%0 Journal Article %J Nat MedNat MedNat Med %D 2014 %T Regulation of astrocyte activation by glycolipids drives chronic CNS inflammation %A Mayo, L. %A Trauger, S. A. %A Blain, M. %A Nadeau, M. %A Patel, B. %A Alvarez, J. I. %A Mascanfroni, I. D. %A Yeste, A. %A Kivisakk, P. %A Kallas, K. %A Ellezam, B. %A Bakshi, R. %A Prat, A. %A Antel, J. P. %A Weiner, H. L. %A Quintana, F. J. %X Astrocytes have complex roles in health and disease, thus it is important to study the pathways that regulate their function. Here we report that lactosylceramide (LacCer) synthesized by beta-1,4-galactosyltransferase 6 (B4GALT6) is upregulated in the central nervous system (CNS) of mice during chronic experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). LacCer acts in an autocrine manner to control astrocyte transcriptional programs that promote neurodegeneration. In addition, LacCer in astrocytes controls the recruitment and activation of microglia and CNS-infiltrating monocytes in a non-cell autonomous manner by regulating production of the chemokine CCL2 and granulocyte-macrophage colony-stimulating factor (GM-CSF), respectively. We also detected high B4GALT6 gene expression and LacCer concentrations in CNS MS lesions. Inhibition of LacCer synthesis in mice suppressed local CNS innate immunity and neurodegeneration in EAE and interfered with the activation of human astrocytes in vitro. Thus, B4GALT6 regulates astrocyte activation and is a potential therapeutic target for MS and other neuroinflammatory disorders. %B Nat MedNat MedNat Med %7 2014/09/14 %8 Sep 14 %@ 1546-170X (Electronic)1078-8956 (Linking) %G Eng %M 25216636 %! Nature medicineNature medicine %0 Journal Article %J BiomaterialsBiomaterialsBiomaterials %D 2014 %T In vivo performance of a drug-eluting contact lens to treat glaucoma for a month %A Ciolino, J. B. %A Stefanescu, C. F. %A Ross, A. E. %A Salvador-Culla, B. %A Cortez, P. %A Ford, E. M. %A Wymbs, K. A. %A Sprague, S. L. %A Mascoop, D. R. %A Rudina, S. S. %A Trauger, S. A. %A Cade, F. %A Kohane, D. S. %K *Contact Lenses %K *Drug Delivery Systems %K Animals %K Drug Stability %K Glaucoma/*drug therapy %K Intraocular Pressure/drug effects %K Prostaglandins F, Synthetic/*administration & dosage/pharmacology/therapeutic use %K Rabbits %X For nearly half a century, contact lenses have been proposed as a means of ocular drug delivery, but achieving controlled drug release has been a significant challenge. We have developed a drug-eluting contact lens designed for prolonged delivery of latanoprost for the treatment of glaucoma, the leading cause of irreversible blindness worldwide. Latanoprost-eluting contact lenses were created by encapsulating latanoprost-poly(lactic-co-glycolic acid) films in methafilcon by ultraviolet light polymerization. In vitro and in vivo studies showed an early burst of drug release followed by sustained release for one month. Contact lenses containing thicker drug-polymer films demonstrated released a greater amount of drug after the initial burst. In vivo, single contact lenses were able to achieve, for at least one month, latanoprost concentrations in the aqueous humor that were comparable to those achieved with topical latanoprost solution, the current first-line treatment for glaucoma. The lenses appeared safe in cell culture and animal studies. This contact lens design can potentially be used as a treatment for glaucoma and as a platform for other ocular drug delivery applications. %B BiomaterialsBiomaterialsBiomaterials %7 2013/10/08 %V 35 %P 432-9 %8 Jan %@ 1878-5905 (Electronic)0142-9612 (Linking) %G eng %9 Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov't %M 24094935 %2 3874329 %! BiomaterialsBiomaterials %0 Journal Article %J NatureNatureNature %D 2014 %T The metabolite alpha-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR %A Chin, R. M. %A Fu, X %A Pai, M. Y. %A Vergnes, L. %A Hwang, H. %A Deng, G. %A Diep, S. %A Lomenick, B. %A Meli, V. S. %A Monsalve, G. C. %A Hu, E. %A Whelan, S. A. %A Wang, J. X. %A Jung, G. %A Solis, G. M. %A Fazlollahi, F. %A Kaweeteerawat, C. %A Quach, A. %A Nili, M. %A Krall, A. S. %A Godwin, H. A. %A Chang, H. R. %A Faull, K. F. %A Guo, F. %A Jiang, M. %A Trauger, S. A. %A Saghatelian, A. %A Braas, D. %A Christofk, H. R. %A Clarke, C. F. %A Teitell, M. A. %A Petrascheck, M. %A Reue, K. %A Jung, M. E. %A Frand, A. R. %A Huang, J. %K Animals %K Caenorhabditis elegans/*drug effects %K Cell Line %K Enzyme Activation/drug effects %K Enzyme Inhibitors/pharmacology %K Gene Knockdown Techniques %K HEK293 Cells %K Humans %K Jurkat Cells %K Ketoglutaric Acids/*pharmacology %K Longevity/drug effects/genetics/*physiology %K Mice %K Mitochondrial Proton-Translocating ATPases/genetics/*metabolism %K Protein Binding %K TOR Serine-Threonine Kinases/*metabolism %X Metabolism and ageing are intimately linked. Compared with ad libitum feeding, dietary restriction consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits. Recently, several metabolites have been identified that modulate ageing; however, the molecular mechanisms underlying this are largely undefined. Here we show that alpha-ketoglutarate (alpha-KG), a tricarboxylic acid cycle intermediate, extends the lifespan of adult Caenorhabditis elegans. ATP synthase subunit beta is identified as a novel binding protein of alpha-KG using a small-molecule target identification strategy termed drug affinity responsive target stability (DARTS). The ATP synthase, also known as complex V of the mitochondrial electron transport chain, is the main cellular energy-generating machinery and is highly conserved throughout evolution. Although complete loss of mitochondrial function is detrimental, partial suppression of the electron transport chain has been shown to extend C. elegans lifespan. We show that alpha-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by alpha-KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells. We provide evidence that the lifespan increase by alpha-KG requires ATP synthase subunit beta and is dependent on target of rapamycin (TOR) downstream. Endogenous alpha-KG levels are increased on starvation and alpha-KG does not extend the lifespan of dietary-restricted animals, indicating that alpha-KG is a key metabolite that mediates longevity by dietary restriction. Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator and dietary restriction in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing and age-related diseases. %B NatureNatureNature %7 2014/05/16 %V 510 %P 397-401 %8 Jun 19 %@ 1476-4687 (Electronic)0028-0836 (Linking) %G eng %9 Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov't %M 24828042 %! NatureNature %0 Journal Article %J Br J HaematolBr J HaematolBr J Haematol %D 2014 %T Warfarin untargeted metabolomics study identifies novel procoagulant ethanolamide plasma lipids %A Deguchi, H. %A Elias, D. J. %A Trauger, S. %A Zhang, H. M. %A Kalisiak, E. %A Siuzdak, G. %A Griffin, J. H. %K Ethanolamine/blood %K Humans %K Lipids/*blood %K Mass Spectrometry/methods %K Metabolomics/methods %K Warfarin/*blood/pharmacology %B Br J HaematolBr J HaematolBr J Haematol %7 2014/01/24 %V 165 %P 409-12 %8 May %@ 1365-2141 (Electronic)0007-1048 (Linking) %G eng %9 LetterResearch Support, N.I.H., Extramural %M 24450944 %2 3984601 %! British journal of haematologyBritish journal of haematology %0 Journal Article %J Nature Medicine %D 2014 %T
Astrocytes have complex roles in health and disease, thus it is important to study the pathways that regulate their function. Here we report that lactosylceramide (LacCer) synthesized by β-1,4-galactosyltransferase 6 (B4GALT6) is upregulated in the central nervous system (CNS) of mice during chronic experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). LacCer acts in an autocrine manner to control astrocyte transcriptional programs that promote neurodegeneration. In addition, LacCer in astrocytes controls the recruitment and activation of microglia and CNS-infiltrating monocytes in a non-cell autonomous manner by regulating production of the chemokine CCL2 and granulocyte-macrophage colony-stimulating factor (GM-CSF), respectively. We also detected high B4GALT6 gene expression and LacCer concentrations in CNS MS lesions. Inhibition of LacCer synthesis in mice suppressed local CNS innate immunity and neurodegeneration in EAE and interfered with the activation of human astrocytes in vitro. Thus, B4GALT6 regulates astrocyte activation and is a potential therapeutic target for MS and other neuroinflammatory disorders.
%B Nature Medicine %V Sep 14 %G eng %0 Journal Article %J AnalystAnalystAnalyst %D 2013 %T Identification of collagen-based materials in cultural heritage %A Kirby, D. P. %A Buckley, M. %A Promise, E. %A Trauger, S. A. %A Holdcraft, T. R. %K *Culture %K Chemistry Techniques, Analytical/*methods %K Collagen/*analysis/chemistry/metabolism %K Humans %K Museums %K Proteolysis %K Skin/chemistry %K Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization %X All stakeholders in cultural heritage share an interest in fabrication methods and material technology. Until now methods for analysis of organic materials, particularly proteins, have not been widely available to researchers at cultural institutions. This paper will describe an analytical method for the identification of collagen-based materials from soft tissue sources and show examples of its application to diverse museum objects. The method, peptide mass fingerprinting (PMF), uses enzymatic digestion of extracted proteins to produce a mixture of peptides. The mass spectrum of the mixture contains characteristic marker ions-a peptide mass fingerprint-which are compared to species-specific markers from references as the basis of identification. Preliminary results indicate that analysis of materials from aged samples, several different tissue types, and tanned or untanned materials yields comparable PMF results. Significantly, PMF is simple, rapid, sensitive and specific, has been implemented in a museum laboratory, and is being practiced successfully by non-specialists. %B AnalystAnalystAnalyst %7 2013/06/29 %V 138 %P 4849-58 %8 Sep 7 %@ 1364-5528 (Electronic)0003-2654 (Linking) %G eng %9 Research Support, Non-U.S. Gov't %M 23807214 %! The AnalystThe Analyst %0 Journal Article %J BiomacromoleculesBiomacromoleculesBiomacromolecules %D 2013 %T Lysine addressability and mammalian cell interactions of bacteriophage lambda procapsids %A Koudelka, K. J. %A Ippoliti, S. %A Medina, E. %A Shriver, L. P. %A Trauger, S. A. %A Catalano, C. E. %A Manchester, M. %K Amino Acid Sequence %K Bacteriophage lambda/*chemistry/metabolism %K Capsid Proteins/chemistry %K Capsid/*chemistry/metabolism %K Drug Carriers/*chemistry/metabolism %K HeLa Cells %K Humans %K Lysine/*chemistry %K Molecular Sequence Data %K Receptors, Transferrin/metabolism %K Tandem Mass Spectrometry %K Transferrin/chemistry/metabolism %K Virus Internalization %X Chemically or genetically modified virus particles, termed viral nanoparticles (VNPs), are being explored in applications such as drug delivery, vaccine development, and materials science. Each virus platform has inherent properties and advantages based on its structure, molecular composition, and biomolecular interactions. Bacteriophage lambda was studied for its lysine addressability, stability, cellular uptake, and the ability to modify its cellular uptake. lambda procapsids could be labeled primarily at a single residue on the gpE capsid protein as determined by tandem mass spectrometry, providing a unique attachment site for further capsid modification. Bioconjugation of transferrin to the procapsids mediated specific interaction with transferrin receptor-expressing cells. These studies demonstrate the utility of bacteriophage lambda procapsids and their potential use as targeted drug delivery vehicles. %B BiomacromoleculesBiomacromoleculesBiomacromolecules %7 2013/11/21 %V 14 %P 4169-76 %8 Dec 9 %@ 1526-4602 (Electronic)1525-7797 (Linking) %G eng %9 Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, Non-P.H.S. %M 24251756 %! BiomacromoleculesBiomacromolecules %0 Journal Article %J Proc Natl Acad Sci U S AProc Natl Acad Sci U S AProc Natl Acad Sci U S A %D 2013 %T ABHD12 controls brain lysophosphatidylserine pathways that are deregulated in a murine model of the neurodegenerative disease PHARC %A Blankman, J. L. %A Long, J. Z. %A Trauger, S. A. %A Siuzdak, G. %A Cravatt, B. F. %K Animals %K Ataxia/*genetics/*metabolism/pathology/physiopathology %K Behavior, Animal/physiology %K Brain/*metabolism/pathology %K Cataract/*genetics/*metabolism/pathology/physiopathology %K Disease Models, Animal %K Humans %K Lipid Metabolism %K Lysophospholipids/*metabolism %K Metabolic Networks and Pathways %K Mice %K Mice, Inbred C57BL %K Mice, Knockout %K Microglia/metabolism %K Models, Neurological %K Monoacylglycerol Lipases/deficiency/*genetics/*metabolism %K Mutation %K Phenotype %K Polyneuropathies/*genetics/*metabolism/pathology/physiopathology %K Retinitis Pigmentosa/*genetics/*metabolism/pathology/physiopathology %X Advances in human genetics are leading to the discovery of new disease-causing mutations at a remarkable rate. Many such mutations, however, occur in genes that encode for proteins of unknown function, which limits our molecular understanding of, and ability to devise treatments for, human disease. Here, we use untargeted metabolomics combined with a genetic mouse model to determine that the poorly characterized serine hydrolase alpha/beta-hydrolase domain-containing (ABHD)12, mutations in which cause the human neurodegenerative disorder PHARC (polyneuropathy, hearing loss, ataxia, retinosis pigmentosa, and cataract), is a principal lysophosphatidylserine (LPS) lipase in the mammalian brain. ABHD12(-/-) mice display massive increases in a rare set of very long chain LPS lipids that have been previously reported as Toll-like receptor 2 activators. We confirm that recombinant ABHD12 protein exhibits robust LPS lipase activity, which is also substantially reduced in ABHD12(-/-) brain tissue. Notably, elevations in brain LPS lipids in ABHD12(-/-) mice