GenNext Open Access Video Resource Library
FOX® TECHNOLOGY & USE CASES
The AutoFox® Protein Footprinting System is a mass spectrometry-enabled platform for mapping protein structures and detecting interaction sites by oxidizing solvent-accessible amino acids. As structural changes or protein interactions affect solvent accessibility, modification levels shift accordingly. The AutoFox System is coupled with bottom-up proteomics and reverse-phase chromatography, to localize and quantify the extent of oxidation. Ion mobility separation further enhances resolution by resolving co-eluted, isobaric peptides before MS/MS fragmentation, improving residue-level precision.
Presented by Dr. Emily Chea, Applied Research Manager of GenNext Technologies, Inc
Dr. Emily Chea received her PhD while studying with Professor Dr. Lisa Jones at the University of Maryland, Baltimore. Emily has made significant contributions to the development of HRPF, with particular emphasis on in-cell FPOP (IC-FPOP) for proteome-wide structural biology characterization of cellular drug–target interactions. Currently, Emily is a key member of the GenNext scientific team in her role as an Applied Research Scientist Manager.
Using the Fox® System, researchers have advanced the understanding of therapeutic HOS by exploring epitope/paratope mapping, aggregation-interface identification, formulation, and small molecule binding. The Fox System, a novel HRPF technique, employs hydroxyl radicals (•OH) to irreversibly modify solvent-exposed amino acid side chains, revealing changes in solvent accessibility and uncovering buried or exposed regions. This presentation will discuss the Fox System, its application in characterizing biotherapeutic HOS and future innovations.
Presented by Dr. Emily Chea, Applied Research Manager of GenNext Technologies, Inc.
Dr. Emily Chea received her PhD while studying with Professor Dr. Lisa Jones at the University of Maryland, Baltimore. Emily has made significant contributions to the development of HRPF, with particular emphasis on in-cell FPOP (IC-FPOP) for proteome-wide structural biology characterization of cellular drug–target interactions. Currently, Emily is a key member of the GenNext scientific team in her role as an Applied Research Scientist Manager.
A study on TNF alpha and its monoclonal antibody adalimumab, using the Fox Protein Footprinting System, revealed oxidation changes in TNF alpha peptides matching the known epitope. Adalimumab’s Fab region showed decreased oxidation due to reduced solvent accessibility when bound to TNF alpha, while the Fc region had increased oxidation, indicating flexibility beyond the crystal structure. This method accurately mapped the TNF alpha epitope and adalimumab paratope, providing insights beyond the crystal structure.
Presented by Dr. Emily Chea, Applied Research Manager of GenNext Technologies, Inc
Dr. Emily Chea received her PhD while studying with Professor Dr. Lisa Jones at the University of Maryland, Baltimore. Emily has made significant contributions to the development of HRPF, with particular emphasis on in-cell FPOP (IC-FPOP) for proteome-wide structural biology characterization of cellular drug–target interactions. Currently, Emily is a key member of the GenNext scientific team in her role as an Applied Research Scientist Manager.
In drug development, characterizing high-order structures (HOS) is essential for understanding protein functionality and stability. Hydroxy Radical Protein Footprinting (HRPF) is a powerful tool for revealing key aspects of protein HOS. It is used to map protein-protein and protein-ligand interactions, including antibody binding regions, aggregation sites, and the characterization of allosteric inhibitors. HRPF provides high-resolution insights while maintaining near-native protein conditions, making it a practical and accessible tool for optimizing therapeutic proteins.
Presented by Dr. Emily Chea, Applied Research Manager of GenNext Technologies, Inc.
Dr. Emily Chea received her PhD while studying with Professor Dr. Lisa Jones at the University of Maryland, Baltimore. Emily has made significant contributions to the development of HRPF, with particular emphasis on in-cell FPOP (IC-FPOP) for proteome-wide structural biology characterization of cellular drug–target interactions. Currently, Emily is a key member of the GenNext scientific team in her role as an Applied Research Scientist Manager.
Dr. Sharp discusses a covalent labeling technique called fast photochemical oxidation of proteins (FPOP), detailing the workflow, its application in research, and how a new platform is designed to enhance user-friendliness. The Fox Oxidation System streamlines the entire process, from radical generation to sample collection, improving safety, reliability, and ease of use compared to traditional FPOP setups. This innovation makes HRPF accessible to more researchers, allowing them to gain valuable insights into protein structures and interactions with greater ease.
