory, which is part of NIH’s Human Health Environmental Analysis Resource.

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

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.

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

Dr. Bryan Vining is the laboratory director for Enthalpy Analytical Ultratrace in Wilmington, NC. Bryan is a 22-year veteran of the laboratory industry. He started his career as a product specialist and later worked in various lab roles before joining Enthalpy Analytical, LLC in 2016. Bryan graduated with a BA in chemistry from Huntingdon College, before proceeding to get both an MBA and PhD from the University of North Carolina Chapel Hill and Florida State University, respectively. Areas of expertise for contaminant testing include isotope dilution, PFAS, HRMS, LC-MS/MS, GC-MS, and persistent organic pollutants in environmental and food matrices.

Dynamite Comes in Small Packages

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.

During this talk you will learn about:

• Isotope dilution is avaiable even for short chain compounds
• CIL is currently providing the most short chain stable-isotope-labelled PFAS compounds available
• Different types of background present difficulties in analyzing short-chain PFAS compounds that require somewhat unusual approaches to deal with them.

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

Dr. Strangman received her PhD from the Scripps Institution of Oceanography, and her postdoctoral research was performed at the University of British Columbia.  Now an assistant professor at UNC Wilmington, her research interests center on the application of mass spectrometry-based analyses for the discovery and detection of toxins and other bioactive compounds produced by harmful algal bloom (HAB) species. Additional research interests include mass spectrometry-based pharmacokinetic assays to assess digestive transformation and permeability of medicinal plant extracts, the discovery of new bioactive natural products produced by marine bacteria from the microbiomes of marine parasites, pathogen-resistant coral microbiomes, and deep arctic sediments, and chemical ecology of Caribbean coral reefs.

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

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.

During this talk you will learn about:

• Urbanization and climate change are resulting in increasing numbers of freshwater harmful algal blooms in North America
• Harmful algal blooms produce a wide variety of potently bioactive compounds
• Analytical tools such as mass spectrometry are critical for accurately assessing toxicity of bloom events

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

Dr. Ortiz Almirall is an adjunct professor at IQS-School of Engineering (Ramon Llull University, Barcelona), and his research focuses on the development of targeted and non-targeted methods for the analysis of cyanotoxins and other environmental pollutants in the environment. In the past he worked at the Ontario Ministry of the Environment as a scientist, where he developed the method E3450 for the automated analysis of microcystins and anatoxin-a using online-SPE combined with LC-QTOFMS instrumental analysis.

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

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.

During this talk you will learn about:

• Isotope dilution method
• Cyanotoxins
• Targeted and non-targeted analysis

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

Dr. Yukari Ishikawa is a Research Associate for the Microplastics Team in the Environmental Research Group of the School of Public Health at Imperial College London. She specializes in the development of analytical methods aimed at analyzing environmental contaminants (SVOCs, VOCs, microplastics) in air, exhaust gases, waste, and biological samples. Yukari's research career began at the National Institute for Environmental Studies in Japan, analyzing PCBs, PCNs, and PBBs in waste-related samples since 2001, followed by VOCs analysis at the University of Tokyo, POPs analysis at the University of Queensland, Australia, and personal air pollution exposure study at King's College London in the UK, to her current position of microplastics analysis at Imperial College London.

Quantitative Analysis of Microplastics Using Isotope-Labelled Polystyrene (Styrene-D8) by Pyrolysis-GCxGC-TOFMS

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.

During this talk you will learn about:

• Quantitative analysis of microplastics
• Analytical methods for microplastics in biological samples
• Airborne microplastics analysis