occur early in life (2-6 mo) and are followed by age-dependent increases in microglial activation and auditory and motor defects that resemble the behavioral phenotypes of human PHARC patients. Taken together, our data provide a molecular model for PHARC, where disruption of ABHD12 causes deregulated LPS metabolism and the accumulation of proinflammatory lipids that promote microglial and neurobehavioral abnormalities. %B Proc Natl Acad Sci U S AProc Natl Acad Sci U S AProc Natl Acad Sci U S A %7 2013/01/09 %V 110 %P 1500-5 %8 Jan 22 %@ 1091-6490 (Electronic)0027-8424 (Linking) %G eng %9 Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov't %M 23297193 %2 3557017 %! Proceedings of the National Academy of Sciences of the United States of AmericaProceedings of the National Academy of Sciences of the United States of America %0 Journal Article %J AIDS Res Hum RetrovirusesAIDS Res Hum RetrovirusesAIDS Res Hum Retroviruses %D 2011 %T Short communication: quantitative proteomic plasma profiling reveals activation of host defense to oxidative stress in chronic SIV and methamphetamine comorbidity %A Pendyala, G. %A Trauger, S. A. %A Siuzdak, G. %A Fox, H. S. %K *Oxidative Stress %K *Proteomics %K Animals %K Blood Proteins/*metabolism %K Chronic Disease %K Humans %K Methamphetamine/*administration & dosage %K Simian Acquired Immunodeficiency Syndrome/*blood %X The double epidemic of substance abuse and HIV infection is a multifaceted problem To investigate mechanistic clues to the effects of substance abuse on infected individuals we preformed quantitative proteomic profiling of plasma in a methamphetamine treated nonhuman primate model for AIDS. A nontargeted quantitative approach identified extracellular superoxide dismutase to be significantly upregulated by SIV and methamphetamine treatment, and targeted studies revealed an increase in expression in the antioxidant glutathione S-transferase, thus pointing to a compensatory response to increased oxidative stress in methamphetamine-treated animals. %B AIDS Res Hum RetrovirusesAIDS Res Hum RetrovirusesAIDS Res Hum Retroviruses %7 2010/10/12 %V 27 %P 179-82 %8 Feb %@ 1931-8405 (Electronic)0889-2229 (Linking) %G eng %9 Research Support, N.I.H., Extramural %M 20929344 %2 3045074 %! AIDS research and human retrovirusesAIDS research and human retroviruses %0 Journal Article %J J BacteriolJ BacteriolJ Bacteriol %D 2011 %T Type I signal peptidase and protein secretion in Staphylococcus epidermidis %A Powers, M. E. %A Smith, P. A. %A Roberts, T. C. %A Fowler, B. J. %A King, C. C. %A Trauger, S. A. %A Siuzdak, G. %A Romesberg, F. E. %K Bacterial Proteins/*secretion %K Chromatography, Liquid %K Electrophoresis, Gel, Two-Dimensional %K Enzyme Inhibitors/chemistry/pharmacology %K Mass Spectrometry %K Membrane Proteins/antagonists & inhibitors/*metabolism %K Molecular Structure %K Oligopeptides/chemistry/pharmacology %K Protein Sorting Signals/genetics %K Serine Endopeptidases/*metabolism %K Staphylococcus epidermidis/*metabolism %X Bacterial protein secretion is a highly orchestrated process that is essential for infection and virulence. Despite extensive efforts to predict or experimentally detect proteins that are secreted, the characterization of the bacterial secretome has remained challenging. A central event in protein secretion is the type I signal peptidase (SPase)-mediated cleavage of the N-terminal signal peptide that targets a protein for secretion via the general secretory pathway, and the arylomycins are a class of natural products that inhibit SPase, suggesting that they may be useful chemical biology tools for characterizing the secretome. Here, using an arylomycin derivative, along with two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identify 11 proteins whose secretion from stationary-phase Staphylococcus epidermidis is dependent on SPase activity, 9 of which are predicted to be translated with canonical N-terminal signal peptides. In addition, we find that the presence of extracellular domains of lipoteichoic acid synthase (LtaS) and the beta-lactam response sensor BlaR1 in the medium is dependent on SPase activity, suggesting that they are cleaved at noncanonical sites within the protein. In all, the data define the proteins whose stationary-phase secretion depends on SPase and also suggest that the arylomycins should be valuable chemical biology tools for the study of protein secretion in a wide variety of different bacteria. %B J BacteriolJ BacteriolJ Bacteriol %7 2010/11/16 %V 193 %P 340-8 %8 Jan %@ 1098-5530 (Electronic)0021-9193 (Linking) %G eng %9 Research Support, N.I.H., ExtramuralResearch Support, U.S. Gov't, Non-P.H.S. %M 21075926 %2 3019839 %! Journal of bacteriologyJournal of bacteriology %0 Journal Article %J BMC BioinformaticsBMC BioinformaticsBMC Bioinformatics %D 2011 %T A computational framework for proteome-wide pursuit and prediction of metalloproteins using ICP-MS and MS/MS data %A Lancaster, W. A. %A Praissman, J. L. %A Poole, F. L., 2nd %A Cvetkovic, A. %A Menon, A. L. %A Scott, J. W. %A Jenney, F. E., Jr. %A Thorgersen, M. P. %A Kalisiak, E. %A Apon, J. V. %A Trauger, S. A. %A Siuzdak, G. %A Tainer, J. A. %A Adams, M. W. %K *Tandem Mass Spectrometry %K Amino Acid Sequence %K Automatic Data Processing/methods %K Bacterial Proteins/analysis/chemistry/isolation & purification %K Computational Biology/*methods %K Databases, Protein %K Metalloproteins/*analysis/chemistry/isolation & purification %K Metals/analysis/chemistry/metabolism %K Molybdenum/chemistry %K Nickel/chemistry %K Protein Interaction Domains and Motifs %K Proteome/*analysis %K Pyrococcus furiosus/metabolism %X BACKGROUND: Metal-containing proteins comprise a diverse and sizable category within the proteomes of organisms, ranging from proteins that use metals to catalyze reactions to proteins in which metals play key structural roles. Unfortunately, reliably predicting that a protein will contain a specific metal from its amino acid sequence is not currently possible. We recently developed a generally-applicable experimental technique for finding metalloproteins on a genome-wide scale. Applying this metal-directed protein purification approach (ICP-MS and MS/MS based) to the prototypical microbe Pyrococcus furiosus conclusively demonstrated the extent and diversity of the uncharacterized portion of microbial metalloproteomes since a majority of the observed metal peaks could not be assigned to known or predicted metalloproteins. However, even using this technique, it is not technically feasible to purify to homogeneity all metalloproteins in an organism. In order to address these limitations and complement the metal-directed protein purification, we developed a computational infrastructure and statistical methodology to aid in the pursuit and identification of novel metalloproteins. RESULTS: We demonstrate that our methodology enables predictions of metal-protein interactions using an experimental data set derived from a chromatography fractionation experiment in which 870 proteins and 10 metals were measured over 2,589 fractions. For each of the 10 metals, cobalt, iron, manganese, molybdenum, nickel, lead, tungsten, uranium, vanadium, and zinc, clusters of proteins frequently occurring in metal peaks (of a specific metal) within the fractionation space were defined. This resulted in predictions that there are from 5 undiscovered vanadium- to 13 undiscovered cobalt-containing proteins in Pyrococcus furiosus. Molybdenum and nickel were chosen for additional assessment producing lists of genes predicted to encode metalloproteins or metalloprotein subunits, 22 for nickel including seven from known nickel-proteins, and 20 for molybdenum including two from known molybdo-proteins. The uncharacterized proteins are prime candidates for metal-based purification or recombinant approaches to validate these predictions. CONCLUSIONS: We conclude that the largely uncharacterized extent of native metalloproteomes can be revealed through analysis of the co-occurrence of metals and proteins across a fractionation space. This can significantly impact our understanding of metallobiochemistry, disease mechanisms, and metal toxicity, with implications for bioremediation, medicine and other fields. %B BMC BioinformaticsBMC BioinformaticsBMC Bioinformatics %7 2011/03/02 %V 12 %P 64 %@ 1471-2105 (Electronic)1471-2105 (Linking) %G eng %9 Research Support, U.S. Gov't, Non-P.H.S. %M 21356119 %2 3058030 %! BMC bioinformaticsBMC bioinformatics %0 Journal Article %J Nat Chem BiolNat Chem BiolNat Chem Biol %D 2011 %T Metabolomics annotates ABHD3 as a physiologic regulator of medium-chain phospholipids %A Long, J. Z. %A Cisar, J. S. %A Milliken, D. %A Niessen, S. %A Wang, C. %A Trauger, S. A. %A Siuzdak, G. %A Cravatt, B. F. %K Animals %K Gene Expression Regulation, Enzymologic %K Gene Expression Regulation/physiology %K HEK293 Cells %K Humans %K Hydrolases/genetics/*metabolism %K Membrane Proteins/genetics/*metabolism %K Metabolomics/*methods %K Mice %K Mice, Knockout %K Phospholipids/chemistry/*metabolism %K Small Molecule Libraries %K Substrate Specificity %X All organisms, including humans, possess a huge number of uncharacterized enzymes. Here we describe a general cell-based screen for enzyme substrate discovery by untargeted metabolomics and its application to identify the protein alpha/beta-hydrolase domain-containing 3 (ABHD3) as a lipase that selectively cleaves medium-chain and oxidatively truncated phospholipids. Abhd3(-/-) mice possess elevated myristoyl (C14)-phospholipids, including the bioactive lipid C14-lysophosphatidylcholine, confirming the physiological relevance of our substrate assignments. %B Nat Chem BiolNat Chem BiolNat Chem Biol %7 2011/09/20 %V 7 %P 763-5 %8 Nov %@ 1552-4469 (Electronic)1552-4450 (Linking) %G eng %9 Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov't %M 21926997 %2 3201731 %! Nature chemical biologyNature chemical biology %0 Journal Article %J AIDS Res Hum Retroviruses %D 2011 %T Short communication: quantitative proteomic plasma profiling reveals activation of host defense to oxidative stress in chronic SIV and methamphetamine comorbidity %A Pendyala, G. %A Trauger, S. A. %A Siuzdak, G. %A Fox, H. S. %XThe double epidemic of substance abuse and HIV infection is a multifaceted problem To investigate mechanistic clues to the effects of substance abuse on infected individuals we preformed quantitative proteomic profiling of plasma in a methamphetamine treated nonhuman primate model for AIDS. A nontargeted quantitative approach identified extracellular superoxide dismutase to be significantly upregulated by SIV and methamphetamine treatment, and targeted studies revealed an increase in expression in the antioxidant glutathione S-transferase, thus pointing to a compensatory response to increased oxidative stress in methamphetamine-treated animals.
%B AIDS Res Hum Retroviruses %7 2010/10/12 %V 27 %P 179-82 %8 Feb %@ 1931-8405 (Electronic)0889-2229 (Linking) %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/20929344 %9 Research Support, N.I.H., Extramural %M 20929344 %2 3045074 %0 Journal Article %J BMC Bioinformatics %D 2011 %T A computational framework for proteome-wide pursuit and prediction of metalloproteins using ICP-MS and MS/MS data %A Lancaster, W. A. %A Praissman, J. L. %A Poole, F. L., 2nd %A Cvetkovic, A. %A Menon, A. L. %A Scott, J. W. %A Jenney, F. E., Jr. %A Thorgersen, M. P. %A Kalisiak, E. %A Apon, J. V. %A Trauger, S. A. %A Siuzdak, G. %A Tainer, J. A. %A Adams, M. W. %XBACKGROUND: Metal-containing proteins comprise a diverse and sizable category within the proteomes of organisms, ranging from proteins that use metals to catalyze reactions to proteins in which metals play key structural roles. Unfortunately, reliably predicting that a protein will contain a specific metal from its amino acid sequence is not currently possible. We recently developed a generally-applicable experimental technique for finding metalloproteins on a genome-wide scale. Applying this metal-directed protein purification approach (ICP-MS and MS/MS based) to the prototypical microbe Pyrococcus furiosus conclusively demonstrated the extent and diversity of the uncharacterized portion of microbial metalloproteomes since a majority of the observed metal peaks could not be assigned to known or predicted metalloproteins. However, even using this technique, it is not technically feasible to purify to homogeneity all metalloproteins in an organism. In order to address these limitations and complement the metal-directed protein purification, we developed a computational infrastructure and statistical methodology to aid in the pursuit and identification of novel metalloproteins. RESULTS: We demonstrate that our methodology enables predictions of metal-protein interactions using an experimental data set derived from a chromatography fractionation experiment in which 870 proteins and 10 metals were measured over 2,589 fractions. For each of the 10 metals, cobalt, iron, manganese, molybdenum, nickel, lead, tungsten, uranium, vanadium, and zinc, clusters of proteins frequently occurring in metal peaks (of a specific metal) within the fractionation space were defined. This resulted in predictions that there are from 5 undiscovered vanadium- to 13 undiscovered cobalt-containing proteins in Pyrococcus furiosus. Molybdenum and nickel were chosen for additional assessment producing lists of genes predicted to encode metalloproteins or metalloprotein subunits, 22 for nickel including seven from known nickel-proteins, and 20 for molybdenum including two from known molybdo-proteins. The uncharacterized proteins are prime candidates for metal-based purification or recombinant approaches to validate these predictions. CONCLUSIONS: We conclude that the largely uncharacterized extent of native metalloproteomes can be revealed through analysis of the co-occurrence of metals and proteins across a fractionation space. This can significantly impact our understanding of metallobiochemistry, disease mechanisms, and metal toxicity, with implications for bioremediation, medicine and other fields.
