2025 Webinars
Isotope Days 2025 | September 9 | Sponsored by CIL
Mass Spec Focus
Quantitative Analysis of Bile Acids Using High-Resolution Mass Spectrometry (HRMS)-based Methods
Thomas Horvath, PhD | Assistant Professor | Department of Pathology & Immunology, Baylor College of Medicine (USA)
Abstract: As interest in bile acid biochemistry and metabolism continues to accelerate in biomedical and life science research, bioanalytical chemists will be charged with the development of more complex methods to perform quantitative analysis on this functionally interesting and ever-expanding class of molecules. The work described in this presentation will focus primarily on several key operational aspects of our newly developed ZenoTOF 7600+-based bioanalytical method and feature some quantitative bile acid data acquired from blood and/or stool specimen extracts. By leveraging the MRMHR scanning capabilities of the ZenoTOF 7600+, we can specify narrow mass-to-charge (m/z) isolation windows in the SCIEX OS Analytics Module to select specific fragment ions or precursor ions in instances where diagnostic fragment ions aren’t produced from full-scan product ion spectra. These narrow m/z isolation windows result in a boost in detection sensitivity (i.e., signal-to-noise) that is comparable to the sensitivity offered by the QTRAP 7500 in my lab for bile acid detection. Together, these data demonstrate the capability of the ZenoTOF 7600+ system to perform highly sensitive and selective analysis of endogenous bile acid content in biological specimen extracts for matrices such as blood plasma or stool.
A transcript of this presentation in English is available here.
From Amino Acid and Protein Requirements to Protein Quality: Knowledge Gained from Applying the Stable Isotope-based Indicator Amino Acid Oxidation (IAAO) Method
Glenda Courtney-Martin, PhD | Senior Associate Scientist & Associate Professor | Hospital for Sick Children (SickKids) (Canada)
Abstract: Protein is an indispensable nutrient due to its content of amino acids which must be dietarily derived. Current amino acid requirements for young and older adults are based on data from studies in young adults using the stable isotope based indicator amino acid oxidation (IAAO) method. However, protein recommendations are based on nitrogen balance data. The Institute of Medicine recommends that nitrogen balance no longer be regarded as the gold standard for estimating protein requirements and that new methods be sought. We applied the stable isotope-based IAAO method to study protein requirements in young and older adults and found them to be higher than current recommendations but not different from each other. However, using the IAAO method we demonstrated that requirements for key indispensable amino acid are higher in older adults. This suggest that protein quality is more important for older than for younger adults. With recent plant-based protein recommendations, the study of protein quality is necessary to determine to capacity of plant proteins to meet amino acid needs. The IAAO method was validated to study protein quality of foods and has been applied in humans to assess metabolic availability of limiting amino acids and to test the effect of protein complementation. The objectives of the talk are:
To preset the concepts and application of the IAAO method to study amino acid requirements across the lifespan and in vulnerable populations;
To present data on the application of the IAAO method to study protein requirements and physiological studies validating the IAAO derived estimates of protein requirements in adults;
To present data on the application of the IAAO method to study protein quality of plant protein foods and to assess the effect of complementary protein sources.
A transcript of this presentation in English is available here.
Stable Isotope Pulse Tracer Method in Human Clinical Research
Nicolaas Deutz, MD, PhD | Professor and Director | The Center for Translational Research in Aging and Longevity | Texas A&M University (USA)
Abstract: Stable isotope infusions in humans have been used for many decades to estimate the production of amino acids and to calculate net protein breakdown. The primed-constant and continuous infusion protocol was most often used and measurements were done in the tracer steady state condition, both in the postabsorptive as well as during and after a meal. The main disadvantages of using this approach are the need for steady state conditions with sometimes long infusion times, the need for large amounts of isotopes and the fact that the appearance of the amino acids was measured in plasma as a proxy for the appearance intracellularly. We used the pulse tracer approach to circumvent these disadvantages and also to enable compartmental analysis to measure the intracellular production. We developed a protocol in which we give a mixture of 27 different isotopes of amino acids and related products to estimate the production and size of the extracellular and intracellular pools. We applied the model in a variety of different conditions like COPD, older age, ICU. We recently recalculated the net protein breakdown in humans and established different rates than was estimated in the past. We also applied the pulse isotope approach during feeding.
A transcript of this presentation in English is available here.
