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Principal Investigator: Leesa Deterding
Organization: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
Fiscal Year: 2020
Award: $1,064,280
Funding agency: National Institute of Environmental Health Sciences
We have analyzed a variety of molecules obtained from various sources to get the quantitative information. Additionally, we are developing methods to improve the quantitative information that can be gained.
1. Method Development. The use of label-free and labeling approaches for quantitative proteomics studies has been implemented. These methodologies involve both data dependent (DDA) and data-independent analyses (Mse). Additionally, we are incorporating ion mobility methods in an effort to gain further information. Ion mobility is a useful tool to aid in mass spectrometry applications because it allows for the measurement of the collisional cross section of a molecule and gives information about the three-dimensional shape of a compound in the gas phase. Ion mobility separates ions based on their differential mobility through a buffer gas based on the ions shape, charge, and mass. We have also put effort into the development of lipidomics and untargeted metabolomics. Both of these projects involve the identification as well as the quantitation of compounds; thus, these are included in both the identification project and the quantitation project.
2. Eicosanoid Studies. Eicosanoids and related fatty acid metabolites serve as signaling molecules and are intricately involved in inflammation and cardiovascular health. The level of eicosanoids and eicosanoid metabolites are thought to be involved in many diseases. We are involved in a variety of projects measuring these compounds using mass spectrometry. We use liquid chromatography tandem mass spectrometry to analyze a panel of 71 of these molecules which has allowed us to collaborate with several intramural and extramural researchers. We are also developing an untargeted approach to analyze these molecules on another instrument.
3. Steroid Studies. Steroid hormones are widely distributed in nature and are potent signaling molecules. As such, they are of interest to several researchers within the institute. Steroids are often present at low concentrations and exhibit low response in electrospray ionization. We have transferred the LC-MS/MS methodology from a quadrupole instrument to a Q-trap platform because it has provided better sensitivity of the analytes. Ion mobility parameters were tuned for steroids on the 6500+. These will be tested in samples from plasma to see if signal-to-noise is improved. We also developed the separation of isobaric steroids by SFC. SFC-MS/MS produces an order of magnitude lower baseline versus LC-MS/MS of standard solutions of these analytes. Experiments with sample matrix are ongoing.
4. Bile acid studies. In collaboration with the Zeldin laboratory, we have profiled bile acids in Cyp2c transgenic mice. For this project, we developed LC-MS/MS and SFC-MS/MS methods for profiling of unconjugated and taurine conjugated bile acids. In addition, we developed a derivatization protocol for unconjugated bile acids allowing for superior separation and lower limits of detection. We determined there was platform-dependent variability in the analyses (i.e. greater variance in measurements was seen on a triple quadrupole platform than on a Q-Trap platform).
5. BALF Studies. Single nucleotide polymorphisms (SNPs) in low-density lipoprotein receptor-related protein 1 (LRP1) are associated with human pulmonary function in GWAS. Using murine models, we investigated the effect of genetic disruption of the LRP1 gene in smooth muscle cells on pulmonary function in nave animals and after exposure to bacterial lipopolysaccharide (LPS) or house dust mite extract (HDME). Disruption of Lrp1 in smooth muscle cells leads to an increase in tissue resistance, elastance, and tissue elastance at baseline. Further, smooth muscle disruption of Lrp1 increases airway responsiveness as measured by increased total lung resistance and airway resistance after methacholine. Immune cell counts in the bronchoalveolar lavage were increased in animals with Lrp1 disruption. The difference in airway responsiveness by genotype observed in nave animals was not observed following LPS or HDME exposure. To further explore the mechanisms contributing to changes in pulmonary function, we identified several ligands dysregulated with Lrp1 disruption in smooth muscle cells. These data suggest dysregulation of LRP1 in smooth muscle cells affects baseline pulmonary function and airway responsiveness. These data help establish LRP1 as the causal gene at this GWAS locus. The specific role of the MSRSG was to perform quantitative proteomics of bronchoalveolar lavage from animals that had tissue specific Lrp1 disruption. A manuscript submitted to the American Journal of Respiratory Cell and Molecular Biology is in revision.