Presented by Dr. Joshua Sharp, Research Institute of Pharmaceutical Sciences at the University of Mississippi.
Dr. Joshua Sharp is an internationally recognized expert in HRPF, pioneering novel MS-based technology to study HOS focusing on the structure-function relationships of proteins and carbohydrates. With more than 21 peer-reviewed articles, Dr. Sharp has also presented numerous invited lectures around the world. Along with acting as the Chief Technology Officer of GenNext, Dr. Sharp is also Associate Professor of Pharmacology, Associate Professor of Chemistry and Biochemistry, and Director of the Glycoscience Center of Research Excellence at the University of Mississippi.
THE SCIENCE OF RADICAL PROTEIN FOOTPRINTING
Many structural biology researchers seek easier and more robust methods for Higher Order Structural (HOS) analysis, recognizing its critical role in biotherapeutic stability, safety, and function. Traditional HOS approaches can be complex, slow, dangerous, and costly, or unreliable and uninformative. With many factors to consider, choosing the best method for your lab can be challenging. Dr. Chea reviews various options, highlighting their pros and cons, and introduces the Flash Oxidation (Fox®) Protein Footprinting System. She explains its applications in epitope mapping, mAb aggregation, and conformational analysis, helping you select the best method for developing safe and effective biotherapeutics.
Presented by Dr. Emily Chea, Applied Research Manager of GenNext Technologies, Inc.
Dr. Emily Chea received her PhD while studying with Professor Dr. Lisa Jones at the University of Maryland, Baltimore. Emily has made significant contributions to the development of HRPF, with particular emphasis on in-cell FPOP (IC-FPOP) for proteome-wide structural biology characterization of cellular drug–target interactions. Currently, Emily is a key member of the GenNext scientific team in her role as an Applied Research Scientist Manager.
FPOP has become a versatile tool in protein chemistry, offering microsecond-speed footprinting to track rapid protein folding and conformational changes, even those missed by slower techniques. It aids in epitope mapping, detects conformational changes in biopharmaceuticals, and studies protein-ligand interactions relevant to diseases like Alzheimer’s. FPOP is effective for membrane proteins and in living cells, providing unique insights. The lecture will compare FPOP with other footprinting methods.
Presented by Dr. Michael Gross, Washington University in St. Louis
At Washington University in St. Louis, the Gross research group focuses mainly on the development of mass spectrometry (MS) in biophysics and structural proteomics, specifically to probe protein-ligand interaction interfaces, affinities, aggregation, and folding/unfolding. The work includes both instrument and method development and application to important proteins and protein complexes. His research group also employs specific chemical reagents to footprint proteins and determines their interfaces and orientations in complex biological settings, hydrogen/deuterium exchange, and native MS.
This hour-long seminar, hosted with The Analytical Scientist, reviews structural biology case studies showcasing the successful application of protein footprinting in solving key problems in biopharmaceutical research. Examples include epitope and paratope mapping, protein interactions, receptor-drug interactions, and protein folding/unfolding. The seminar highlights the benefits of using protein footprinting technology in advancing biopharmaceutical research.
Presented by Professor Michael Gross of the Washington University in St. Louis
At Washington University in St. Louis, the Gross research group focuses mainly on the development of mass spectrometry (MS) in biophysics and structural proteomics, specifically to probe protein-ligand interaction interfaces, affinities, aggregation, and folding/unfolding. The work includes both instrument and method development and application to important proteins and protein complexes. His research group also employs specific chemical reagents to footprint proteins and determines their interfaces and orientations in complex biological settings, hydrogen/deuterium exchange, and native MS.
This 60-minute webinar, in collaboration with Cambridge Healthtech Institute, offers a comprehensive review and instruction in protein footprinting techniques and applications. Learn about various benchtop covalent modification methods and rapid labeling techniques like X-ray synchrotron and Fast Photo-Oxidation of Protein (FPOP) hydroxyl radical protein footprinting (HRPF). Explore their practical implications for biopharmaceutical research, protein interactions, biosimilar development, epitope and paratope mapping, ligand-binding, and conformational change analysis.