Precision with Purr-pose: Overcoming the Challenges of PCATs Assay Development and Validation
Stacy Dee | Research & Development Scientist | Labcorp (USA)
Abstract: Plasma catecholamine (dopamine, epinephrine, and norepinephrine) measurement helps physicians diagnose the presence of catecholamine-secreting tumors such as pheochromocytomas and paragangliomas when plasma metanephrines testing is inconclusive. Developing a clinical assay for plasma catecholamines (PCATs) requires measuring low concentrations. This presents several challenges. First, PCAT levels in a patient can be affected by medication, food, and even physical activity, therefore patient preparation is crucial. Next, catecholamines are grossly unstable so samples must be handled carefully during collection, shipment, and extraction. Further, separation and quantification are complicated by analyte polarity and the potential presence of interfering medications and metabolites. This presentation will highlight solutions to these challenges in the development and rigorous validation of our high-throughput PCATs assay.
A transcript of this presentation in English is available here.
Accurate Quantification of Protein Conformations with the Isotope Label-based Bottom-up Proteomics Approach ‘Covalent Protein Painting’
Tom Casimir Bamberger, PhD | Research Scientist | Department of Molecular & Structural Biology | The Scripps Research Institute (USA)
Abstract: This seminar will introduce covalent protein painting (CPP), a stable isotope-based protein footprinting technology designed to detect and quantify protein conformational changes using bottom-up mass spectrometry. CPP leverages a rapid, site-specific chemical modification of lysine residues with dimethyl labels to assess the structural accessibility of proteins in complex biological samples, including cells, tissues, and whole organisms. We will present the underlying principles of CPP, discuss experimental workflows, and explore data analysis strategies in detail. Through selected case studies, we will demonstrate the utility of CPP in probing conformational dynamics associated with cancer, neurodegeneration, and protein misfolding disorders such as cystic fibrosis. The seminar will highlight how CPP enables quantitative insights into disease-relevant structural changes at the proteome level.
A transcript of this presentation in English is available here.
Isotope Days 2025 | September 18 | Sponsored by CIL
NMR Focus
Fluorine as Atomic Beacons for Probing the Structure and Dynamics of Large Biomolecules
Haribabu Arthanari, PhD | Associate Professor | Department of Biological Chemistry & Molecular Pharmacology | Harvard Medical School and Dana-Farber Cancer Institute (USA)
Abstract: NMR spectroscopy provides atomic-level insights into the structure, dynamics, and interactions of biomolecules. However, in large macromolecular systems, its application is often limited by signal attenuation arising from line broadening due to fast relaxation and spectral overlap. The rarity of fluorine atoms in biological systems offers a unique opportunity to employ them as atomic beacons for reporting on structure and dynamics. While the 19F chemical shift is highly sensitive to its chemical environment, CSA-induced relaxation has restricted its utility in high–molecular weight systems. To overcome this limitation, we exploit the slow relaxation properties of 13C nuclei covalently attached to 19F in aromatic 19F–13C spin pairs, enabling the effective use of fluorine as an atomic probe in large biomolecules.
A transcript of the presentation in English is available here.
Advances in Deuterium Metabolic Imaging: Paving the Way to Clinical Translation
Jeanine Prompers, PhD | Professor | Department of Human Biology | NUTRIM Institute of Nutrition and Translational Research in Metabolism | Maastricht University Medical Centre (Netherlands)
Abstract: Deuterium (2H) magnetic resonance spectroscopy (MRS) and spectroscopic imaging (i.e., deuterium metabolic imaging; DMI) provide powerful, non-invasive means to study tissue metabolism in vivo. In particular, DMI enables real-time tracking of metabolic conversions such as the transformation of deuterated glucose into lactate, offering direct insights into altered metabolic pathways like the Warburg effect in tumors. This lecture will highlight recent advances in deuterium metabolic imaging in humans, particularly at ultra-high magnetic field strengths. Examples from both healthy volunteers and patient studies will illustrate the clinical potential of this emerging technique.
Recording and transcript not available.