6. NAD Studies. NAD is a cofactor for hundreds of metabolic reactions in all cell types, and plays an essential role in metabolism, DNA repair, and aging. How NAD metabolism is impacted by the environment remains unclear. Per a request from the laboratory of Dr. Xiaoling Li, the MSRSG has recently developed an LC-MS-based panel for the relative quantitation of metabolites in the NAD pathway. The method was developed to include 8 compounds in the pathway and has been optimized for the analyses of samples arising from cell culture, media, serum, feces, and multiple tissues. We were able to use this approach in the discovery of cooperation between bacteria and mammalian cells wherein bacteria contribute to host NAD biosynthesis. Mechanistically, a microbial nicotinamidase (PncA) can convert nicotinamide to nicotinic acid, a precursor in the alternative deamidated NAD salvage pathway. Using stable isotope tracing and microbiota-depleted mice, we demonstrate that this bacteria-mediated deamidation contributes substantially to the NAD-boosting effect of oral nicotinamide and nicotinamide riboside supplementation in several tissues. Collectively, our findings reveal an important role of bacteria-enabled deamidated pathway in host NAD metabolism. Specifically, the MSRSG did all the measurements of NAD and its precursors including all the isotope tracing experiments.
7. Comparison Proteomics Study. In collaboration with the Doetsch lab, we are comparing the proteins expressed in HBEC lung epithelial cells and MCF10A breast epithelial cells before and after overexpression of Endonuclease III-like protein 1 (NTHL1), a bifunctional DNA glycosylase.
8. UDP-Hexose In collaboration with the laboratory of Dr. Don Cook, the MSRSG developed a method for the relative quantitation of UDP-hexose from bronchoalveolar lavage fluid. This method was used to measure UDP-hexose under a variety of challenge conditions. We studied the role of UDP-glucose (UDP-G), a nucleotide sugar that we found to be selectively released into the airways of allergen-sensitized mice upon their subsequent challenge with that same allergen. A manuscript has been submitted to Journal of Clinical Investigation and is in the revision process.
Additionally, the MSRSG has performed UDP-hexose quantitation from lavage fluid from mice that have been challenged with polyIC as a surrogate/mimetic of viral and/or SARS-CoV-2 infection.
9. TMT studies. We have used this strategy to investigate possible correlation between plasma proteins and airway dysfunction in established mouse models of LPS-enhanced asthma. These analyses showed that a few proteins of interest were significantly altered in plasma
This project involves research on human coronavirus, novel coronavirus, COVID-19, Severe Acute Respiratory Syndrome coronavirus disease, SARS coronavirus, SARS-coronavirus-2, SARS-cov-2, SARS-cov2, SARS-related coronavirus 2, Severe acute respiratory syndrome coronavirus 2, SARS-Associated Coronavirus, SARS-cov, or SARS-Related Coronavirus.
Terms: <2-amino-ethanesulfonic acid><2019 novel coronavirus><2019-nCoV><3-D><3-Dimensional><3-Pyridinecarboxamide><3-Pyridinecarboxylic Acid><3D><Adopted><Affect><Aging><Airway Resistance><Allergens><American><Amino Acids><Anabolism><Animals><Apo E Receptor><ApoE Receptor><Apolipoprotein E Receptor><Area><Asthma><Bacteria><Bile Acids><Biological><Blood Plasma><Blood Serum><Body Tissues><Breast Epithelial Cells><Bronchial Asthma><Bronchioalveolar Lavage><Bronchoalveolar Lavage><Bronchoalveolar Lavage Fluid><Bronchopulmonary Lavage><Buffers><COVID-19><COVID19><Cell Communication and Signaling><Cell Count><Cell Culture Techniques><Cell Number><Cell Signaling><Cellular biology><Charge><CoV emergence><Collaborations><Culture Media><D-Glucose><DNA Damage Repair><DNA Molecular Biology><DNA N-glycosidase><DNA Repair><DNA glycosylase><Data><Data Analyses><Data Analysis><Detection><Development><Dextrose><Disease><Disorder><Dysfunction><E coli DNA endonuclease III><E coli DNA repair endonuclease III><E coli NTH endonuclease><E coli NTH protein><E coli endonuclease III><E. coli DNA endonuclease III><E. coli DNA repair endonuclease III><E. coli NTH endonuclease><E. coli NTH protein><E. coli