Articles by Robert C. Parry in JoVE
LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism Andrew J. Worth*1,2, Dylan M. Marchione*2,3, Robert C. Parry1,2, Qingqing Wang2,3, Kevin P. Gillespie2,3, Noelle N. Saillant4, Carrie Sims4, Clementina Mesaros1,2, Nathaniel W. Snyder5, Ian A. Blair1,2 1Center for Cancer Pharmacology, University of Pennsylvania, 2Center for Excellence in Environmental Toxicology, University of Pennsylvania, 3Penn SRP and Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, 4Division of Traumatology, Department of Surgery, Critical Care and Acute Care Surgery, University of Pennsylvania, 5A.J. Drexel Autism Institute, Drexel University Here we show isolated human platelets can be used as an accessible ex vivo model to study metabolic adaptations in response to the complex I inhibitor rotenone. This approach employs isotopic tracing and relative quantification by liquid chromatography-mass spectrometry and can be applied to a variety of study designs.
Other articles by Robert C. Parry on PubMed
Production of Stable Isotope-labeled Acyl-coenzyme A Thioesters by Yeast Stable Isotope Labeling by Essential Nutrients in Cell Culture Analytical Biochemistry. Apr, 2015 | Pubmed ID: 25572876 Acyl-coenzyme A (CoA) thioesters are key metabolites in numerous anabolic and catabolic pathways, including fatty acid biosynthesis and β-oxidation, the Krebs cycle, and cholesterol and isoprenoid biosynthesis. Stable isotope dilution-based methodology is the "gold standard" for quantitative analyses by mass spectrometry. However, chemical synthesis of families of stable isotope-labeled metabolites such as acyl-CoA thioesters is impractical. Previously, we biosynthetically generated a library of stable isotope internal standard analogs of acyl-CoA thioesters by exploiting the essential requirement in mammals and insects for pantothenic acid (vitamin B5) as a metabolic precursor for the CoA backbone. By replacing pantothenic acid in the cell medium with commercially available [(13)C3(15)N1]-pantothenic acid, mammalian cells exclusively incorporated [(13)C3(15)N1]-pantothenate into the biosynthesis of acyl-CoA and acyl-CoA thioesters. We have now developed a much more efficient method for generating stable isotope-labeled CoA and acyl-CoAs from [(13)C3(15)N1]-pantothenate using stable isotope labeling by essential nutrients in cell culture (SILEC) in Pan6-deficient yeast cells. Efficiency and consistency of labeling were also increased, likely due to the stringently defined and reproducible conditions used for yeast culture. The yeast SILEC method greatly enhances the ease of use and accessibility of labeled CoA thioesters and also provides proof of concept for generating other labeled metabolites in yeast mutants.
Comprehensive Curation and Analysis of Fungal Biosynthetic Gene Clusters of Published Natural Products Fungal Genetics and Biology : FG & B. Apr, 2016 | Pubmed ID: 26808821 Microorganisms produce a wide range of natural products (NPs) with clinically and agriculturally relevant biological activities. In bacteria and fungi, genes encoding successive steps in a biosynthetic pathway tend to be clustered on the chromosome as biosynthetic gene clusters (BGCs). Historically, "activity-guided" approaches to NP discovery have focused on bioactivity screening of NPs produced by culturable microbes. In contrast, recent "genome mining" approaches first identify candidate BGCs, express these biosynthetic genes using synthetic biology methods, and finally test for the production of NPs. Fungal genome mining efforts and the exploration of novel sequence and NP space are limited, however, by the lack of a comprehensive catalog of BGCs encoding experimentally-validated products. In this study, we generated a comprehensive reference set of fungal NPs whose biosynthetic gene clusters are described in the published literature. To generate this dataset, we first identified NCBI records that included both a peer-reviewed article and an associated nucleotide record. We filtered these records by text and homology criteria to identify putative NP-related articles and BGCs. Next, we manually curated the resulting articles, chemical structures, and protein sequences. The resulting catalog contains 197 unique NP compounds covering several major classes of fungal NPs, including polyketides, non-ribosomal peptides, terpenoids, and alkaloids. The distribution of articles published per compound shows a bias toward the study of certain popular compounds, such as the aflatoxins. Phylogenetic analysis of biosynthetic genes suggests that much chemical and enzymatic diversity remains to be discovered in fungi. Our catalog was incorporated into the recently launched Minimum Information about Biosynthetic Gene cluster (MIBiG) repository to create the largest known set of fungal BGCs and associated NPs, a resource that we anticipate will guide future genome mining and synthetic biology efforts toward discovering novel fungal enzymes and metabolites.