Protocol for the Preparation of Fab and Fc Fragments from Therapeutic Monoclonal Antibodies Using Escherichia coli to Speed up Complete Backbone and Near-complete Side-chain Methyl Assignment
Yves Aubin, PhD | Research Scientist | Regulatory Research Division, Biologics and Radiopharmaceutical Drug Directorate | Health Canada (Canada)
Abstract: Characterization of therapeutic monoclonal antibodies (mAbs) using NMR spectroscopy has benefited from recent advances in sample preparation methods for isotopically labeled fragments. These have opened new possibilities for method development for the detailed assessment of the structure of innovator and biosimilar products, the study mAb-excipients interactions using protein dynamics, and others. Here, we propose a simple protocol that facilitates and accelerates the resonance assignment step that is required to fully exploit the strengths of NMR spectroscopy. The proposed approach is based on a simplified version of our single chain strategy, where the heavy and the light chains are produced separately in E. coli and mixed together prior to refolding. This offers the advantage of performing differential labelling where only one chain (either the heavy or the light) is isotopically labelled, thereby providing simpler NMR spectra that are straightforward to analyze and assign by NMR, either by computer-assisted manual assignment, or fully automatic assignment software packages. The protocol was demonstrated on bevacizumab, infliximab, rituximab, and trastuzumab monoclonal antibodies. Near complete assignments (>90%) of the backbone resonance of bevacizumab was accomplished in only a few hours. Moreover, use of 13C-glucose while allowing comparable results in terms of backbone assignment completeness, also allowed near complete assignments of side-chain methyl groups of alanine, threonine-γ1 and isoleucine-γ2.
A transcript of the presentation in English is available here.
Isotopic Labeling Strategies for RNA-Targeted Drug Discovery
Yaqiang Wang, PhD | Assistant Professor | Department of Biophysics | Medical College of Wisconsin (USA)
Abstract: RNA has emerged as a promising therapeutic target, driven by growing insights into its diverse biological roles. Structural studies of RNA and RNA-protein complexes are essential for elucidating molecular recognition mechanisms and advancing RNA-targeted drug discovery. Isotopic labeling enables atomic-resolution characterization of RNA structure, dynamics, and ligand interactions, particularly through NMR spectroscopy. In this presentation, I will explore isotopic labeling strategies for RNA in the context of drug discovery, addressing the unique challenges of RNA-targeted therapeutics, comparing labeling approaches, and highlighting practical considerations for their implementation.
A transcript of this presentation in English is available here.
Isotope Days 2025 | October 1 | Sponsored by CIL
Environmental Analysis
Children’s Environmental Epidemiology and Human Biomonitoring in Japan
Shoji Nakayama, MD, PhD | Deputy Director | Japan Environment and Children’s Study Programme Office (Japan)
Abstract: Recent environmental health research including epidemiological studies have focused on the impact of chemical exposure on children’s health and development. Japan has been conducting a large-scale birth cohort study, involving >100,000 mother-child pairs, since 2011, namely the Japan Environment and Children’s Study (JECS). Along side the longitudinal study, the Ministry of the Environment, Japan launched a nationwide human biomonitoring (HBM) program this year. In this presentation, the outcomes and future of JECS as well as details about ongoing HBM will be introduced.
A transcript of this presentation in English is available here.
Ion Mobility Spectrometry of Quaternary Ammonium Disinfectants
Adrian Covaci, PhD | Professor | Department of Pharmaceutical Sciences | University of Antwerp (Belgium)
Abstract: Collision cross section (CCS) values derived from ion mobility high-resolution mass spectrometry (IM-HRMS) can serve as an additional identification parameter in suspect and non-target screening analyses of contaminants of emerging concern (CECs), such as quaternary ammonium compounds (QACs). We present the advantages of using CCS-m/z trendlines (i.e., the relationship between CCS values and mass-to-charge (m/z) ratios) for the annotation of suspect QACs. Indoor dust samples (n=46) from Flanders (Belgium) were extracted and analysed by liquid chromatography (LC) coupled to IM-HRMS. QACs were identified as two relevant contaminant classes, with several compounds showing high abundances and detection frequencies. Twenty-one QACs were identified at confidence level (CL) 1[2] and another 17 QACs could be assigned to CL 3, as no library mass spectra were available and/or possible isomeric structures could not be narrowed down to a single candidate. Therefore, CCS values for the assigned suspects were matched against m/z-CCS trendlines established for QACs for which reference standards were available, respectively[3]. This approach allowed the identification of extensive homologue series for both classes containing structural modifications barely described in literature so far. For QACs, in contrast to available standards, homologues with uneven numbered side chains were identified. For all suspect QACs, experimental CCS values fell within the 95th percent confidence interval calculated for the reference CCS-m/z trendlines, vastly contributing to the confidence in the compound annotation. References: [1] Belova, L., Celma, A., et al., Anal Chim Acta, 2022; 1229: 340361. [2] Schymanski, E L, Jeon J, Gulde R et al., Environ Sci Technol, 2014; 48(4): 2097-8. [3] Belova, L., Caballero-Casero, N., et al., Anal Chem, 2021; 93(16): 6428-6436.