endonuclease III><EXTMR><Eicosanoids><Electrospray Ionization><Environment><Epithelial Cells><Evaluation><Exhibits><Exposure to><Extramural><Extramural Activities><Fatty Acids><Feces><Functional disorder><G(s), alpha Subunit><G(s), α Subunit><G(s)alpha><G(s)α><GTP-Binding Protein alpha Subunits, Gs><GTP-Binding Protein α Subunits, Gs><GWA study><GWAS><Gases><Genes><Genetic><Genotype><Glucose><Goals><Gs alpha Family G-Protein><Gsα><Gαs><HCoV><Hexoses><House Dust Mites><Housedust Mites><Human><Immune><Immunes><Infection><Inflammation><Institutes><Intermediary Metabolism><Intracellular Communication and Signaling><Investigators><Involuntary Muscle><Ions><Irrigation><Isotopes><Journals><LDL-Receptor Related Protein 1><Label><Laboratories><Lavage><Leiomyocyte><Ligands><Link><Lipopolysaccharides><Lipoprotein Receptor><Liquid Chromatography><Liquid substance><Low Density Lipoprotein Receptor-Related Protein><Low-Density-Lipoprotein Receptor-Related Protein-1><Lung><Lung Lavage><Lung Respiratory System><MCF-10A><MCF10A><MCF10A cells><Magazine><Mammalian Cell><Manuscripts><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Measurement><Measures><Mediating><Metabolic><Metabolic Processes><Metabolism><Methodology><Methods><Mice><Mice Mammals><Modern Man><Molecular Biology><Murine><Mus><NIEHS><National Institute of Environmental Health Sciences><Nature><Niacin><Niacinamide><Nicotinamidase><Nicotinamide><Nicotinamide Amidohydrolase><Nicotinamide Deamidase><Nicotinamidum><Nicotinic Acids><Nicotinic acid amide><Nicotylamide><Noise><Nonvaginal irrigation><Nonvaginal lavage><Oral><Pathway interactions><Pellagra-Preventing Factor><Peptides><Performance><Phase><Physiopathology><Plasma><Plasma Proteins><Plasma Serum><Play><Process><Proteins><Proteomics><Protocol><Protocols documentation><Pyroglyphidae><Reaction><Regulatory Ns Protein><Research><Research Personnel><Researchers><Resistance><Reticuloendothelial System, Serum, Plasma><Role><SARS><SARS Virus><SARS corona virus><SARS coronavirus><SARS coronavirus disease><SARS-Associated Coronavirus><SARS-CoV><SARS-CoV disease><SARS-CoV-2><SARS-CoV2><SARS-Related Coronavirus><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related coronavirus 2><Sampling><Scanning><Science><Series><Serum><Severe Acute Respiratory Syndrome><Severe Acute Respiratory Syndrome CoV disease><Severe Acute Respiratory Syndrome Virus><Severe Acute Respiratory Syndrome corona virus><Severe Acute Respiratory Syndrome coronavirus><Severe Acute Respiratory Syndrome coronavirus disease><Severe acute respiratory syndrome coronavirus 2><Shapes><Signal Transduction><Signal Transduction Systems><Signaling><Signaling Molecule><Single Base Polymorphism><Single Nucleotide Polymorphism><Smooth Muscle><Smooth Muscle Cells><Smooth Muscle Myocytes><Smooth Muscle Tissue Cell><Source><Stable Isotope Labeling><Steroid Compound><Steroids><Stimulatory Gs G-Protein><Sum><Tauphon><Taurine><Testing><Therapeutic Steroid Hormone><Tissues><Transgenic Mice><Unscheduled DNA Synthesis><Viral><Vitamin B 3><Vitamin B3><Vitamin PP><Work><Wuhan coronavirus><alpha Subunit Stimulatory GTP-Binding Protein><alpha-2-Macroglobulin Receptor><alpha-Gs><alpha2-Macroglobulin Signaling Receptor><aminoacid><base><biological signal transduction><biosynthesis><bronchopulmonary lavage therapy><cardiovascular health><causal allele><causal gene><causal mutation><causal variant><causative mutation><causative variant><cell biology><cell culture><cell type><clinical investigation><cofactor><cooking><corona virus disease 2019><coronavirus disease 2019><coronavirus emergence><cost efficient><data acquisition><data 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supplementation><nicotinamide-beta-riboside><novel CoV><novel coronavirus><other than vaginal Douching><other than vaginal Irrigation><overexpress><overexpression><pathophysiology><pathway><pulmonary><pulmonary function><resistant><respiratory><response><severe acute respiratory syndrome-CoV><single nucleotide variant><social role><stable isotope><steroid hormone><stool><sugar nucleotide><tandem mass spectrometry><three dimensional><tool><whole genome association analysis><whole genome association studies><whole genome association study><α-Gs>