%B BMC Bioinformatics %7 2011/03/02 %V 12 %P 64 %@ 1471-2105 (Electronic)1471-2105 (Linking) %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/21356119 %9 Research Support, U.S. Gov't, Non-P.H.S. %M 21356119 %2 3058030 %0 Journal Article %J J Bacteriol %D 2011 %T Type I signal peptidase and protein secretion in Staphylococcus epidermidis %A Powers, M. E. %A Smith, P. A. %A Roberts, T. C. %A Fowler, B. J. %A King, C. C. %A Trauger, S. A. %A Siuzdak, G. %A Romesberg, F. E. %XBacterial protein secretion is a highly orchestrated process that is essential for infection and virulence. Despite extensive efforts to predict or experimentally detect proteins that are secreted, the characterization of the bacterial secretome has remained challenging. A central event in protein secretion is the type I signal peptidase (SPase)-mediated cleavage of the N-terminal signal peptide that targets a protein for secretion via the general secretory pathway, and the arylomycins are a class of natural products that inhibit SPase, suggesting that they may be useful chemical biology tools for characterizing the secretome. Here, using an arylomycin derivative, along with two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identify 11 proteins whose secretion from stationary-phase Staphylococcus epidermidis is dependent on SPase activity, 9 of which are predicted to be translated with canonical N-terminal signal peptides. In addition, we find that the presence of extracellular domains of lipoteichoic acid synthase (LtaS) and the beta-lactam response sensor BlaR1 in the medium is dependent on SPase activity, suggesting that they are cleaved at noncanonical sites within the protein. In all, the data define the proteins whose stationary-phase secretion depends on SPase and also suggest that the arylomycins should be valuable chemical biology tools for the study of protein secretion in a wide variety of different bacteria.
%B J Bacteriol %7 2010/11/16 %V 193 %P 340-8 %8 Jan %@ 1098-5530 (Electronic)0021-9193 (Linking) %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/21075926 %9 Research Support, N.I.H., ExtramuralResearch Support, U.S. Gov't, Non-P.H.S. %M 21075926 %2 3019839 %0 Journal Article %J Nat Chem Biol %D 2011 %T Metabolomics annotates ABHD3 as a physiologic regulator of medium-chain phospholipids %A Long, J. Z. %A Cisar, J. S. %A Milliken, D. %A Niessen, S. %A Wang, C. %A Trauger, S. A. %A Siuzdak, G. %A Cravatt, B. F. %XAll organisms, including humans, possess a huge number of uncharacterized enzymes. Here we describe a general cell-based screen for enzyme substrate discovery by untargeted metabolomics and its application to identify the protein alpha/beta-hydrolase domain-containing 3 (ABHD3) as a lipase that selectively cleaves medium-chain and oxidatively truncated phospholipids. Abhd3(-/-) mice possess elevated myristoyl (C14)-phospholipids, including the bioactive lipid C14-lysophosphatidylcholine, confirming the physiological relevance of our substrate assignments.
%B Nat Chem Biol %7 2011/09/20 %V 7 %P 763-5 %8 Nov %@ 1552-4469 (Electronic)1552-4450 (Linking) %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/21926997 %9 Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov't %M 21926997 %2 3201731 %0 Journal Article %J Nat Chem BiolNat Chem BiolNat Chem Biol %D 2010 %T Metabolic oxidation regulates embryonic stem cell differentiation %A Yanes, O. %A Clark, J. %A Wong, D. M. %A Patti, G. J. %A Sanchez-Ruiz, A. %A Benton, H. P. %A Trauger, S. A. %A Desponts, C. %A Ding, S. %A Siuzdak, G. %K Amino Acids/metabolism %K Carboxylic Acids/metabolism %K Carnitine/metabolism %K Cell Differentiation %K Eicosanoids/metabolism %K Embryonic Stem Cells/*cytology/*metabolism %K Gene Expression Regulation %K Glutathione/metabolism %K Humans %K Oxidation-Reduction %K Phenotype %K Proteome/metabolism %K Software %K Stem Cells/cytology/metabolism %X Metabolites offer an important unexplored complementary approach to understanding the pluripotency of stem cells. Using MS-based metabolomics, we show that embryonic stem cells are characterized by abundant metabolites with highly unsaturated structures whose levels decrease upon differentiation. By monitoring the reduced and oxidized glutathione ratio as well as ascorbic acid levels, we demonstrate that the stem cell redox status is regulated during differentiation. On the basis of the oxidative biochemistry of the unsaturated metabolites, we experimentally manipulated specific pathways in embryonic stem cells while monitoring the effects on differentiation. Inhibition of the eicosanoid signaling pathway promoted pluripotency and maintained levels of unsaturated fatty acids. In contrast, downstream oxidized metabolites (for example, neuroprotectin D1) and substrates of pro-oxidative reactions (for example, acyl-carnitines), promoted neuronal and cardiac differentiation. We postulate that the highly unsaturated metabolome sustained by stem cells allows them to differentiate in response to in vivo oxidative processes such as inflammation. %B Nat Chem BiolNat Chem BiolNat Chem Biol %7 2010/05/04 %V 6 %P 411-7 %8 Jun %@ 1552-4469 (Electronic)1552-4450 (Linking) %G eng %9 Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, Non-P.H.S. %M 20436487 %2 2873061 %! Nature chemical biologyNature chemical biology %0 Journal Article %J J Proteome ResJ Proteome ResJ Proteome Res %D 2010 %T Quantitative plasma proteomic profiling identifies the vitamin E binding protein afamin as a potential pathogenic factor in SIV induced CNS disease %A Pendyala, G. %A Trauger, S. A. %A Siuzdak, G. %A Fox, H. S. %K Animals %K Carrier Proteins/*blood %K Encephalitis, Viral/*blood/virology %K Glycoproteins/*blood %K Macaca mulatta %K Peptide Mapping %K Proteome %K Proteomics/*methods %K Reproducibility of Results %K Serum Albumin/*analysis %K Simian Acquired Immunodeficiency Syndrome/*blood %K Simian immunodeficiency virus %K Tandem Mass Spectrometry %K Vitamin E/metabolism %X Investigating, predicting, diagnosing, and treating HIV-1-associated neurocognitive disorder (HAND) has been hindered by the lack of disease-related molecular markers. In this study, plasma from rhesus monkeys (n = 6), before and after infection with simian immunodeficiency virus (SIV), was profiled to obtain differential fingerprints in protein expression during SIV-induced central nervous system (CNS) disease. A quantitative proteomic analysis was performed by means of isobaric tag for relative and absolute quantification (iTRAQ) labeling, using multidimensional liquid chromatography tandem mass spectrometry (LC-MS/MS) run on a linear ion trap mass spectrometer in an integrated mode comprising pulsed-Q-dissociation (PQD) and CID. Among a panel of proteins showing differential expression following SIV infection, we identified afamin, a member of the albumin superfamily, to be significantly down regulated after infection. Validation by Western blot confirmed this observation and, given its potential implication in neuroprotection by transport of alpha-tocopherol (alphaTocH), provides new avenues into further understanding HIV induced CNS disease. iTRAQ-based LC-MS/MS provides a valuable platform for plasma protein profiling and has important implications in identifying molecular markers relevant for the pathogenesis of neurodegenerative diseases. Using such an approach, we show its successful application in identifying differential fingerprints in SIV/HIV induced CNS disease. %B J Proteome ResJ Proteome ResJ Proteome Res %7 2009/11/17 %V 9 %P 352-8 %8 Jan %@ 1535-3907 (Electronic)1535-3893 (Linking) %G eng %9 Research Support, N.I.H., Extramural %M 19908921 %2 2801767 %! Journal of proteome researchJournal of proteome research %0 Journal Article %J Chem BiolChem BiolChem Biol %D 2010 %T The glycerophospho metabolome and its influence on amino acid homeostasis revealed by brain metabolomics of GDE1(-/-) mice %A Kopp, F. %A Komatsu, T. %A Nomura, D. K. %A Trauger, S. A. %A Thomas, J. R. %A Siuzdak, G. %A Simon, G. M. %A Cravatt, B. F. %K *Homeostasis %K *Metabolome %K Amino Acids/*metabolism %K Animals %K Brain/cytology/*metabolism %K Inositol Phosphates/*metabolism %K Male %K Mass Spectrometry %K Metabolomics/*methods %K Mice %K Phosphoric Diester Hydrolases/*deficiency/metabolism %K Serine/metabolism %K Solubility %K Water/chemistry %X GDE1 is a mammalian glycerophosphodiesterase (GDE) implicated by in vitro studies in the regulation of glycerophophoinositol (GroPIns) and possibly other glycerophospho (GroP) metabolites. Here, we show using untargeted metabolomics that GroPIns is profoundly (>20-fold) elevated in brain tissue from GDE1(-/-) mice. Furthermore, two additional GroP metabolites not previously identified in eukaryotic cells, glycerophosphoserine (GroPSer) and glycerophosphoglycerate (GroPGate), were also highly elevated in GDE1(-/-) brains. Enzyme assays with synthetic GroP metabolites confirmed that GroPSer and GroPGate are direct substrates of GDE1. Interestingly, our metabolomic profiles also revealed that serine (both L-and D-) levels were significantly reduced in brains of GDE1(-/-) mice. These findings designate GroPSer as a previously unappreciated reservoir for free serine in the nervous system and suggest that GDE1, through recycling serine from GroPSer, may impact D-serine-dependent neural signaling processes in vivo. %B Chem BiolChem BiolChem Biol %7 2010/08/28 %V 17 %P 831-40 %8 Aug 27 %@ 1879-1301 (Electronic)1074-5521 (Linking) %G eng %9 Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov't %M 20797612 %2 2931592 %! Chemistry & biologyChemistry & biology %0 Journal Article %J BMC Syst BiolBMC Syst BiolBMC Syst Biol %D 2010 %T Large scale physiological readjustment during growth enables rapid, comprehensive and inexpensive systems analysis %A Facciotti, M. T. %A Pang, W. L. %A Lo, F. Y. %A Whitehead, K. %A Koide, T. %A Masumura, K. %A Pan, M. %A Kaur, A. %A Larsen, D. J. %A Reiss, D. J. %A Hoang, L. %A Kalisiak, E. %A Northen, T. %A Trauger, S. A. %A Siuzdak, G. %A Baliga, N. S. %K *Systems Analysis %K *Systems Biology %K Chromatography, Liquid/methods %K Escherichia coli/metabolism %K Gene Expression Profiling %K Gene Regulatory Networks %K Halobacterium salinarum/*genetics/metabolism %K Mass Spectrometry/methods %K Models, Biological %K Models, Genetic %K Oligonucleotide Array Sequence Analysis %K Phenotype %K RNA, Messenger/metabolism %K Transcription, Genetic %X BACKGROUND: Rapidly characterizing the operational interrelationships among all genes in a given organism is a critical bottleneck to significantly advancing our understanding of thousands of newly sequenced microbial and eukaryotic species. While evolving technologies for global profiling of transcripts, proteins, and metabolites are making it possible to comprehensively survey cellular physiology in newly sequenced organisms, these experimental techniques have not kept pace with sequencing efforts. Compounding these technological challenges is the fact that individual experiments typically only stimulate relatively small-scale cellular responses, thus requiring numerous expensive experiments to survey the operational relationships among nearly all genetic elements. Therefore, a relatively quick and inexpensive strategy for observing changes in large fractions of the genetic elements is highly desirable. RESULTS: We have discovered in the model organism Halobacterium salinarum NRC-1 that batch culturing in complex medium stimulates meaningful changes in the expression of approximately two thirds of all genes. While the majority of these changes occur during transition from rapid exponential growth to the stationary phase, several transient physiological states were detected beyond what has been previously observed. In sum, integrated analysis of transcript and metabolite changes has helped uncover growth phase-associated physiologies, operational interrelationships among two thirds of all genes, specialized functions for gene family members, waves of transcription factor activities, and growth phase associated cell morphology control. CONCLUSIONS: Simple laboratory culturing in complex medium can be enormously informative regarding the activities of and interrelationships among a large fraction of all genes in an organism. This also yields important baseline physiological context for designing specific perturbation experiments at different phases of growth. The integration of such growth and perturbation studies with measurements of associated environmental factor changes is a practical and economical route for the elucidation of comprehensive systems-level models of biological systems. %B BMC Syst BiolBMC Syst BiolBMC Syst Biol %7 2010/05/18 %V 4 %P 64 %@ 1752-0509 (Electronic)1752-0509 (Linking) %G eng %9 Research Support, N.I.H., ExtramuralResearch Support, U.S. Gov't, Non-P.H.S. %M 20470417 %2 2880973 %! BMC systems biologyBMC systems biology %0 Journal Article %J GliaGliaGlia %D 2010 %T Maintaining retinal astrocytes normalizes revascularization and prevents vascular pathology associated with oxygen-induced retinopathy %A Dorrell, M. I. %A Aguilar, E. %A Jacobson, R. %A Trauger, S. A. %A Friedlander, J. %A Siuzdak, G. %A Friedlander, M. %K Age Factors %K Animals %K Animals, Newborn %K Antigens, CD11b/metabolism %K Astrocytes/chemistry/pathology/*physiology %K Bone Marrow Cells/physiology %K Bone Marrow Transplantation/methods %K Cells, Cultured %K Cerebral Cortex/cytology %K Culture Media, Conditioned/pharmacology %K Disease Models, Animal %K Fibroblast Growth Factors/therapeutic use %K Glial Fibrillary Acidic Protein/metabolism %K Humans %K induced/*complications/*pathology/*prevention & control %K Infant, Newborn %K Injections, Intraventricular/methods %K Mice %K Mice, Inbred BALB C %K Mice, Inbred C57BL %K Myeloid Cells/physiology %K Neovascularization, Physiologic/*physiology %K Oxygen/adverse effects %K Proteomics/methods %K Retinal Neovascularization/*etiology %K Retinopathy of Prematurity/chemically %K Vascular Endothelial Growth Factor A/therapeutic use %X Astrocytes are well known modulators of normal developmental retinal vascularization. However, relatively little is known about the role of glial cells during pathological retinal neovascularization (NV), a leading contributor to vision loss in industrialized nations. We demonstrate that the loss of astrocytes and microglia directly correlates with the development of pathological NV in a mouse model of oxygen-induced retinopathy (OIR). These two distinct glial cell populations were found to have