Dave Muddiman

Mass spectrometry is an extraordinarily powerful bioanalytical technique that has had a profound impact on our molecular understanding of human health and disease. Major advances in mass analyzer technology, dissociation techniques, lasers, and ionization methods are largely attributed to the central role that mass spectrometry plays in the field of systems biology. While mass spectrometry has evolved over the last century into a highly effective analytical tool, there remain significant opportunities for innovation, allowing an even more diverse array of biological questions to be addressed. This proposal is centered on the development of new ionization methods for biological mass spectrometry to enable tissue imaging across several classes of biological molecules. The short term objective of this proposal is to further develop and fundamentally understand this innovative ionization method using real biological systems. These results will provide a solid foundation from which biological applications will directly benefit. In this mindset, we will develop and apply these new ionization methods to tissue imaging in model organisms to gain mechanistic insights into, 1) ischemic stroke; 2) wound healing; and 3) cardiometabolic disease. The long-term objective is to establish these new ionization methods as an enabling bioanalytical technology to effectively address questions in human health and disease. Public Description of Proposed Research Mass spectrometry (MS), the science related to the �������weighing of molecules�������, has had a profound impact on the study of human health and disease including cancer, heart disease, neurodegenerative diseases, neural development, and auto-immune diseases. A prerequisite of MS is to convert neutral molecules into charged species (ions) such that they can be �������weighed������� by the mass spectrometer and identified by advanced analytical techniques. The focus of this research is to develop new ionization methods allowing a more diverse array of contemporary biomedical questions to be addressed. This will include the imaging of tissues to ultimately provide new biological insights into stroke, wound healing and cardiometabolic disease.

AIMS. Tailoring AAVs to specific patient cohorts via gene or capsid protein engineering results in a broad spectrum of biomolecular properties and hence therapeutically-relevant CQAs. This poses the need for processing technologies and design principles that are capsid-agnostic. Our bioprocess-focused hypothesis is that if (1) we elucidate the biomolecular-level rules governing AAV expression by HEK293 cells in industrial bioreactors and leverage them to identify optimal harvest conditions, (2) implement a flow-through purification strategy that does not rely on adsorption and harsh elution to isolate and concentrate ����������������full��������������� AAVs, and (3) integrate these AAV-tailored upstream (1) and downstream (2) technologies in a process equipped with in/at- line biosensors real-time monitoring, then a platform AAV bioprocess for affordable manufacture of GTPs to small patient cohorts is possible.

The mission of the Center for Human Health and the Environment (CHHE) is to advance understanding of environmental impacts on human health. Through a systems biology framework integrating all levels of biological organization, CHHE aims to elucidate the fundamental mechanisms through which environmental exposures/stressors interface with biomolecules, pathways, the genome, and epigenome to influence human disease. CHHE will develop three interdisciplinary research teams that represent NC State������������������s distinctive strengths. CHHE will implement specific mechanisms to promote intra- and inter-team interactions and build interdisciplinary bridges to advance basic science discovery and translational research in environmental health science along the continuum from genes to population. These teams are; - The Molecular/Cellular-Based Systems and Model Organisms Team will utilize cutting edge molecular/cellular-based systems and powerful vertebrate and invertebrate model organisms to define mechanisms, pathways, GxE interactions, and individual susceptibility to environmental agents. - The Human Population Science Team will integrate expertise on environmental exposures, epidemiology, genomics and epigenomics to identify key human pathways and link exposure and disease across populations. - Bioinformatics Team will develop novel analytics and computational tools to translate Big Data generated across high-throughput and multiscale experiments into systems-level discoveries To further increase the impact and translational capacity of these teams, CHHE will develop three new facility cores that will provide instrumentation, expertise, and training to facilitate basic mechanism- to population-based research. - The Integrative Health Sciences Facility Core will expand the ability of CHHE members to translate basic science discoveries across species and provide mechanistic insights into epidemiological studies by partnering with: a) NC State������������������s Comparative Toxicogenomics Database (CTD); b) East Carolina University Brody School of Medicine and c) NC Dept. of Health and Human Services. - The Comparative Pathobiology Core will be located at NC State������������������s top-ranked College of Veterinary Medicine and its nationally recognized veterinary pathology group to facilitate assessment of the effects of environmental stressors in the many model organisms utilized by CHHE members. - The Systems Technologies Core will introduce state-of-the-art proteomics capabilities and dedicated bioinformatics support to expand the ability of CHHE members to analyze the Next Generation Sequencing data involving the genome, transcriptome and epigenome. As a land-grant university, NC State has an extensive and active Cooperative Extension Service network throughout North Carolina. CHHE will utilize this unique network to develop a highly effective, multi-directional Community Outreach and Engagement Core to disseminate findings that will contribute to addressing disparity in exposures and health outcomes and to educate communities about environmental influences on health. A strong Career Development Core for early stage scientists that is coordinated with a robust Pilot Project Program will support cutting-edge, collaborative and multidisciplinary environmental health projects to enhance the research success and impact of our membership. Through these activities and the purposeful interfacing of different disciplines CHHE will build on NC State������������������s unique research and community outreach strengths to become a premier transformative and synergistic EHS Core Center.

Mass spectrometry imaging (MSI) provides unprecedented detail of molecular distributions across ex vivo tissue samples. Although MSI has the capability to map molecules ranging from large proteins to small metabolites, these data only describe the tissue status at a single time point and lack any information on how these molecules interact to provide metabolic function. Typical functional studies derive kinetic data from administration of isotope-labeled substrates and monitoring label transit in tissue samples harvested over a specified time-course. Isotope kinetics tracked by MSI, however, have some unique requirements to in order to perform a similar functional study and exploit the full power of the technique to provide location-specific metabolite flux. This proposal will develop an MSI-based method to track isotope metabolic activity through the use of a timed infusion of isotopologues of glycine to measure the rate of glutathione and serine flux within each 50 x 50 x 10 ������������m image voxel. This timed infusion allows a voxel-by-voxel determination of metabolic rate that is not possible by other methods. In addition, since metabolic flux and pathway selection is influenced both by tissue perfusion and oxygenation, we also will develop MSI-based methods to measure tissue perfusion and hypoxia in the same samples. Isotopologues of pimonidazole will be used to map regions of both chronic and cycling hypoxia that can be tied directly to metabolic activity. To demonstrate the feasibility of these techniques in different tissue types, we will map functional heterogeneity across mouse liver and mammary 4T1 tumors. As the MSI method uses frozen thin-sections, histochemical data from adjacent sections can be used to tie traditional tissue markers to metabolic function. The methods described herein will be demonstrated on the glutathione and serine pathways but can be used to study functional heterogeneity in any tissue and any metabolic network with proper selection of substrates.

This project at North Carolina State University will be led by David C. Muddiman. This project is part of a larger small-group study to evaluate the variability associated with operator-associated variability in measuring released glycans from a monoclonal antibody sample. This project will procure an integrated LC-SLIM-QTOF-MS from Agilent Technologies. This approach will be used to measure and quantitatively evaluate the glycan profile of a reference monoclonal antibody using glycan standards as well as the glycan profile of a reference monoclonal antibody where the release was performed at a central laboratory. After this project is completed, the Agilent/MobilIon LC-SLIM-QTOF-MS and GlycoHunter will be available on a priority basis for NIIMBL-related technology and workforce activities, within any necessary constraints of the host institution, to be negotiated with NIIMBL.