2021 Webinars

MS 'Omics Webinar Series | Sponsored by CIL

Deep Proteome Sequence Analysis, Increasing Isobaric Tag Reporter Ion Signal Using IR Photons, and Coupling MS to Electron Microscopy

December 1, 2021
Josh Coon, PhD | Professor of Chemistry and Biomolecular Chemistry | University of Wisconsin-Madison

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.

Metabolomics for Molecular Diagnostics in Urinary Cancers

November 17, 2021
Tim Garrett, PhD | Associate Professor | University of Florida

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.

Combining Multidimensional Measurements with Standards and Isotopologue Workflows to Detect, Identify and Validate Molecules in Omic Studies

October 28, 2021
Erin Baker, PhD | Associate Professor | North Carolina State University

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.

Isotope Day | June 2, 2021 | Sponsored by CIL

During this symposium, experience a variety of live presentations from top-ranked scientists on the wide-ranging applications of stable isotopes. These include metabolic tracing, quantification, qualification, structure determination, imaging, and more! 

Speakers – NMR

Kevin Millis, PhD | Cambridge Isotope Laboratories, Inc.

Stable Isotopes in Biomolecular Nuclear Magnetic Resonance Research: A critical technology

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. 

Robert G. Griffin, PhD | MIT

Atomic Resolution Structures of Amyloid Fibrils: Magic Angle Spinning (MAS), Dynamic Nuclear Polarization (DNP) ¹H Detected NMR

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.

Jan Marchant, PhD | University of Maryland

Structural Characterization of Large RNAs from HIV-1 Using NMR

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.

Haribabu Arthanari, PhD | Harvard Medical School and Dana Farber Cancer Institute

Leveraging Tailored Isotope Labeling and Novel Pulse Design to Encode Amino Acid Selective Line Shapes

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.

Lewis Kay, PhD | University of Toronto

Without Isotopes There Is No Fancy NMR

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.

Christoffer Laustsen, PhD | Aarhus University Hospital

Translation of Hyperpolarized Carbon-13 and Deuterium Imaging

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.

Speakers – Mass Spectrometry

Andrew Percy, PhD | Cambridge Isotope Laboratories, Inc.

Example MS Applications of Stable Isotope Standards and their Mixtures

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.

Matt Vander Heiden, MD, PhD | MIT

Using Stable Isotopes to Study Proliferating Cell Metabolism

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.

David Muddiman, PhD | North Carolina State University

Quantifying Human Exposure to Emerging Contaminants of Concern Using Mass Spectrometry

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.

Lingjun Li, PhD | University of Wisconsin

High Resolution FTMS Enabled Mass Defect-based Chemical Tags for Multiplex Quantitative Omics

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.

John Yates, PhD | Scripps Research Institute

Quantitative Bioorthogonal Chemistry for Proteomics

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.

Gary Patti, PhD | Washington University

Big Data from Heavy Molecules

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.