cooperative survival effects in vitro and in vivo. The intravitreal injection of myeloid progenitor cells, astrocytes, or astrocyte-conditioned media rescued endogenous astrocytes from degeneration that normally occurs within the hypoxic, vaso-obliterated retina following return to normoxia. Protection of the retinal astrocytes and microglia was directly correlated with accelerated revascularization of the normal retinal plexuses and reduction of pathological intravitreal NV normally associated with OIR. Using astrocyte-conditioned media, several factors were identified that may contribute to the observed astrocytic protection and subsequent normalization of the retinal vasculature, including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Injection of VEGF or bFGF at specific doses rescued the retinas from developing OIR-associated pathology, an effect that was also preceded by protection of endogenous glia from hypoxia-induced degeneration. Together, these data suggest that vascular-associated glia are also required for normalized revascularization of the hypoxic retina. Methods developed to target and protect glial cells may provide a novel strategy by which normalized revascularization can be promoted and the consequences of abnormal NV in retinal vascular diseases can be prevented. %B GliaGliaGlia %7 2009/06/23 %V 58 %P 43-54 %8 Jan 1 %@ 1098-1136 (Electronic)0894-1491 (Linking) %G eng %9 Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov't %M 19544395 %2 2814838 %! GliaGlia %0 Journal Article %J NatureNatureNature %D 2010 %T Microbial metalloproteomes are largely uncharacterized %A Cvetkovic, A. %A Menon, A. L. %A Thorgersen, M. P. %A Scott, J. W. %A Poole, F. L., 2nd %A Jenney, F. E., Jr. %A Lancaster, W. A. %A Praissman, J. L. %A Shanmukh, S. %A Vaccaro, B. J. %A Trauger, S. A. %A Kalisiak, E. %A Apon, J. V. %A Siuzdak, G. %A Yannone, S. M. %A Tainer, J. A. %A Adams, M. W. %K Bacterial Proteins/*analysis/chemistry %K Chromatography, Liquid %K Escherichia coli/chemistry %K Metalloproteins/*analysis/*chemistry %K Metals/*analysis/chemistry/metabolism %K Proteome/*analysis/chemistry %K Proteomics %K Pyrococcus furiosus/*chemistry/metabolism %K Sulfolobus solfataricus/chemistry %K Tandem Mass Spectrometry %X Metal ion cofactors afford proteins virtually unlimited catalytic potential, enable electron transfer reactions and have a great impact on protein stability. Consequently, metalloproteins have key roles in most biological processes, including respiration (iron and copper), photosynthesis (manganese) and drug metabolism (iron). Yet, predicting from genome sequence the numbers and types of metal an organism assimilates from its environment or uses in its metalloproteome is currently impossible because metal coordination sites are diverse and poorly recognized. We present here a robust, metal-based approach to determine all metals an organism assimilates and identify its metalloproteins on a genome-wide scale. This shifts the focus from classical protein-based purification to metal-based identification and purification by liquid chromatography, high-throughput tandem mass spectrometry (HT-MS/MS) and inductively coupled plasma mass spectrometry (ICP-MS) to characterize cytoplasmic metalloproteins from an exemplary microorganism (Pyrococcus furiosus). Of 343 metal peaks in chromatography fractions, 158 did not match any predicted metalloprotein. Unassigned peaks included metals known to be used (cobalt, iron, nickel, tungsten and zinc; 83 peaks) plus metals the organism was not thought to assimilate (lead, manganese, molybdenum, uranium and vanadium; 75 peaks). Purification of eight of 158 unexpected metal peaks yielded four novel nickel- and molybdenum-containing proteins, whereas four purified proteins contained sub-stoichiometric amounts of misincorporated lead and uranium. Analyses of two additional microorganisms (Escherichia coli and Sulfolobus solfataricus) revealed species-specific assimilation of yet more unexpected metals. Metalloproteomes are therefore much more extensive and diverse than previously recognized, and promise to provide key insights for cell biology, microbial growth and toxicity mechanisms. %B NatureNatureNature %7 2010/07/20 %V 466 %P 779-82 %8 Aug 5 %@ 1476-4687 (Electronic)0028-0836 (Linking) %G eng %9 Research Support, U.S. Gov't, Non-P.H.S. %M 20639861 %! NatureNature %0 Journal Article %J Glia %D 2010 %T Maintaining retinal astrocytes normalizes revascularization and prevents vascular pathology associated with oxygen-induced retinopathy %A Dorrell, M. I. %A Aguilar, E. %A Jacobson, R. %A Trauger, S. A. %A Friedlander, J. %A Siuzdak, G. %A Friedlander, M. %XAstrocytes are well known modulators of normal developmental retinal vascularization. However, relatively little is known about the role of glial cells during pathological retinal neovascularization (NV), a leading contributor to vision loss in industrialized nations. We demonstrate that the loss of astrocytes and microglia directly correlates with the development of pathological NV in a mouse model of oxygen-induced retinopathy (OIR). These two distinct glial cell populations were found to have cooperative survival effects in vitro and in vivo. The intravitreal injection of myeloid progenitor cells, astrocytes, or astrocyte-conditioned media rescued endogenous astrocytes from degeneration that normally occurs within the hypoxic, vaso-obliterated retina following return to normoxia. Protection of the retinal astrocytes and microglia was directly correlated with accelerated revascularization of the normal retinal plexuses and reduction of pathological intravitreal NV normally associated with OIR. Using astrocyte-conditioned media, several factors were identified that may contribute to the observed astrocytic protection and subsequent normalization of the retinal vasculature, including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Injection of VEGF or bFGF at specific doses rescued the retinas from developing OIR-associated pathology, an effect that was also preceded by protection of endogenous glia from hypoxia-induced degeneration. Together, these data suggest that vascular-associated glia are also required for normalized revascularization of the hypoxic retina. Methods developed to target and protect glial cells may provide a novel strategy by which normalized revascularization can be promoted and the consequences of abnormal NV in retinal vascular diseases can be prevented.
%B Glia %7 2009/06/23 %V 58 %P 43-54 %8 Jan 1 %@ 1098-1136 (Electronic)0894-1491 (Linking) %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/19544395 %9 Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov't %M 19544395 %2 2814838 %0 Journal Article %J BMC Syst Biol %D 2010 %T Large scale physiological readjustment during growth enables rapid, comprehensive and inexpensive systems analysis %A Facciotti, M. T. %A Pang, W. L. %A Lo, F. Y. %A Whitehead, K. %A Koide, T. %A Masumura, K. %A Pan, M. %A Kaur, A. %A Larsen, D. J. %A Reiss, D. J. %A Hoang, L. %A Kalisiak, E. %A Northen, T. %A Trauger, S. A. %A Siuzdak, G. %A Baliga, N. S. %XBACKGROUND: Rapidly characterizing the operational interrelationships among all genes in a given organism is a critical bottleneck to significantly advancing our understanding of thousands of newly sequenced microbial and eukaryotic species. While evolving technologies for global profiling of transcripts, proteins, and metabolites are making it possible to comprehensively survey cellular physiology in newly sequenced organisms, these experimental techniques have not kept pace with sequencing efforts. Compounding these technological challenges is the fact that individual experiments typically only stimulate relatively small-scale cellular responses, thus requiring numerous expensive experiments to survey the operational relationships among nearly all genetic elements. Therefore, a relatively quick and inexpensive strategy for observing changes in large fractions of the genetic elements is highly desirable. RESULTS: We have discovered in the model organism Halobacterium salinarum NRC-1 that batch culturing in complex medium stimulates meaningful changes in the expression of approximately two thirds of all genes. While the majority of these changes occur during transition from rapid exponential growth to the stationary phase, several transient physiological states were detected beyond what has been previously observed. In sum, integrated analysis of transcript and metabolite changes has helped uncover growth phase-associated physiologies, operational interrelationships among two thirds of all genes, specialized functions for gene family members, waves of transcription factor activities, and growth phase associated cell morphology control. CONCLUSIONS: Simple laboratory culturing in complex medium can be enormously informative regarding the activities of and interrelationships among a large fraction of all genes in an organism. This also yields important baseline physiological context for designing specific perturbation experiments at different phases of growth. The integration of such growth and perturbation studies with measurements of associated environmental factor changes is a practical and economical route for the elucidation of comprehensive systems-level models of biological systems.
%B BMC Syst Biol %7 2010/05/18 %V 4 %P 64 %@ 1752-0509 (Electronic)1752-0509 (Linking) %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/20470417 %9 Research Support, N.I.H., ExtramuralResearch Support, U.S. Gov't, Non-P.H.S. %M 20470417 %2 2880973 %0 Journal Article %J Nat Chem Biol %D 2010 %T Metabolic oxidation regulates embryonic stem cell differentiation %A Yanes, O. %A Clark, J. %A Wong, D. M. %A Patti, G. J. %A Sanchez-Ruiz, A. %A Benton, H. P. %A Trauger, S. A. %A Desponts, C. %A Ding, S. %A Siuzdak, G. %XMetabolites offer an important unexplored complementary approach to understanding the pluripotency of stem cells. Using MS-based metabolomics, we show that embryonic stem cells are characterized by abundant metabolites with highly unsaturated structures whose levels decrease upon differentiation. By monitoring the reduced and oxidized glutathione ratio as well as ascorbic acid levels, we demonstrate that the stem cell redox status is regulated during differentiation. On the basis of the oxidative biochemistry of the unsaturated metabolites, we experimentally manipulated specific pathways in embryonic stem cells while monitoring the effects on differentiation. Inhibition of the eicosanoid signaling pathway promoted pluripotency and maintained levels of unsaturated fatty acids. In contrast, downstream oxidized metabolites (for example, neuroprotectin D1) and substrates of pro-oxidative reactions (for example, acyl-carnitines), promoted neuronal and cardiac differentiation. We postulate that the highly unsaturated metabolome sustained by stem cells allows them to differentiate in response to in vivo oxidative processes such as inflammation.
%B Nat Chem Biol %7 2010/05/04 %V 6 %P 411-7 %8 Jun %@ 1552-4469 (Electronic)1552-4450 (Linking) %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/20436487 %9 Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, Non-P.H.S. %M 20436487 %2 2873061 %0 Journal Article %J J Proteome Res %D 2010 %T Quantitative plasma proteomic profiling identifies the vitamin E binding protein afamin as a potential pathogenic factor in SIV induced CNS disease %A Pendyala, G. %A Trauger, S. A. %A Siuzdak, G. %A Fox, H. S. %XInvestigating, predicting, diagnosing, and treating HIV-1-associated neurocognitive disorder (HAND) has been hindered by the lack of disease-related molecular markers. In this study, plasma from rhesus monkeys (n = 6), before and after infection with simian immunodeficiency virus (SIV), was profiled to obtain differential fingerprints in protein expression during SIV-induced central nervous system (CNS) disease. A quantitative proteomic analysis was performed by means of isobaric tag for relative and absolute quantification (iTRAQ) labeling, using multidimensional liquid chromatography tandem mass spectrometry (LC-MS/MS) run on a linear ion trap mass spectrometer in an integrated mode comprising pulsed-Q-dissociation (PQD) and CID. Among a panel of proteins showing differential expression following SIV infection, we identified afamin, a member of the albumin superfamily, to be significantly down regulated after infection. Validation by Western blot confirmed this observation and, given its potential implication in neuroprotection by transport of alpha-tocopherol (alphaTocH), provides new avenues into further understanding HIV induced CNS disease. iTRAQ-based LC-MS/MS provides a valuable platform for plasma protein profiling and has important implications in identifying molecular markers relevant for the pathogenesis of neurodegenerative diseases. Using such an approach, we show its successful application in identifying differential fingerprints in SIV/HIV induced CNS disease.
%B J Proteome Res %7 2009/11/17 %V 9 %P 352-8 %8 Jan %@ 1535-3907 (Electronic)1535-3893 (Linking) %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/19908921 %9 Research Support, N.I.H., Extramural %M 19908921 %2 2801767 %0 Journal Article %J Chem Biol %D 2010 %T The glycerophospho metabolome and its influence on amino acid homeostasis revealed by brain metabolomics of GDE1(-/-) mice %A Kopp, F. %A Komatsu, T. %A Nomura, D. K. %A Trauger, S. A. %A Thomas, J. R. %A Siuzdak, G. %A Simon, G. M. %A Cravatt, B. F. %XGDE1 is a mammalian glycerophosphodiesterase (GDE) implicated by in vitro studies in the regulation of glycerophophoinositol (GroPIns) and possibly other glycerophospho (GroP) metabolites. Here, we show using untargeted metabolomics that GroPIns is profoundly (>20-fold) elevated in brain tissue from GDE1(-/-) mice. Furthermore, two additional GroP metabolites not previously identified in eukaryotic cells, glycerophosphoserine (GroPSer) and glycerophosphoglycerate (GroPGate), were also highly elevated in GDE1(-/-) brains. Enzyme assays with synthetic GroP metabolites confirmed that GroPSer and GroPGate are direct substrates of GDE1. Interestingly, our metabolomic profiles also revealed that serine (both L-and D-) levels were significantly reduced in brains of GDE1(-/-) mice. These findings designate GroPSer as a previously unappreciated reservoir for free serine in the nervous system and suggest that GDE1, through recycling serine from GroPSer, may impact D-serine-dependent neural signaling processes in vivo.
