Peter Nemes, Ph.D. - US grants

? DESCRIPTION: Formation of a heterogeneous population of cells is critical to embryogenesis and normal development; cell heterogeneity gives rise to different types of tissues and is also implicated in the onset and progression of diseases. Understanding cell heterogeneity holds important implications in human health, but requires specialized approaches, such as mass spectrometry (MS), that deliver high detection selectivity and sensitivity. A substantial portion o bioanalytical methodologies including single-cell MS, however, work ex vivo or rely on long-term cell cultures. These conditions potentially change the proteomic and, especially, the metabolomic composition and function of cells and complicate the interpretation of results on cell-to-cell differences. Herein we propose to introduce an in situ single-cell analysis platform based on MS and uncover metabolomic and proteomic differences among single cell that form in the actual, live, freely developing embryo of the South African clawed frog (Xenopus laevis) and the zebrafish (Danio rerio), both of which are well-established vertebrate models in cell and developmental biology and human disease research. Key aspects of this platform are in situ and high-throughput operation to identify and measure any given cell of interest directly in the specimen, a capability for repeated measurement of cell morphology and biochemical composition, label-free identification of diverse types of metabolites and peptides without having to know their presence before experiments, and scalability to different cell dimensions; thus, culturing and isolation of single cells are avoided. The platform is validated using single cells i the 16- and 32-cell Xenopus embryo that have highly reproducible tissue fates and exhibit known transcriptomic cell heterogeneity both in the horizontal and vertical body plan. Metabolites and peptides are measured in strategically selected identified cells along the dorso- ventral and animal-vegetal axes, and the resulting complex chemical information is mathematically evaluated to uncover similarity between cells that have the same identity in different embryos. Furthermore, we propose to extend single-cell investigations to the 1- to 16-cell zebrafish embryo, where cells are inherently smaller and cell heterogeneity is less understood during early development. Besides developing a new technology, the anticipated results provide new information on the spatiotemporal dynamics of cell heterogeneity in the actual developing embryo, providing important biochemical data for cell and developmental biology and human health. These outcomes are matched well with the goals of RFA-RM-13-021, Exceptionally Innovative Tools and Technologies for Single Cell Analysis. The proposed approach is adaptable to different physical and temporal resolutions, broad types of biomolecules, and different model systems to aid health research and the development of next-generation pharmaceuticals.

An award is made to the George Washington University to develop a device that will be able to measure the production of broad types of biomolecules in multiple, individual cells of the developing embryo. This project will enhance education by integrating biology and chemistry, and provide new investigative tools to raise creative research opportunities in basic and applied research. Development of the single-cell mass spectrometer will require regular interactions between analytical chemists, biologists, mass spectrometrists, and curators of data repositories, essentially creating an interdisciplinary environment for students and researchers to accomplish training beyond the classical curriculum in these disciplines. By demonstrating the device at the George Washington University and discussing its design, performance, and use at national conferences and publications, this work will broaden scientific literacy and inform of the availability of the device to a broader base of users. Data resulting from measurements on the production of biomolecules during embryogenesis will be disseminated in publicly accessible data repositories, providing a larger number of users with access to facilitate research and education in cell and developmental biology and neuroscience. Notably, the combination of these scientific and outreach elements, including participation of local high-school students in the project, will enhance research and education at the interface of biology, instrument development, and analytical chemistry, providing interdisciplinary solutions to current and future challenges in science and education.

Characterization of biomolecular expression in single cells of the embryo will provide new insights into basic biochemical mechanisms that orchestrate embryonic development, the complex suite of processes by which a fertilized egg gives rise to an entire, fully functioning organism such as the frog, fish, or human. Although it has been technologically feasible to measure genes and transcripts in single embryonic cells, a lack of analytical technology has so far hindered the characterization of proteins, peptides, and metabolites in single embryonic cells. This project will provide one such technological innovation, a single-cell mass spectrometer, by combining traditional tools in cell and developmental biology and neuroscience with next-generation instrumentation from bioanalytical chemistry. Specifically, optical microscopy, microinjection, microcapillary electrophoresis, and nanoelectrospray ionization will be adapted to high-resolution tandem mass spectrometry to determine the production of biomolecules, proteins to metabolites, in multiple cells of the embryo using the frog Xenopus laevis and zebrafish as models. The instrument will be developed in collaboration with leading mass spectrometric industry, biologists, and students, and will be tested by biologists and students working with these important developmental models.