A transcript of this presentation in English is available here.
Dirty Water, Clean Data: How We Measure and Detect PFAS Using Stable Isotope Standards
Rebecca Weed, PhD | Research Scholar | North Carolina State University (USA)
Abstract: Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants found in various matrices. This presentation will cover the challenges of measuring PFAS and will investigate two examples for the quantitation and suspect screening of PFAS in different types of water samples. The first project focuses on a community engaged research (CER) approach that provided an exposure assessment of PFAS in North Carolina residential drinking and recreational water. Working in concert with community partners, who acted as liaisons to local residents, samples were collected by North Carolina residents from three different locations along the Cape Fear River basin: upper, middle, and lower areas of the river. Residents collected either drinking water samples from their homes or recreational water samples from near their residence that were then submitted by the community partners for PFAS analysis. The non-targeted approach, which utilized a suspect screening list (obtained from EPA CompTox database) identified several PFAS compounds at a level two confidence rating (Schymanski scale); compounds identified included a fluorinated insecticide, a fluorinated herbicide, a PFAS used in polymer chemistry, and another that is used in battery production. Notably, at several locations, PFOA (39.8 ng/L) and PFOS (205.3 ng/L) were at levels that exceeded the mandatory EPA maximum contaminant level (MCL) of 4 ng/L. Additionally, several sites had detectable levels of PFAS that are unique to a local chemical manufacturer. These findings were communicated back to the community partners who then disseminated this information to the local residents to help empower and aid in making decisions for reducing their PFAS exposure. The second project focuses on PFAS that are present in North Carolina seawater and sea foam. This study utilized both quantitative and non-targeted analysis to investigate PFAS contamination in sea foam, bulk water, and blank samples as previous work suggested PFAS can accumulate in higher concentrations in sea foam than the bulk water. Quantitative analysis detected up to 42 PFAS compounds, with particularly high concentrations in condensed sea foam samples, highlighting their role in accumulating PFAS. In one foam sample, PFOS, was observed at ca. 8,000,000 ng/L whereas, PFO5DoA, a compound predominantly found in North Carolina, was detected at levels exceeding 20,000 ng/L in some samples. Notably, emerging and regionally relevant PFAS – including unsaturated perfluorooctane sulfonic acid (UPFOS), hexafluoropropylene oxide trimer acid (HFPO-TeA), and 3-oxaperfluorononane sulfonic acid (F53) – were identified through nontargeted analysis. Comparisons between bulk water and sea foam samples revealed distinct PFAS concentration patterns, and significantly higher concentrations in the sea foam. The study underscores the need for continued monitoring of PFAS in coastal environments to assess potential human and ecological exposure risks.
A transcript of this presentation in English is available here.
Advancing TOP Assay for PFAS Assessments: Incorporating a Novel Reverse Surrogate to Track Oxidization Efficiency
Sarah Choyke, PhD | Technical Director | Eurofins Environment Testing – Denver (USA)
Abstract: The total oxidizable precursor (TOP) assay has been widely used to assess and manage the burden of per- and polyfluoroalkyl substances (PFAS) at contaminated sites. A limitation of the method is the ability to measure for complete oxidation or over oxidation in a manner that does not impact the targeted analytical results. An improvement to the TOP Assay incorporates an isotopically labeled sulfonamide reverse surrogate and its isotopically labeled carboxylate transformation product to quantify oxidation efficiency without interfering with the 1633A Extracted Internal Standards (EIS) and quantitation scheme. The reverse surrogate was designed to mimic a PFAS precursor while remaining analytically distinct from target PFAS, thereby enabling accurate tracking of oxidative transformation. These validation studies demonstrate that the reverse surrogates undergo complete transformation under standard TOP conditions, monitoring both the loss of the isotopically labeled sulfonamide and the creation of the isotopically labeled carboxylate. This approach enhances method robustness, ensures compatibility with EPA Method 1633A, and provides a more effective measurement for precursor oxidation efficiency. The integration of these reverse surrogates represents a significant advancement in the TOP assay supporting more accurate environmental assessments.
A transcript of this presentation in English is available here.