%B Chem Biol %7 2010/08/28 %V 17 %P 831-40 %8 Aug 27 %@ 1879-1301 (Electronic)1074-5521 (Linking) %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/20797612 %9 Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov't %M 20797612 %2 2931592 %0 Journal Article %J Nature %D 2010 %T Microbial metalloproteomes are largely uncharacterized %A Cvetkovic, A. %A Menon, A. L. %A Thorgersen, M. P. %A Scott, J. W. %A Poole, F. L., 2nd %A Jenney, F. E., Jr. %A Lancaster, W. A. %A Praissman, J. L. %A Shanmukh, S. %A Vaccaro, B. J. %A Trauger, S. A. %A Kalisiak, E. %A Apon, J. V. %A Siuzdak, G. %A Yannone, S. M. %A Tainer, J. A. %A Adams, M. W. %XMetal ion cofactors afford proteins virtually unlimited catalytic potential, enable electron transfer reactions and have a great impact on protein stability. Consequently, metalloproteins have key roles in most biological processes, including respiration (iron and copper), photosynthesis (manganese) and drug metabolism (iron). Yet, predicting from genome sequence the numbers and types of metal an organism assimilates from its environment or uses in its metalloproteome is currently impossible because metal coordination sites are diverse and poorly recognized. We present here a robust, metal-based approach to determine all metals an organism assimilates and identify its metalloproteins on a genome-wide scale. This shifts the focus from classical protein-based purification to metal-based identification and purification by liquid chromatography, high-throughput tandem mass spectrometry (HT-MS/MS) and inductively coupled plasma mass spectrometry (ICP-MS) to characterize cytoplasmic metalloproteins from an exemplary microorganism (Pyrococcus furiosus). Of 343 metal peaks in chromatography fractions, 158 did not match any predicted metalloprotein. Unassigned peaks included metals known to be used (cobalt, iron, nickel, tungsten and zinc; 83 peaks) plus metals the organism was not thought to assimilate (lead, manganese, molybdenum, uranium and vanadium; 75 peaks). Purification of eight of 158 unexpected metal peaks yielded four novel nickel- and molybdenum-containing proteins, whereas four purified proteins contained sub-stoichiometric amounts of misincorporated lead and uranium. Analyses of two additional microorganisms (Escherichia coli and Sulfolobus solfataricus) revealed species-specific assimilation of yet more unexpected metals. Metalloproteomes are therefore much more extensive and diverse than previously recognized, and promise to provide key insights for cell biology, microbial growth and toxicity mechanisms.
%B Nature %7 2010/07/20 %V 466 %P 779-82 %8 Aug 5 %@ 1476-4687 (Electronic)0028-0836 (Linking) %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/20639861 %9 Research Support, U.S. Gov't, Non-P.H.S. %M 20639861 %0 Journal Article %J Cell Stem CellCell Stem CellCell Stem Cell %D 2009 %T Generation of induced pluripotent stem cells using recombinant proteins %A Zhou, H. %A Wu, S. %A Joo, J. Y. %A Zhu, S. %A Han, D. W. %A Lin, T. %A Trauger, S. %A Bien, G. %A Yao, S. %A Zhu, Y. %A Siuzdak, G. %A Scholer, H. R. %A Duan, L. %A Ding, S. %K Animals %K Mice %K Pluripotent Stem Cells/*cytology/metabolism %K Recombinant Proteins/genetics/*metabolism %K Transcription Factors/genetics/metabolism %B Cell Stem CellCell Stem CellCell Stem Cell %7 2009/04/29 %V 4 %P 381-4 %8 May 8 %@ 1875-9777 (Electronic) %G eng %9 Research Support, Non-U.S. Gov't %M 19398399 %! Cell stem cellCell stem cell %0 Journal Article %J Rapid Commun Mass SpectromRapid Commun Mass SpectromRapid Commun Mass Spectrom %D 2009 %T Phosphonium labeling for increasing metabolomic coverage of neutral lipids using electrospray ionization mass spectrometry %A Woo, H. K. %A Go, E. P. %A Hoang, L. %A Trauger, S. A. %A Bowen, B. %A Siuzdak, G. %A Northen, T. R. %K *Lipid Metabolism %K *Metabolomics %K Humans %K Lipids/blood/*chemistry %K Male %K Organophosphorus Compounds/chemical synthesis/*chemistry %K Sensitivity and Specificity %K Spectrometry, Mass, Electrospray Ionization/*methods %K Staining and Labeling %X Mass spectrometry has become an indispensable tool for the global study of metabolites (metabolomics), primarily using electrospray ionization mass spectrometry (ESI-MS). However, many important classes of molecules such as neutral lipids do not ionize well by ESI and go undetected. Chemical derivatization of metabolites can enhance ionization for increased sensitivity and metabolomic coverage. Here we describe the use of tris(2,4,6,-trimethoxyphenyl)phosphonium acetic acid (TMPP-AA) to improve liquid chromatography (LC)/ESI-MS detection of hydroxylated metabolites (i.e. lipids) from serum extracts. Cholesterol which is not normally detected from serum using ESI is observed with attomole sensitivity. This approach was applied to identify four endogenous lipids (hexadecanoyl-sn-glycerol, dihydrotachysterol, octadecanol, and alpha-tocopherol) from human serum. Overall, this approach extends the types of metabolites which can be detected using standard ESI-MS instrumentation and demonstrates the potential for targeted metabolomics analysis. %B Rapid Commun Mass SpectromRapid Commun Mass SpectromRapid Commun Mass Spectrom %7 2009/05/19 %V 23 %P 1849-55 %8 Jun %@ 1097-0231 (Electronic)0951-4198 (Linking) %G eng %9 Evaluation StudiesResearch Support, N.I.H., ExtramuralResearch Support, U.S. Gov't, Non-P.H.S. %M 19449318 %2 3052201 %! Rapid communications in mass spectrometry : RCMRapid communications in mass spectrometry : RCM %0 Journal Article %J J Proteome ResJ Proteome ResJ Proteome Res %D 2009 %T Response and recovery in the plasma metabolome tracks the acute LCMV-induced immune response %A Wikoff, W. R. %A Kalisak, E. %A Trauger, S. %A Manchester, M. %A Siuzdak, G. %K Animals %K Blood Proteins/*chemistry %K Immunity, Innate %K Interferon-gamma/metabolism %K Kinetics %K Lymphocytic choriomeningitis virus/*metabolism %K Male %K Mass Spectrometry/methods %K Metabolome %K Metabolomics/*methods %K Mice %K Mice, Inbred C57BL %K Models, Biological %K Models, Chemical %K Systems Biology/methods %X Lymphocytic choriomeningitis virus (LCMV) infection of mice is noncytopathic, producing well-characterized changes reflecting the host immune response. Untargeted metabolomics using mass spectrometry identified endogenous small molecule changes in blood from mice inoculated with LCMV, sampled at days 1, 3, 7, and 14 post infection. These time points correspond to well characterized events during acute LCMV infection and the immune response. Diverse pathways were altered, including TCA cycle intermediates, gamma-glutamyl dipeptides, lysophosphatidyl cholines, and fatty acids. The kynurenine pathway was activated, surprising because it is stimulated by IFN-gamma, which LCMV suppresses, thus, suggesting alternative activators. In contrast, biopterin/neopterin, another IFN-gamma stimulated pathway, was not activated. Many metabolites followed "response and recovery" kinetics, decreasing after infection to a minimum at days 3-7, and returning to normal by day 14. The TCA pathway followed this pattern, including citrate, cis-aconitate and alpha-ketoglutarate, intriguing because succinate has been shown to mediate cellular immunity. This response and recovery dynamic tracks the immune response, including the rise and fall of natural killer cell populations, serum TNF receptor concentration, and viral clearance. Metabolomics can provide target pathways for molecular diagnostics or therapeutics of viral infection and immunity. %B J Proteome ResJ Proteome ResJ Proteome Res %7 2009/06/06 %V 8 %P 3578-87 %8 Jul %@ 1535-3893 (Print)1535-3893 (Linking) %G eng %9 Research Support, N.I.H., Extramural %M 19496611 %2 3437991 %! Journal of proteome researchJournal of proteome research %0 Journal Article %J J Proteome ResJ Proteome ResJ Proteome Res %D 2009 %T Cerebrospinal fluid proteomics reveals potential pathogenic changes in the brains of SIV-infected monkeys %A Pendyala, G. %A Trauger, S. A. %A Kalisiak, E. %A Ellis, R. J. %A Siuzdak, G. %A Fox, H. S. %K Animals %K Brain Diseases/*cerebrospinal fluid/virology %K Brain/metabolism/pathology/virology %K Chromatography, Liquid %K Complement C3/genetics/metabolism %K Hemopexin/genetics/metabolism %K Host-Pathogen Interactions %K Humans %K Immunoglobulin Heavy Chains/genetics/metabolism %K Immunoglobulin M/genetics/metabolism %K Immunohistochemistry %K Macaca mulatta %K Plasminogen/genetics/metabolism %K Proteins/*analysis/genetics %K Proteomics/*methods %K Reverse Transcriptase Polymerase Chain Reaction %K Simian Acquired Immunodeficiency Syndrome/*cerebrospinal fluid/virology %K Simian immunodeficiency virus/physiology %K Tandem Mass Spectrometry %X The HIV-1-associated neurocognitive disorder occurs in approximately one-third of infected individuals. It has persisted in the current era of antiretroviral therapy, and its study is complicated by the lack of biomarkers for this condition. Since the cerebrospinal fluid is the most proximal biofluid to the site of pathology, we studied the cerebrospinal fluid in a nonhuman primate model for HIV-1-associated neurocognitive disorder. Here we present a simple and efficient liquid chromatography-coupled mass spectrometry-based proteomics approach that utilizes small amounts of cerebrospinal fluid. First, we demonstrate the validity of the methodology using human cerebrospinal fluid. Next, using the simian immunodeficiency virus-infected monkey model, we show its efficacy in identifying proteins such as alpha-1-antitrypsin, complement C3, hemopexin, IgM heavy chain, and plasminogen, whose increased expression is linked to disease. Finally, we find that the increase in cerebrospinal fluid proteins is linked to increased expression of their genes in the brain parenchyma, revealing that the cerebrospinal fluid alterations identified reflect changes in the brain itself and not merely leakage of the blood-brain or blood-cerebrospinal fluid barriers. This study reveals new central nervous system alterations in lentivirus-induced neurological disease, and this technique can be applied to other systems in which limited amounts of biofluids can be obtained. %B J Proteome ResJ Proteome ResJ Proteome Res %7 2009/03/14 %V 8 %P 2253-60 %8 May %@ 1535-3893 (Print)1535-3893 (Linking) %G eng %9 Research Support, N.I.H., Extramural %M 19281240 %2 2682714 %! Journal of proteome researchJournal of proteome research %0 Journal Article %J Mol Cell ProteomicsMol Cell ProteomicsMol Cell Proteomics %D 2009 %T Novel multiprotein complexes identified in the hyperthermophilic archaeon Pyrococcus furiosus by non-denaturing fractionation of the native proteome %A Menon, A. L. %A Poole, F. L., 2nd %A Cvetkovic, A. %A Trauger, S. A. %A Kalisiak, E. %A Scott, J. W. %A Shanmukh, S. %A Praissman, J. %A Jenney, F. E., Jr. %A Wikoff, W. R. %A Apon, J. V. %A Siuzdak, G. %A Adams, M. W. %K Amino Acids/metabolism %K Archaeal Proteins/*analysis/isolation & purification %K Chemical Fractionation/*methods %K Cytoplasm/metabolism %K Multiprotein Complexes/*analysis %K Protein Denaturation %K Protein Multimerization %K Proteome/*analysis %K Pyrococcus furiosus/*metabolism %X Virtually all cellular processes are carried out by dynamic molecular assemblies or multiprotein complexes, the compositions of which are largely undefined. They cannot be predicted solely from bioinformatics analyses nor are there well defined techniques currently available to unequivocally identify protein complexes (PCs). To address this issue, we attempted to directly determine the identity of PCs from native microbial biomass using Pyrococcus furiosus, a hyperthermophilic archaeon that grows optimally at 100 degrees C, as the model organism. Novel PCs were identified by large scale fractionation of the native proteome using non-denaturing, sequential column chromatography under anaerobic, reducing conditions. A total of 967 distinct P. furiosus proteins were identified by mass spectrometry (nano LC-ESI-MS/MS), representing approximately 80% of the cytoplasmic proteins. Based on the co-fractionation of proteins that are encoded by adjacent genes on the chromosome, 106 potential heteromeric PCs containing 243 proteins were identified, only 20 of which were known or expected. In addition to those of unknown function, novel and uncharacterized PCs were identified that are proposed to be involved in the metabolism of amino acids (10), carbohydrates (four), lipids (two), vitamins and metals (three), and DNA and RNA (nine). A further 30 potential PCs were classified as tentative, and the remaining potential PCs (13) were classified as weakly interacting. Some major advantages of native biomass fractionation for PC identification are that it provides a road map for the (partial) purification of native forms of novel and uncharacterized PCs, and the results can be utilized for the recombinant production of low abundance PCs to provide enough material for detailed structural and biochemical analyses. %B Mol Cell ProteomicsMol Cell ProteomicsMol Cell Proteomics %7 2008/12/02 %V 8 %P 735-51 %8 Apr %@ 1535-9484 (Electronic)1535-9476 (Linking) %G eng %9 Research Support, U.S. Gov't, Non-P.H.S. %M 19043064 %2 2668238 %! Molecular & cellular proteomics : MCPMolecular & cellular proteomics : MCP %0 Journal Article %J PLoS PathogPLoS PathogPLoS Pathog %D 2009 %T Endothelial targeting of cowpea mosaic virus (CPMV) via surface vimentin %A Koudelka, K. J. %A Destito, G. %A Plummer, E. M. %A Trauger, S. A. %A Siuzdak, G. %A Manchester, M. %K Animals %K Aorta/metabolism %K Cell Line %K Cell Membrane/metabolism %K Chromatography, Liquid %K Comovirus/*metabolism %K Endothelium, Vascular/*metabolism/*virology %K HeLa Cells %K Humans %K Male %K Mice %K Protein Binding %K Proteomics %K Rats %K Rats, Sprague-Dawley %K Tandem Mass