Abstract The metabolome provides a unique window to monitor a system's molecular state as a result of both intrinsic and extrinsic events. Using ultrahigh-performance liquid chromatography (UPLC) high-resolution mass spectrometry (HRMS), the gold standard technology of metabolomics, it is possible to measure hundreds of metabolites in thousands-to-millions of cells to enhance the signal for trace-level compounds. Recent advances in HRMS technology have extended these measurements to also trace-level detection for rare or precious samples, where averaging is not feasible or hinders results interpretation. Despite the availability of high-sensitivity HRMS, a bottleneck in metabolomics is a lack of software tools capable of detecting trace-level signals in the resulting complex metabolomics data. The proposed work fills this technological gap by developing a software suite that surveys HRMS data sets for trace-level signals (Specific Aim 1) and helps find correlations between metabolite variances (Specific Aim 2). The approach stems from manual data processing protocols that have been established and validated for high-sensitivity analyses as well as critical transition models in physics that efficiently indicate transition points in a network. The software is validated using HRMS data sets that have been acquired for differentiating cells in the early developing embryo of the South African clawed frog (Xenopus laevis), a powerful model in cell and developmental studies, and functional experiments that test the developmental significance of select metabolites. The work includes tests designed to ensure the compatibility of the software to HRMS data from broad types of mass spectrometry instrumentation and different types of metabolomics studies, including neuroscience and drug metabolism and data deposited in MetabolomicsWorkbench, a public metabolomics data repository. The final product is metabolomics software that is applicable to broad types of metabolomics investigations. Besides providing new software, the data that are obtained during this work provide new information on metabolomic changes underlying cell differentiation in the developing embryo. The outcomes of the proposed work are matched with the goals of RFA-RM-15-021, ?Metabolomics Data Analysis (R03).? The proposed software is scalable to HRMS data from diverse instrument vendors, aids the identification of trace-level metabolite signals, and facilitates the analysis of metabolite- metabolite correlations in the system, which in turn facilitates the design of hypothesis-driven studies to help better understand health.

Non-Technical Paragraph
During normal development of the vertebrate embryo, cells of the embryo must develop into all the different types of tissues of the organism, but the complete set of biomolecules contributing to tissue formation is unknown. This project utilizes advanced instruments that have only recently been developed in analytical chemistry to determine changes in small molecules as individual cells form different types of tissues in the early developing frog embryo, which is an important model of vertebrate embryo development. The resulting data will provide previously unavailable insights into basic biological processes important for the formation of cells, tissues, organs, and organisms. These research efforts serve as the foundation for interdisciplinary training of diverse participants at the host university, as well as conferences, seminars, and national training centers such as Cold Spring Harbor Laboratory, NY. This work will train a new generation of scientists in both biology and chemistry, including many traditionally underrepresented populations in science, to allow them to address current challenges but also to ask new questions in these fields to better understand normal vertebrate embryo development and diseases.

Technical Paragraph
Decades of research has uncovered many genes and gene products with critical roles during development of the vertebrate embryo, but how small molecules (called metabolites) participate in cell developmental processes is not fully known. The PI's laboratory recently discovered metabolites capable of altering the normal dorsal-ventral fate of select stem cells in the early frog (Xenopus laevis) embryo, demonstrating that these molecules, too, are active players during patterning of the vertebrate body. The overall goal of this work is to determine the mechanism of action underlying metabolite-induced cell fate decisions. This will be accomplished through a systems cell biology approach, in which the molecular state of metabolite-injected cells will be characterized using unique single-cell mass spectrometry technologies that were developed and validated in the PI's laboratory. The project will identify how cell-fate altering metabolites perturb close-proximity metabolic networks as well as key proteins of metabolism and known signaling pathways of dorsal-ventral specification. Two single-cell mass spectrometry instruments will be used to perform flux analysis for the injected metabolites and to measure the relative translation of targeted proteins in fluorescently tracked cell clones that form from the metabolite-injected cells in the living frog embryo. The resulting data will identify gene candidates for functional tests via gene knock-down experiments to validate the proposed mechanism of action for metabolite-induced cell fate changes. Understanding small-molecule effects on cell fate commitment raises broad implications in diverse areas of the life sciences. The work will also train underrepresented groups in techniques bridging biology and analytical chemistry.