Presented by Professor Mark Chance of Case Western Reserve University and Professor Joshua Sharp of the University of Mississippi
Professor Chance is an internationally recognized expert in proteomics and structural biology who has built multiple science and training programs over a 30-year career in academia. He has published over 300 papers with over 15,000 citations. He is currently Vice Dean for Research and holds the titles of Distinguished University Professor, the Charles W. and Iona A. Mathias Professor of Cancer Research, as well as Professor appointments in the Departments of Nutrition, Genetics & Genome Sciences, and General Medical Sciences at the School of Medicine of Case Western Reserve University (CWRU). He is the founding director (since 2005) of the Center for Proteomics and Bioinformatics and founding director of the Center for Synchrotron Biosciences (since 1994). Prior to his appointment at CWRU, Dr. Chance was professor of Physiology and Biophysics at Albert Einstein College of Medicine.
Dr. Joshua Sharp is an internationally recognized expert in HRPF, pioneering novel MS-based technology to study HOS focusing on the structure-function relationships of proteins and carbohydrates. With more than 21 peer-reviewed articles, Dr. Sharp has also presented numerous invited lectures around the world. Along with acting as the Chief Technology Officer of GenNext, Dr. Sharp is also Associate Professor of Pharmacology, Associate Professor of Chemistry and Biochemistry, and Director of the Glycoscience Center of Research Excellence at the University of Mississippi.
This 60-minute webinar offers in-depth instruction on the theory, experimental design, and practical execution of FPOP HRPF experiments. The focus will be on achieving robust and reproducible labeling through real-time radical dosimetry, with a strong emphasis on exemplary applications.
Presented by Professor Michael Gross of the Washington University in St. Louis
At Washington University in St. Louis, the Gross research group focuses mainly on the development of mass spectrometry (MS) in biophysics and structural proteomics, specifically to probe protein-ligand interaction interfaces, affinities, aggregation, and folding/unfolding. The work includes both instrument and method development and application to important proteins and protein complexes. His research group also employs specific chemical reagents to footprint proteins and determines their interfaces and orientations in complex biological settings, hydrogen/deuterium exchange, and native MS.
This presentation provides a detailed discussion on data processing and analytics for HRPF studies. It emphasizes effective methods for peptide and residue-level differential analysis and explores the practical implications of changes in protein higher-order structure due to ligand binding, antibody-antigen interactions, and protein interaction dynamics.
Presented Professor Joshua Sharp of the University of Mississippi
Dr. Joshua Sharp is an internationally recognized expert in HRPF, pioneering novel MS-based technology to study HOS focusing on the structure-function relationships of proteins and carbohydrates. With more than 21 peer-reviewed articles, Dr. Sharp has also presented numerous invited lectures around the world. Along with acting as the Chief Technology Officer of GenNext, Dr. Sharp is also Associate Professor of Pharmacology, Associate Professor of Chemistry and Biochemistry, and Director of the Glycoscience Center of Research Excellence at the University of Mississippi.
EMERGING APPLICATIONS OF RADICAL PROTEIN FOOTPRINTING
Radical protein footprinting (RPF) is a structural biology technique used in cells and organisms. This presentation describes the first example of radical protein footprinting in mammalian blood using sulfate radicals. It shows the first oxidation of proteins by sodium persulfate using a Fox Protein Footprinting System, and the suitability of adenine dosimetry for measuring sulfate radical dose. The research also introduces improved mixing and quenching methods to reduce background oxidation, successfully applying RPF to stabilized whole mouse blood.
Presented by Dr. Joshua Sharp, Research Institute of Pharmaceutical Sciences at the University of Mississippi.
Dr. Joshua Sharp is an internationally recognized expert in HRPF, pioneering novel MS-based technology to study HOS focusing on the structure-function relationships of proteins and carbohydrates. With more than 21 peer-reviewed articles, Dr. Sharp has also presented numerous invited lectures around the world. Along with acting as the Chief Technology Officer of GenNext, Dr. Sharp is also Associate Professor of Pharmacology, Associate Professor of Chemistry and Biochemistry, and Director of the Glycoscience Center of Research Excellence at the University of Mississippi.