Spectrometry %K Vimentin/*metabolism %K Virion/metabolism %X Cowpea mosaic virus (CPMV) is a plant comovirus in the picornavirus superfamily, and is used for a wide variety of biomedical and material science applications. Although its replication is restricted to plants, CPMV binds to and enters mammalian cells, including endothelial cells and particularly tumor neovascular endothelium in vivo. This natural capacity has lead to the use of CPMV as a sensor for intravital imaging of vascular development. Binding of CPMV to endothelial cells occurs via interaction with a 54 kD cell-surface protein, but this protein has not previously been identified. Here we identify the CPMV binding protein as a cell-surface form of the intermediate filament vimentin. The CPMV-vimentin interaction was established using proteomic screens and confirmed by direct interaction of CPMV with purified vimentin, as well as inhibition in a vimentin-knockout cell line. Vimentin and CPMV were also co-localized in vascular endothelium of mouse and rat in vivo. Together these studies indicate that surface vimentin mediates binding and may lead to internalization of CPMV in vivo, establishing surface vimentin as an important vascular endothelial ligand for nanoparticle targeting to tumors. These results also establish vimentin as a ligand for picornaviruses in both the plant and animal kingdoms of life. Since bacterial pathogens and several other classes of viruses also bind to surface vimentin, these studies suggest a common role for surface vimentin in pathogen transmission. %B PLoS PathogPLoS PathogPLoS Pathog %7 2009/05/05 %V 5 %P e1000417 %8 May %@ 1553-7374 (Electronic)1553-7366 (Linking) %G eng %9 Research Support, N.I.H., Extramural %M 19412526 %2 2670497 %! PLoS pathogensPLoS pathogens %0 Journal Article %J J Am Chem SocJ Am Chem SocJ Am Chem Soc %D 2009 %T Identification of a new endogenous metabolite and the characterization of its protein interactions through an immobilization approach %A Kalisiak, J. %A Trauger, S. A. %A Kalisiak, E. %A Morita, H. %A Fokin, V. V. %A Adams, M. W. %A Sharpless, K. B. %A Siuzdak, G. %K Metabolomics/*methods %K Proteins/*analysis/metabolism %K Pyrococcus furiosus/chemistry/metabolism %K Spermidine/*analogs & derivatives %K Tandem Mass Spectrometry/*methods %X The emerging field of global mass-based metabolomics provides a platform for discovering unknown metabolites and their specific biochemical pathways. We report the identification of a new endogenous metabolite, N(4)-(N-acetylaminopropyl)spermidine and the use of a novel proteomics based method for the investigation of its protein interaction using metabolite immobilization on agarose beads. The metabolite was isolated from the organism Pyrococcus furiosus, and structurally characterized through an iterative process of synthesizing candidate molecules and comparative analysis using accurate mass LC-MS/MS. An approach developed for the selective preparation of N(1)-acetylthermospermine, one of the possible structures of the unknown metabolite, provides a convenient route to new polyamine derivatives through methylation on the N(8) and N(4) of the thermospermine scaffold. The biochemical role of the novel metabolite as well as that of two other polyamines: spermidine and agmatine is investigated through metabolite immobilization and incubation with native proteins. The identification of eleven proteins that uniquely bind with N(4)-(N-acetylaminopropyl)spermidine, provides information on the role of this novel metabolite in the native organism. Identified proteins included hypothetical ones such as PF0607 and PF1199, and those involved in translation, DNA synthesis and the urea cycle like translation initiation factor IF-2, 50S ribosomal protein L14e, DNA-directed RNA polymerase, and ornithine carbamoyltransferase. The immobilization approach demonstrated here has the potential for application to other newly discovered endogenous metabolites found through untargeted metabolomics, as a preliminary screen for generating a list of proteins that could be further investigated for specific activity. %B J Am Chem SocJ Am Chem SocJ Am Chem Soc %7 2008/12/06 %V 131 %P 378-86 %8 Jan 14 %@ 1520-5126 (Electronic)0002-7863 (Linking) %G eng %9 Research Support, N.I.H., ExtramuralResearch Support, U.S. Gov't, Non-P.H.S. %M 19055353 %2 2735399 %! Journal of the American Chemical SocietyJournal of the American Chemical Society %0 Journal Article %J J Biol ChemJ Biol ChemJ Biol Chem %D 2009 %T Increased enzymatic O-GlcNAcylation of mitochondrial proteins impairs mitochondrial function in cardiac myocytes exposed to high glucose %A Hu, Y. %A Suarez, J. %A Fricovsky, E. %A Wang, H. %A Scott, B. T. %A Trauger, S. A. %A Han, W. %A Oyeleye, M. O. %A Dillmann, W. H. %K Acetylglucosamine/genetics/metabolism %K Animals %K Cyclooxygenase 1/metabolism %K Diabetes Mellitus/*enzymology/pathology %K Gene Expression Regulation, Enzymologic/drug effects %K Glucose/*pharmacology %K Glucosyltransferases/*biosynthesis %K Glycosylation/drug effects %K Membrane Proteins/metabolism %K Mice %K Mitochondria, Heart/*enzymology/pathology %K Mitochondrial Proteins/*biosynthesis %K Myocytes, Cardiac/*enzymology/pathology %K NADH Dehydrogenase/metabolism %K Protein Processing, Post-Translational/*drug effects %K Rats %K Sweetening Agents/*pharmacology %X Increased nuclear protein O-linked beta-N-acetylglucosamine glycosylation (O-GlcNAcylation) mediated by high glucose treatment or the hyperglycemia of diabetes mellitus contributes to cardiac myocyte dysfunction. However, whether mitochondrial proteins in cardiac myocytes are also submitted to O-GlcNAcylation or excessive O-GlcNAcylation alters mitochondrial function is unknown. In this study, we determined if mitochondrial proteins are O-GlcNAcylated and explored if increased O-GlcNAcylation is linked to high glucose-induced mitochondrial dysfunction in neonatal rat cardiomyocytes. By immunoprecipitation, we found that several mitochondrial proteins, which are members of complexes of the respiratory chain, like subunit NDUFA9 of complex I, subunits core 1 and core 2 of complex III, and the mitochondrial DNA-encoded subunit I of complex IV (COX I) are O-GlcNAcylated. By mass spectrometry, we identified that serine 156 on NDUFA9 is O-GlcNAcylated. High glucose treatment (30 mm glucose) increases mitochondrial protein O-GlcNAcylation, including those of COX I and NDUFA9 which are reduced by expression of O-GlcNAcase (GCA). Increased mitochondrial O-GlcNAcylation is associated with impaired activity of complex I, III, and IV in addition to lower mitochondrial calcium and cellular ATP content. When the excessive O-GlcNAc modification is reduced by GCA expression, mitochondrial function improves; the activity of complex I, III, and IV increases to normal and mitochondrial calcium and cellular ATP content are returned to control levels. From these results we conclude that specific mitochondrial proteins of cardiac myocytes are O-GlcNAcylated and that exposure to high glucose increases mitochondrial protein O-GlcNAcylation, which in turn contributes to impaired mitochondrial function. %B J Biol ChemJ Biol ChemJ Biol Chem %7 2008/11/14 %V 284 %P 547-55 %8 Jan 2 %@ 0021-9258 (Print)0021-9258 (Linking) %G eng %9 Research Support, N.I.H., Extramural %M 19004814 %2 2610513 %! The Journal of biological chemistryThe Journal of biological chemistry %0 Journal Article %J J Biol Chem %D 2009 %T Increased enzymatic O-GlcNAcylation of mitochondrial proteins impairs mitochondrial function in cardiac myocytes exposed to high glucose %A Hu, Y. %A Suarez, J. %A Fricovsky, E. %A Wang, H. %A Scott, B. T. %A Trauger, S. A. %A Han, W. %A Oyeleye, M. O. %A Dillmann, W. H. %XIncreased nuclear protein O-linked beta-N-acetylglucosamine glycosylation (O-GlcNAcylation) mediated by high glucose treatment or the hyperglycemia of diabetes mellitus contributes to cardiac myocyte dysfunction. However, whether mitochondrial proteins in cardiac myocytes are also submitted to O-GlcNAcylation or excessive O-GlcNAcylation alters mitochondrial function is unknown. In this study, we determined if mitochondrial proteins are O-GlcNAcylated and explored if increased O-GlcNAcylation is linked to high glucose-induced mitochondrial dysfunction in neonatal rat cardiomyocytes. By immunoprecipitation, we found that several mitochondrial proteins, which are members of complexes of the respiratory chain, like subunit NDUFA9 of complex I, subunits core 1 and core 2 of complex III, and the mitochondrial DNA-encoded subunit I of complex IV (COX I) are O-GlcNAcylated. By mass spectrometry, we identified that serine 156 on NDUFA9 is O-GlcNAcylated. High glucose treatment (30 mm glucose) increases mitochondrial protein O-GlcNAcylation, including those of COX I and NDUFA9 which are reduced by expression of O-GlcNAcase (GCA). Increased mitochondrial O-GlcNAcylation is associated with impaired activity of complex I, III, and IV in addition to lower mitochondrial calcium and cellular ATP content. When the excessive O-GlcNAc modification is reduced by GCA expression, mitochondrial function improves; the activity of complex I, III, and IV increases to normal and mitochondrial calcium and cellular ATP content are returned to control levels. From these results we conclude that specific mitochondrial proteins of cardiac myocytes are O-GlcNAcylated and that exposure to high glucose increases mitochondrial protein O-GlcNAcylation, which in turn contributes to impaired mitochondrial function.
%B J Biol Chem %7 2008/11/14 %V 284 %P 547-55 %8 Jan 2 %@ 0021-9258 (Print)0021-9258 (Linking) %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/19004814 %9 Research Support, N.I.H., Extramural %M 19004814 %2 2610513 %0 Journal Article %J J Proteome Res %D 2009 %T Cerebrospinal fluid proteomics reveals potential pathogenic changes in the brains of SIV-infected monkeys %A Pendyala, G. %A Trauger, S. A. %A Kalisiak, E. %A Ellis, R. J. %A Siuzdak, G. %A Fox, H. S. %XThe HIV-1-associated neurocognitive disorder occurs in approximately one-third of infected individuals. It has persisted in the current era of antiretroviral therapy, and its study is complicated by the lack of biomarkers for this condition. Since the cerebrospinal fluid is the most proximal biofluid to the site of pathology, we studied the cerebrospinal fluid in a nonhuman primate model for HIV-1-associated neurocognitive disorder. Here we present a simple and efficient liquid chromatography-coupled mass spectrometry-based proteomics approach that utilizes small amounts of cerebrospinal fluid. First, we demonstrate the validity of the methodology using human cerebrospinal fluid. Next, using the simian immunodeficiency virus-infected monkey model, we show its efficacy in identifying proteins such as alpha-1-antitrypsin, complement C3, hemopexin, IgM heavy chain, and plasminogen, whose increased expression is linked to disease. Finally, we find that the increase in cerebrospinal fluid proteins is linked to increased expression of their genes in the brain parenchyma, revealing that the cerebrospinal fluid alterations identified reflect changes in the brain itself and not merely leakage of the blood-brain or blood-cerebrospinal fluid barriers. This study reveals new central nervous system alterations in lentivirus-induced neurological disease, and this technique can be applied to other systems in which limited amounts of biofluids can be obtained.
%B J Proteome Res %7 2009/03/14 %V 8 %P 2253-60 %8 May %@ 1535-3893 (Print)1535-3893 (Linking) %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/19281240 %9 Research Support, N.I.H., Extramural %M 19281240 %2 2682714 %0 Journal Article %J J Am Chem Soc %D 2009 %T Identification of a new endogenous metabolite and the characterization of its protein interactions through an immobilization approach %A Kalisiak, J. %A Trauger, S. A. %A Kalisiak, E. %A Morita, H. %A Fokin, V. V. %A Adams, M. W. %A Sharpless, K. B. %A Siuzdak, G. %XThe emerging field of global mass-based metabolomics provides a platform for discovering unknown metabolites and their specific biochemical pathways. We report the identification of a new endogenous metabolite, N(4)-(N-acetylaminopropyl)spermidine and the use of a novel proteomics based method for the investigation of its protein interaction using metabolite immobilization on agarose beads. The metabolite was isolated from the organism Pyrococcus furiosus, and structurally characterized through an iterative process of synthesizing candidate molecules and comparative analysis using accurate mass LC-MS/MS. An approach developed for the selective preparation of N(1)-acetylthermospermine, one of the possible structures of the unknown metabolite, provides a convenient route to new polyamine derivatives through methylation on the N(8) and N(4) of the thermospermine scaffold. The biochemical role of the novel metabolite as well as that of two other polyamines: spermidine and agmatine is investigated through metabolite immobilization and incubation with native proteins. The identification of eleven proteins that uniquely bind with N(4)-(N-acetylaminopropyl)spermidine, provides information on the role of this novel metabolite in the native organism. Identified proteins included hypothetical ones such as PF0607 and PF1199, and those involved in translation, DNA synthesis and the urea cycle like translation initiation factor IF-2, 50S ribosomal protein L14e, DNA-directed RNA polymerase, and ornithine carbamoyltransferase. The immobilization approach demonstrated here has the potential for application to other newly discovered endogenous metabolites found through untargeted metabolomics, as a preliminary screen for generating a list of proteins that could be further investigated for specific activity.