Abstract Understanding embryonic development requires knowledge of all the molecules produced as the zygote differentiates into the three primary germ layers of the embryo. Four decades of innovative embryological manipulations, testing of gene functions one gene at a time, and recently, Next-Generation Sequencing have identified multiple transcripts and abundant proteins that are essential to the patterning of the vertebrate embryo. However, very little is known about the total array of proteins and their post-translational modifications that contribute to the formation of the germ layers, and next to nothing is known about the contribution of small molecules (called metabolites) to these processes. To date, systems biology has defined the spatial and temporal changes of mRNAs, abundant proteins, and metabolites in the whole embryo, but it has been technologically impossible to utilize high-resolution mass spectrometry (HRMS), the gold standard technology for small molecules, to study hundreds-to-thousands of metabolites and proteins in single embryonic cells in the vertebrate embryo. The proposed research program fills this enormous knowledge and technological gap by utilizing novel single-cell mass spectrometry technologies to understand cell molecular processes that contribute to the formation of the three germ layers required for the successful patterning of the vertebrate frog (Xenopus laevis) embryo, a favorite model in cell/developmental biology. Most recently, single-cell mass spectrometry discovered metabolites capable of altering the normal cell fates of embryonic cells, suggesting that the complete molecular players are not yet fully identified or understood for germ layer induction. The proposed research program will determine this missing link in the understanding of molecular mechanisms governing vertebrate development. This work will integrate quantitative single-cell mass spectrometry, cell fate tracking, and gene knock-down experiments to determine how a targeted set of small-molecular reactions impact the formation of signaling centers required for dorsal axis specification. The outcomes of this interdisciplinary approach will help illuminate the role of the proteome and metabolome for the establishment of these important precursors. Because these molecular processes are highly conserved across vertebrates, the data collected from Xenopus are likely to have high relevance to human structural birth defects. The new biochemical information that will be obtained in individual embryonic cells and their progeny (cell lineage) at several critical developmental time points will also advance other research fields that involve cell differentiation (e.g., of stem cells) and the developmental origins of adult disease.

This award is supported by the Major Research Instrumentation and the Chemistry Research Instrumentation programs. Professor Peter Nemes from the University of Maryland College Park and colleagues Daniel Falvey, Lyle Isaacs, Lisa Taneyhill and Scott Juntti are acquiring a high-resolution, high-pressure liquid chromatograph mass spectrometer with electrospray ionization capabilities (HR-HPLC-ESI-MS). In general, mass spectrometry (MS) is one of the key analytical methods used to identify and characterize small quantities of chemical species embedded in complex samples. In a typical experiment, the components flow into a mass spectrometer where they are ionized into ions and the ions' masses are measured. This highly sensitive technique allows the structure of molecules in complex mixtures to be studied. An instrument with a liquid chromatograph can separate mixtures of compounds before they reach the mass spectrometer. In the electrospray technique a high voltage is applied to a liquid to create an aerosol. This voltage is useful to produce ions from large molecules by avoiding the propensity of macromolecules to fragment when ionized. The acquisition strengthens the research infrastructure at the University and regional area. The instrument broadens participation by involving diverse groups of students in research and research training using this modern analytical technique. The acquisition also provides training opportunities to many undergraduate and graduate students as well as postdoctoral fellows at this institution. The new capability to measure both small biological and organic molecules in a shared Mass Spectrometry Facility has a broad impact on scientists and students in the District of Columbia-Maryland-Virginia region through workshops as well as curriculum modernization and collaborations with Bowie State University.