Professor Sharp discusses advancements in protein footprinting technology, including the ability to perform experiments on benchtops using UV-catalyzed hydroxyl radicals for protein labeling. He explains how this technique can address challenges in studying dynamic, heterogeneous systems like glycoproteins, aggregation processes, and phosphorylation. Applications include studying HIV glycoproteins, malaria proteins, and monoclonal antibody aggregation.
Presented by Dr. Joshua Sharp, Research Institute of Pharmaceutical Sciences at the University of Mississippi.
Dr. Joshua Sharp is an internationally recognized expert in HRPF, pioneering novel MS-based technology to study HOS focusing on the structure-function relationships of proteins and carbohydrates. With more than 21 peer-reviewed articles, Dr. Sharp has also presented numerous invited lectures around the world. Along with acting as the Chief Technology Officer of GenNext, Dr. Sharp is also Associate Professor of Pharmacology, Associate Professor of Chemistry and Biochemistry, and Director of the Glycoscience Center of Research Excellence at the University of Mississippi.
In-cell and in vivo hydroxyl radical protein footprinting (FPOP) enables studying protein structure and interactions directly within cells or live organisms, crucial for understanding molecular crowding’s effects. The method, using UV lasers to split hydrogen peroxide, labels proteins in situ. Recent developments, including single-cell flow systems and the PIXI platform, increase throughput and enable analysis of complex models like 3D cultures.
Presented by Professor Lisa Jones, University of San Diego.
In the Department of Chemistry and Biochemistry at UCSD, the Jones Lab is a structural proteomics group that uses biochemical, analytical, and biophysical approaches to study protein interactions important in biological processes. Their research focuses on protein footprinting methods coupled with mass spectrometry to identify these interactions. A major focus of their lab is extending FPOP as an in-cell method for monitoring proteins in their native cellular environment. This method would be especially useful for membrane proteins, the largest class of drug targets, which are challenging to study in vitro owing to the difficulty of purifying these proteins. The Jones lab has further expanded FPOP for in vivo analysis in C. elegans, an animal model for human disease.
This hour-long seminar reviews recent advancements in in-cell and whole organism FPOP. It offers a detailed discussion on experimental design and format, highlighting the strengths and benefits of these methods compared to in vitro FPOP. The seminar also covers the implications for protein HOS research and drug development, featuring real research examples from eukaryotic cells and C. elegans.
Presented by Professor Lisa Jones, University of San Diego.
In the Department of Chemistry and Biochemistry at UCSD, the Jones Lab is a structural proteomics group that uses biochemical, analytical, and biophysical approaches to study protein interactions important in biological processes. Their research focuses on protein footprinting methods coupled with mass spectrometry to identify these interactions. A major focus of their lab is extending FPOP as an in-cell method for monitoring proteins in their native cellular environment. This method would be especially useful for membrane proteins, the largest class of drug targets, which are challenging to study in vitro owing to the difficulty of purifying these proteins. The Jones lab has further expanded FPOP for in vivo analysis in C. elegans, an animal model for human disease.
Mark Chance discussed his vision for proteomics and structural biology, focusing on megadalton protein complexes and the technologies needed to study them. He highlighted the importance of combining techniques like NMR, crystallography, cryo-EM, and mass spectrometry for protein interaction insights. He explained hydroxyl radical footprinting, a method for mapping protein surfaces and how it has advanced drug development. He shared examples from Rodeo Therapeutics, CASMA, and Foghorn Therapeutics, showcasing its practical applications.
Presented by Professor Mark Chance, Case Western Reserve University.
Professor Chance is an internationally recognized expert in proteomics and structural biology who has built multiple science and training programs over a 30-year career in academia. He has published over 300 papers with over 15,000 citations. He is currently Vice Dean for Research and holds the titles of Distinguished University Professor, the Charles W. and Iona A. Mathias Professor of Cancer Research, as well as Professor appointments in the Departments of Nutrition, Genetics & Genome Sciences, and General Medical Sciences at the School of Medicine of Case Western Reserve University (CWRU). He is the founding director (since 2005) of the Center for Proteomics and Bioinformatics and founding director of the Center for Synchrotron Biosciences (since 1994). Prior to his appointment at CWRU, Dr. Chance was professor of Physiology and Biophysics at Albert Einstein College of Medicine.