%B J Am Chem Soc %7 2008/12/06 %V 131 %P 378-86 %8 Jan 14 %@ 1520-5126 (Electronic)0002-7863 (Linking) %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/19055353 %9 Research Support, N.I.H., ExtramuralResearch Support, U.S. Gov't, Non-P.H.S. %M 19055353 %2 2735399 %0 Journal Article %J Mol Cell Proteomics %D 2009 %T Novel multiprotein complexes identified in the hyperthermophilic archaeon Pyrococcus furiosus by non-denaturing fractionation of the native proteome %A Menon, A. L. %A Poole, F. L., 2nd %A Cvetkovic, A. %A Trauger, S. A. %A Kalisiak, E. %A Scott, J. W. %A Shanmukh, S. %A Praissman, J. %A Jenney, F. E., Jr. %A Wikoff, W. R. %A Apon, J. V. %A Siuzdak, G. %A Adams, M. W. %XVirtually all cellular processes are carried out by dynamic molecular assemblies or multiprotein complexes, the compositions of which are largely undefined. They cannot be predicted solely from bioinformatics analyses nor are there well defined techniques currently available to unequivocally identify protein complexes (PCs). To address this issue, we attempted to directly determine the identity of PCs from native microbial biomass using Pyrococcus furiosus, a hyperthermophilic archaeon that grows optimally at 100 degrees C, as the model organism. Novel PCs were identified by large scale fractionation of the native proteome using non-denaturing, sequential column chromatography under anaerobic, reducing conditions. A total of 967 distinct P. furiosus proteins were identified by mass spectrometry (nano LC-ESI-MS/MS), representing approximately 80% of the cytoplasmic proteins. Based on the co-fractionation of proteins that are encoded by adjacent genes on the chromosome, 106 potential heteromeric PCs containing 243 proteins were identified, only 20 of which were known or expected. In addition to those of unknown function, novel and uncharacterized PCs were identified that are proposed to be involved in the metabolism of amino acids (10), carbohydrates (four), lipids (two), vitamins and metals (three), and DNA and RNA (nine). A further 30 potential PCs were classified as tentative, and the remaining potential PCs (13) were classified as weakly interacting. Some major advantages of native biomass fractionation for PC identification are that it provides a road map for the (partial) purification of native forms of novel and uncharacterized PCs, and the results can be utilized for the recombinant production of low abundance PCs to provide enough material for detailed structural and biochemical analyses.
%B Mol Cell Proteomics %7 2008/12/02 %V 8 %P 735-51 %8 Apr %@ 1535-9484 (Electronic)1535-9476 (Linking) %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/19043064 %9 Research Support, U.S. Gov't, Non-P.H.S. %M 19043064 %2 2668238 %0 Journal Article %J PLoS Pathog %D 2009 %T Endothelial targeting of cowpea mosaic virus (CPMV) via surface vimentin %A Koudelka, K. J. %A Destito, G. %A Plummer, E. M. %A Trauger, S. A. %A Siuzdak, G. %A Manchester, M. %XCowpea mosaic virus (CPMV) is a plant comovirus in the picornavirus superfamily, and is used for a wide variety of biomedical and material science applications. Although its replication is restricted to plants, CPMV binds to and enters mammalian cells, including endothelial cells and particularly tumor neovascular endothelium in vivo. This natural capacity has lead to the use of CPMV as a sensor for intravital imaging of vascular development. Binding of CPMV to endothelial cells occurs via interaction with a 54 kD cell-surface protein, but this protein has not previously been identified. Here we identify the CPMV binding protein as a cell-surface form of the intermediate filament vimentin. The CPMV-vimentin interaction was established using proteomic screens and confirmed by direct interaction of CPMV with purified vimentin, as well as inhibition in a vimentin-knockout cell line. Vimentin and CPMV were also co-localized in vascular endothelium of mouse and rat in vivo. Together these studies indicate that surface vimentin mediates binding and may lead to internalization of CPMV in vivo, establishing surface vimentin as an important vascular endothelial ligand for nanoparticle targeting to tumors. These results also establish vimentin as a ligand for picornaviruses in both the plant and animal kingdoms of life. Since bacterial pathogens and several other classes of viruses also bind to surface vimentin, these studies suggest a common role for surface vimentin in pathogen transmission.
%B PLoS Pathog %7 2009/05/05 %V 5 %P e1000417 %8 May %@ 1553-7374 (Electronic)1553-7366 (Linking) %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/19412526 %9 Research Support, N.I.H., Extramural %M 19412526 %2 2670497 %0 Journal Article %J Cell Stem Cell %D 2009 %T Generation of induced pluripotent stem cells using recombinant proteins %A Zhou, H. %A Wu, S. %A Joo, J. Y. %A Zhu, S. %A Han, D. W. %A Lin, T. %A Trauger, S. %A Bien, G. %A Yao, S. %A Zhu, Y. %A Siuzdak, G. %A Scholer, H. R. %A Duan, L. %A Ding, S. %X n/a %B Cell Stem Cell %7 2009/04/29 %V 4 %P 381-4 %8 May 8 %@ 1875-9777 (Electronic) %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/19398399 %9 Research Support, Non-U.S. Gov't %M 19398399 %0 Journal Article %J Rapid Commun Mass Spectrom %D 2009 %T Phosphonium labeling for increasing metabolomic coverage of neutral lipids using electrospray ionization mass spectrometry %A Woo, H. K. %A Go, E. P. %A Hoang, L. %A Trauger, S. A. %A Bowen, B. %A Siuzdak, G. %A Northen, T. R. %XMass spectrometry has become an indispensable tool for the global study of metabolites (metabolomics), primarily using electrospray ionization mass spectrometry (ESI-MS). However, many important classes of molecules such as neutral lipids do not ionize well by ESI and go undetected. Chemical derivatization of metabolites can enhance ionization for increased sensitivity and metabolomic coverage. Here we describe the use of tris(2,4,6,-trimethoxyphenyl)phosphonium acetic acid (TMPP-AA) to improve liquid chromatography (LC)/ESI-MS detection of hydroxylated metabolites (i.e. lipids) from serum extracts. Cholesterol which is not normally detected from serum using ESI is observed with attomole sensitivity. This approach was applied to identify four endogenous lipids (hexadecanoyl-sn-glycerol, dihydrotachysterol, octadecanol, and alpha-tocopherol) from human serum. Overall, this approach extends the types of metabolites which can be detected using standard ESI-MS instrumentation and demonstrates the potential for targeted metabolomics analysis.
%B Rapid Commun Mass Spectrom %7 2009/05/19 %V 23 %P 1849-55 %8 Jun %@ 1097-0231 (Electronic)0951-4198 (Linking) %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/19449318 %9 Evaluation StudiesResearch Support, N.I.H., ExtramuralResearch Support, U.S. Gov't, Non-P.H.S. %M 19449318 %2 3052201 %0 Journal Article %J J Proteome Res %D 2009 %T Response and recovery in the plasma metabolome tracks the acute LCMV-induced immune response %A Wikoff, W. R. %A Kalisak, E. %A Trauger, S. %A Manchester, M. %A Siuzdak, G. %XLymphocytic choriomeningitis virus (LCMV) infection of mice is noncytopathic, producing well-characterized changes reflecting the host immune response. Untargeted metabolomics using mass spectrometry identified endogenous small molecule changes in blood from mice inoculated with LCMV, sampled at days 1, 3, 7, and 14 post infection. These time points correspond to well characterized events during acute LCMV infection and the immune response. Diverse pathways were altered, including TCA cycle intermediates, gamma-glutamyl dipeptides, lysophosphatidyl cholines, and fatty acids. The kynurenine pathway was activated, surprising because it is stimulated by IFN-gamma, which LCMV suppresses, thus, suggesting alternative activators. In contrast, biopterin/neopterin, another IFN-gamma stimulated pathway, was not activated. Many metabolites followed "response and recovery" kinetics, decreasing after infection to a minimum at days 3-7, and returning to normal by day 14. The TCA pathway followed this pattern, including citrate, cis-aconitate and alpha-ketoglutarate, intriguing because succinate has been shown to mediate cellular immunity. This response and recovery dynamic tracks the immune response, including the rise and fall of natural killer cell populations, serum TNF receptor concentration, and viral clearance. Metabolomics can provide target pathways for molecular diagnostics or therapeutics of viral infection and immunity.
%B J Proteome Res %7 2009/06/06 %V 8 %P 3578-87 %8 Jul %@ 1535-3893 (Print)1535-3893 (Linking) %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/19496611 %9 Research Support, N.I.H., Extramural %M 19496611 %0 Journal Article %J J Proteome ResJ Proteome ResJ Proteome Res %D 2008 %T Correlating the transcriptome, proteome, and metabolome in the environmental adaptation of a hyperthermophile %A Trauger, S. A. %A Kalisak, E. %A Kalisiak, J. %A Morita, H. %A Weinberg, M. V. %A Menon, A. L. %A Poole, F. L., 2nd %A Adams, M. W. %A Siuzdak, G. %K *Adaptation, Physiological %K *Proteome %K Bacterial Proteins/chemistry/genetics/*metabolism %K Oligonucleotide Array Sequence Analysis %K Peptides/isolation & purification %K RNA, Messenger/*genetics %K Spectrometry, Mass, Electrospray Ionization %K Thermus thermophilus/genetics/*metabolism/physiology %X We have performed a comprehensive characterization of global molecular changes for a model organism Pyrococcus furiosus using transcriptomic (DNA microarray), proteomic, and metabolomic analysis as it undergoes a cold adaptation response from its optimal 95 to 72 degrees C. Metabolic profiling on the same set of samples shows the down-regulation of many metabolites. However, some metabolites are found to be strongly up-regulated. An approach using accurate mass, isotopic pattern, database searching, and retention time is used to putatively identify several metabolites of interest. Many of the up-regulated metabolites are part of an alternative polyamine biosynthesis pathway previously established in a thermophilic bacterium Thermus thermophilus. Arginine, agmatine, spermidine, and branched polyamines N4-aminopropylspermidine and N4-( N-acetylaminopropyl)spermidine were unambiguously identified based on their accurate mass, isotopic pattern, and matching of MS/MS data acquired under identical conditions for the natural metabolite and a high purity standard. Both DNA microarray and semiquantitative proteomic analysis using a label-free spectral counting approach indicate the down-regulation of a large majority of genes with diverse predicted functions related to growth such as transcription, amino acid biosynthesis, and translation. Some genes are, however, found to be up-regulated through the measurement of their relative mRNA and protein levels. The complimentary information obtained by the various "omics" techniques is used to catalogue and correlate the overall molecular changes. %B J Proteome ResJ Proteome ResJ Proteome Res %7 2008/02/06 %V 7 %P 1027-35 %8 Mar %@ 1535-3893 (Print)1535-3893 (Linking) %G eng %9 Research Support, U.S. Gov't, Non-P.H.S. %M 18247545 %! Journal of proteome researchJournal of proteome research %0 Journal Article %J Anal ChemAnal ChemAnal Chem %D 2008 %T Quantitative ESI-TOF analysis of macromolecular assembly kinetics %A Bunner, A. E. %A Trauger, S. A. %A Siuzdak, G. %A Williamson, J. R. %K *Spectrometry, Mass, Electrospray Ionization %K *Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization %K Escherichia coli/genetics/metabolism %K Kinetics %K Macromolecular Substances/chemistry/*metabolism %K Protein Subunits %K Ribosomal Proteins/genetics/*metabolism %K Ribosomes/*chemistry/genetics/*metabolism %K RNA, Ribosomal, 16S/genetics/metabolism %X The Escherichia coli small (30S) ribosomal subunit is a particularly well-characterized model system for studying in vitro self-assembly. A previously developed pulse-chase monitored by quantitative mass spectrometry (PC/QMS) approach to measuring kinetics of in vitro 30S assembly suffered from poor signal-to-noise and was unable to observe some ribosomal proteins. We have developed an improved LC-MS based method using quantitative ESI-TOF analysis of isotope-labeled tryptic peptides. Binding rates for 18 of the 20 ribosomal proteins are reported, and exchange of proteins S2 and S21 between bound and unbound states prevented measurement of their binding kinetics. Multiphasic kinetics of 3' domain proteins S7 and S9 are reported, which support an assembly mechanism that utilizes multiple parallel pathways. This quantitative ESI-TOF approach should be widely applicable to study the assembly of other macromolecular complexes and to quantitative proteomics experiments in general. %B Anal ChemAnal ChemAnal Chem %7 2008/11/15 %V 80 %P 9379-86 %8 Dec 15 %@ 1520-6882 (Electronic)0003-2700 (Linking) %G eng %9 Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov't %M 19007188 %2 2735594 %! Analytical chemistryAnalytical chemistry %0 Journal Article %J Anal ChemAnal ChemAnal Chem %D 2008 %T XCMS2: processing tandem mass spectrometry data for metabolite identification and structural characterization %A Benton, H. P. %A Wong, D. M. %A Trauger, S. A. %A Siuzdak, G. %K *Algorithms %K *Databases as Topic %K *Software %K *Tandem Mass Spectrometry %K Biological Markers/analysis/*chemistry %K Chromatography, Liquid %K Computational Biology/*methods %K Databases, Protein %K Humans %K Molecular Structure %K Peptide Mapping/methods %K Sequence Analysis, Protein/methods %K Serum/*chemistry %X Mass spectrometry based metabolomics represents a new area for bioinformatics technology development. While the computational tools currently available such as XCMS statistically assess and rank LC-MS features, they do not provide information about their structural identity. XCMS(2) is an open source software package which has been developed to automatically search tandem mass spectrometry (MS/MS) data against high quality experimental MS/MS data from known metabolites contained in a reference library (METLIN). Scoring of hits is based on a "shared peak count" method that identifies masses of fragment ions shared between the analytical and reference MS/MS spectra. Another functional component of XCMS(2) is the capability of providing structural information for unknown metabolites, which are not in the METLIN database. This "similarity search" algorithm has been developed to detect possible structural motifs in the unknown metabolite which may produce characteristic fragment ions and neutral losses to related reference compounds contained in METLIN, even if the precursor masses are not the same. %B Anal ChemAnal ChemAnal Chem %7 2008/07/17 %V 80 %P 6382-9 %8 Aug 15 %@ 1520-6882 (Electronic)0003-2700 (Linking) %G eng %9 Research Support, N.I.H., ExtramuralResearch Support, U.S. Gov't, Non-P.H.S. %M 18627180 %2 2728033 %! Analytical chemistryAnalytical chemistry %0 Journal Article %J J Proteome Res %D 2008 %T Correlating the transcriptome, proteome, and metabolome in the environmental adaptation of a hyperthermophile %A Trauger, S. A. %A Kalisak, E. %A Kalisiak, J. %A Morita, H. %A Weinberg, M. V. %A Menon, A. L. %A Poole, F. L., 2nd %A Adams, M. W. %A Siuzdak, G. %XWe have performed a comprehensive characterization of global molecular changes for a model organism Pyrococcus furiosus using transcriptomic (DNA microarray), proteomic, and metabolomic analysis as it undergoes a cold adaptation response from its optimal 95 to 72 degrees C. Metabolic profiling on the same set of samples shows the down-regulation of many metabolites. However, some metabolites are found to be strongly up-regulated. An approach using accurate mass, isotopic pattern, database searching, and retention time is used to putatively identify several metabolites of interest. Many of the up-regulated metabolites are part of an alternative polyamine biosynthesis pathway previously established in a thermophilic bacterium Thermus thermophilus. Arginine, agmatine, spermidine, and branched polyamines N4-aminopropylspermidine and N4-( N-acetylaminopropyl)spermidine were unambiguously identified based on their accurate mass, isotopic pattern, and matching of MS/MS data acquired under identical conditions for the natural metabolite and a high purity standard. Both DNA microarray and semiquantitative proteomic analysis using a label-free spectral counting approach indicate the down-regulation of a large majority of genes with diverse predicted functions related to growth such as transcription, amino acid biosynthesis, and translation. Some genes are, however, found to be up-regulated through the measurement of their relative mRNA and protein levels. The complimentary information obtained by the various "omics" techniques is used to catalogue and correlate the overall molecular changes.