The award of the mass spectrometer is aimed at enhancing research and education at all levels. It especially impacts studies of metabolic effectors of embryonic development and hormone metabolite activity in nervous system functions. The instrumentation is also used for research on mitochondrial metabolism in the liver as well as molecular mechanisms in neural crest and dental placode cells. In addition, the MS provides information useful for the search of the next generation enzymatic kinetic isotope effects and for studying binders for the valosine-containing protein (VCP) that segregate protein molecules from large cellular structures. The mass spectrometer is also used to inform the design of efficient syntheses for molecular guest compounds. It is also utilized in investigations of electrophilic nitrogen containing species and their reactivity with proteins and nucleic acids.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Abstract Understanding embryonic development requires knowledge of all the molecules produced as the zygote differentiates into the three primary germ layers of the embryo. Four decades of innovative embryological manipulations, testing of gene functions one gene at a time, and recently, Next-Generation Sequencing have identified multiple transcripts and abundant proteins that are essential to the patterning of the vertebrate embryo. However, very little is known about the total array of proteins and their post-translational modifications that contribute to the formation of the germ layers, and next to nothing is known about the contribution of small molecules (called metabolites) to these processes. To date, systems biology has defined the spatial and temporal changes of mRNAs, abundant proteins, and metabolites in the whole embryo, but it has been technologically impossible to utilize high-resolution mass spectrometry (HRMS), the gold standard technology for small molecules, to study hundreds-to-thousands of metabolites and proteins in single embryonic cells in the vertebrate embryo. The proposed research program fills this enormous knowledge and technological gap by utilizing novel single-cell mass spectrometry technologies to understand cell molecular processes that contribute to the formation of the three germ layers required for the successful patterning of the vertebrate frog (Xenopus laevis) embryo, a favorite model in cell/developmental biology. Most recently, single-cell mass spectrometry discovered metabolites capable of altering the normal cell fates of embryonic cells, suggesting that the complete molecular players are not yet fully identified or understood for germ layer induction. The proposed research program will determine this missing link in the understanding of molecular mechanisms governing vertebrate development. This work will integrate quantitative single-cell mass spectrometry, cell fate tracking, and gene knock-down experiments to determine how a targeted set of small-molecular reactions impact the formation of signaling centers required for dorsal axis specification. The outcomes of this interdisciplinary approach will help illuminate the role of the proteome and metabolome for the establishment of these important precursors. Because these molecular processes are highly conserved across vertebrates, the data collected from Xenopus are likely to have high relevance to human structural birth defects. The new biochemical information that will be obtained in individual embryonic cells and their progeny (cell lineage) at several critical developmental time points will also advance other research fields that involve cell differentiation (e.g., of stem cells) and the developmental origins of adult disease.

Abstract The goal of this project is to enhance the diversity of the biomedical research workforce by training a PhD Graduate Student from underrepresented backgrounds to enable their original research and career development at the frontiers of chemistry and biology. The student will learn advanced bioanalytical chemistry and vertebrate embryology while elucidating the mechanism of action underlying cell fate changes by small molecules called metabolites, which the Nemes Research Laboratory has recently discovered. Understanding embryogenesis requires knowledge of all the molecules produced as the zygote differentiates into the three primary germ layers of the embryo. Four decades of innovative embryological manipulations, testing of gene functions one gene at a time, and recently, Next-Generation Sequencing have identified multiple transcripts and abundant proteins that are essential to the patterning of the vertebrate embryo. However, very little is known about the contribution of small molecules called metabolites to the formation of the germ layers and the long-term development and functioning of the embryo. The proposed training?research program fills this knowledge gap in technology and biology by empowering the PhD Graduate Student to conduct original research at the chemistry-biology interface. The student will develop skills in bioanalytical chemistry, specifically quantitative metabolomics by capillary electrophoresis and ultrasensitive electrospray ionization mass spectrometry to enable the characterization of the metabolomic states of cells and tissues. Further, the student will also develop the required biomedical?biological skills to study the developmental and cognitive implications of cell fate decisions, including classical embryological manipulations, cell fate tracking, Xenopus laevis biology, and behavioral assays. The outcomes of this interdisciplinary approach will help illuminate the role of the metabolome for the establishment of these important precursor cells and tissues. Because these molecular processes are highly conserved across vertebrates, the data collected from Xenopus are likely to have high relevance to human structural birth defects. The new biochemical information that will be obtained in individual embryonic cells and their progeny (cell lineage) at several critical developmental time points will also advance other research fields that involve cell differentiation (e.g., of stem cells) and the developmental origins of adult disease. This project will provide immersive cross-disciplinary training?research experience to enable the student to pursue an independent career while diversifying the biomedical research workforce. Nemes-Abstract-1|1