%B J Proteome Res %7 2008/02/06 %V 7 %P 1027-35 %8 Mar %@ 1535-3893 (Print)1535-3893 (Linking) %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/18247545 %9 Research Support, U.S. Gov't, Non-P.H.S. %M 18247545 %0 Journal Article %J Anal Chem %D 2008 %T Quantitative ESI-TOF analysis of macromolecular assembly kinetics %A Bunner, A. E. %A Trauger, S. A. %A Siuzdak, G. %A Williamson, J. R. %XThe Escherichia coli small (30S) ribosomal subunit is a particularly well-characterized model system for studying in vitro self-assembly. A previously developed pulse-chase monitored by quantitative mass spectrometry (PC/QMS) approach to measuring kinetics of in vitro 30S assembly suffered from poor signal-to-noise and was unable to observe some ribosomal proteins. We have developed an improved LC-MS based method using quantitative ESI-TOF analysis of isotope-labeled tryptic peptides. Binding rates for 18 of the 20 ribosomal proteins are reported, and exchange of proteins S2 and S21 between bound and unbound states prevented measurement of their binding kinetics. Multiphasic kinetics of 3' domain proteins S7 and S9 are reported, which support an assembly mechanism that utilizes multiple parallel pathways. This quantitative ESI-TOF approach should be widely applicable to study the assembly of other macromolecular complexes and to quantitative proteomics experiments in general.
%B Anal Chem %7 2008/11/15 %V 80 %P 9379-86 %8 Dec 15 %@ 1520-6882 (Electronic)0003-2700 (Linking) %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/19007188 %9 Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov't %M 19007188 %2 2735594 %0 Journal Article %J Anal Chem %D 2008 %T XCMS2: processing tandem mass spectrometry data for metabolite identification and structural characterization %A Benton, H. P. %A Wong, D. M. %A Trauger, S. A. %A Siuzdak, G. %XMass spectrometry based metabolomics represents a new area for bioinformatics technology development. While the computational tools currently available such as XCMS statistically assess and rank LC-MS features, they do not provide information about their structural identity. XCMS(2) is an open source software package which has been developed to automatically search tandem mass spectrometry (MS/MS) data against high quality experimental MS/MS data from known metabolites contained in a reference library (METLIN). Scoring of hits is based on a "shared peak count" method that identifies masses of fragment ions shared between the analytical and reference MS/MS spectra. Another functional component of XCMS(2) is the capability of providing structural information for unknown metabolites, which are not in the METLIN database. This "similarity search" algorithm has been developed to detect possible structural motifs in the unknown metabolite which may produce characteristic fragment ions and neutral losses to related reference compounds contained in METLIN, even if the precursor masses are not the same.
%B Anal Chem %7 2008/07/17 %V 80 %P 6382-9 %8 Aug 15 %@ 1520-6882 (Electronic)0003-2700 (Linking) %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/18627180 %9 Research Support, N.I.H., ExtramuralResearch Support, U.S. Gov't, Non-P.H.S. %M 18627180 %2 2728033 %0 Journal Article %J J Biol ChemJ Biol ChemJ Biol Chem %D 2007 %T A metalloantibody that irreversibly binds a protein antigen %A Trisler, K. %A Looger, L. L. %A Sharma, V. %A Baker, M. %A Benson, D. E. %A Trauger, S. %A Schultz, P. G. %A Smider, V. V. %K *Antibody Affinity/genetics %K Antibodies, Monoclonal/*chemistry/diagnostic use/genetics/metabolism/therapeutic %K Antigens/*chemistry %K Humans %K Kinetics %K Metals/*chemistry/metabolism %K Protein Binding/genetics %K Tumor Necrosis Factor-alpha/*chemistry/metabolism %K use %X Antibody affinity is critically important in therapeutic applications, as well as steady state diagnostic assays. Picomolar affinity antibodies, approaching the association limit of protein-protein interactions, have been discovered for highly potent antigens, but even such high-affinity binders have off-rates sufficient to negate therapeutic efficacy. To cross this affinity threshold, antibodies that tether their targets in a manner other than reversible non-covalent interaction will be required. Here we report the design and construction of an antibody that forms an irreversible complex with a protein antigen in a metal-dependent reaction. The complex resists thermal and chemical denaturation, as well as attempts to remove the coordinating metal ion. Such irreversibly binding antibodies could facilitate the development of next generation "reactive antibody" therapeutics and diagnostics. %B J Biol ChemJ Biol ChemJ Biol Chem %7 2007/07/10 %V 282 %P 26344-53 %8 Sep 7 %@ 0021-9258 (Print)0021-9258 (Linking) %G eng %M 17617633 %! The Journal of biological chemistryThe Journal of biological chemistry %0 Journal Article %J J Proteome ResJ Proteome ResJ Proteome Res %D 2007 %T Selective metabolite and peptide capture/mass detection using fluorous affinity tags %A Go, E. P. %A Uritboonthai, W. %A Apon, J. V. %A Trauger, S. A. %A Nordstrom, A. %A O'Maille, G. %A Brittain, S. M. %A Peters, E. C. %A Siuzdak, G. %K Affinity Labels/*chemistry %K Amino Acid Sequence %K Amino Acids/*blood %K Humans %K Hydrocarbons, Fluorinated/*chemistry %K Mass Spectrometry/*methods %K Molecular Sequence Data %K Peptides/*analysis %K Silicon/chemistry %K Surface Properties %X A new and general methodology is described for the targeted enrichment and subsequent direct mass spectrometric characterization of sample subsets bearing various chemical functionalities from highly complex mixtures of biological origin. Specifically, sample components containing a chemical moiety of interest are first selectively labeled with perfluoroalkyl groups, and the entire sample is then applied to a perfluoroalkyl-silylated porous silicon (pSi) surface. Due to the unique hydrophobic and lipophobic nature of the perfluorinated tags, unlabeled sample components are readily removed using simple surface washes, and the enriched sample fraction can then directly be analyzed by desorption/ionization on silicon mass spectrometry (DIOS-MS). Importantly, this fluorous-based enrichment methodology provides a single platform that is equally applicable to both peptide as well as small molecule focused applications. The utility of this technique is demonstrated by the enrichment and mass spectrometric analysis of both various peptide subsets from protein digests as well as amino acids from serum. %B J Proteome ResJ Proteome ResJ Proteome Res %7 2007/03/09 %V 6 %P 1492-9 %8 Apr %@ 1535-3893 (Print)1535-3893 (Linking) %G eng %9 Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, Non-P.H.S. %M 17343404 %2 2530906 %! Journal of proteome researchJournal of proteome research %0 Journal Article %J J Biol Chem %D 2007 %T A metalloantibody that irreversibly binds a protein antigen %A Trisler, K. %A Looger, L. L. %A Sharma, V. %A Baker, M. %A Benson, D. E. %A Trauger, S. %A Schultz, P. G. %A Smider, V. V. %XAntibody affinity is critically important in therapeutic applications, as well as steady state diagnostic assays. Picomolar affinity antibodies, approaching the association limit of protein-protein interactions, have been discovered for highly potent antigens, but even such high-affinity binders have off-rates sufficient to negate therapeutic efficacy. To cross this affinity threshold, antibodies that tether their targets in a manner other than reversible non-covalent interaction will be required. Here we report the design and construction of an antibody that forms an irreversible complex with a protein antigen in a metal-dependent reaction. The complex resists thermal and chemical denaturation, as well as attempts to remove the coordinating metal ion. Such irreversibly binding antibodies could facilitate the development of next generation "reactive antibody" therapeutics and diagnostics.
%B J Biol Chem %7 2007/07/10 %V 282 %P 26344-53 %8 Sep 7 %@ 0021-9258 (Print)0021-9258 (Linking) %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/17617633 %M 17617633 %0 Journal Article %J J Proteome Res %D 2007 %T Selective metabolite and peptide capture/mass detection using fluorous affinity tags %A Go, E. P. %A Uritboonthai, W. %A Apon, J. V. %A Trauger, S. A. %A Nordstrom, A. %A O'Maille, G. %A Brittain, S. M. %A Peters, E. C. %A Siuzdak, G. %XA new and general methodology is described for the targeted enrichment and subsequent direct mass spectrometric characterization of sample subsets bearing various chemical functionalities from highly complex mixtures of biological origin. Specifically, sample components containing a chemical moiety of interest are first selectively labeled with perfluoroalkyl groups, and the entire sample is then applied to a perfluoroalkyl-silylated porous silicon (pSi) surface. Due to the unique hydrophobic and lipophobic nature of the perfluorinated tags, unlabeled sample components are readily removed using simple surface washes, and the enriched sample fraction can then directly be analyzed by desorption/ionization on silicon mass spectrometry (DIOS-MS). Importantly, this fluorous-based enrichment methodology provides a single platform that is equally applicable to both peptide as well as small molecule focused applications. The utility of this technique is demonstrated by the enrichment and mass spectrometric analysis of both various peptide subsets from protein digests as well as amino acids from serum.
%B J Proteome Res %7 2007/03/09 %V 6 %P 1492-9 %8 Apr %@ 1535-3893 (Print)1535-3893 (Linking) %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/17343404 %9 Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, Non-P.H.S. %M 17343404 %2 2530906 %0 Journal Article %J Anal ChemAnal ChemAnal Chem %D 2006 %T Solvent-dependent metabolite distribution, clustering, and protein extraction for serum profiling with mass spectrometry %A Want, E. J. %A O'Maille, G. %A Smith, C. A. %A Brandon, T. R. %A Uritboonthai, W. %A Qin, C. %A Trauger, S. A. %A Siuzdak, G. %K Blood Proteins/*analysis/isolation & purification %K Chromatography, Liquid/methods %K Humans %K Male %K Sensitivity and Specificity %K Serum/*chemistry %K Solvents/chemistry %K Spectrometry, Mass, Electrospray Ionization/*methods %X The aim of metabolite profiling is to monitor all metabolites within a biological sample for applications in basic biochemical research as well as pharmacokinetic studies and biomarker discovery. Here, novel data analysis software, XCMS, was used to monitor all metabolite features detected from an array of serum extraction methods, with application to metabolite profiling using electrospray liquid chromatography/mass spectrometry (ESI-LC/MS). The XCMS software enabled the comparison of methods with regard to reproducibility, the number and type of metabolite features detected, and the similarity of these features between different extraction methods. Extraction efficiency with regard to metabolite feature hydrophobicity was examined through the generation of unique feature density distribution plots, displaying feature distribution along chromatographic time. Hierarchical clustering was performed to highlight similarities in the metabolite features observed between the extraction methods. Protein extraction efficiency was determined using the Bradford assay, and the residual proteins were identified using nano-LC/MS/MS. Additionally, the identification of four of the most intensely ionized serum metabolites using FTMS and tandem mass spectrometry was reported. The extraction methods, ranging from organic solvents and acids to heat denaturation, varied widely in both protein removal efficiency and the number of mass spectral features detected. Methanol protein precipitation followed by centrifugation was found to be the most effective, straightforward, and reproducible approach, resulting in serum extracts containing over 2000 detected metabolite features and less than 2% residual protein. Interestingly, the combination of all approaches produced over 10,000 unique metabolite features, a number that is indicative of the complexity of the human metabolome and the potential of metabolomics in biomarker discovery. %B Anal ChemAnal ChemAnal Chem %7 2006/02/02 %V 78 %P 743-52 %8 Feb 1 %@ 0003-2700 (Print)0003-2700 (Linking) %G eng %M 16448047 %! Analytical chemistryAnalytical chemistry %0 Journal Article %J J Proteome ResJ Proteome ResJ Proteome Res %D 2006 %T Mass spectrometry reveals specific and global molecular transformations during viral infection %A Go, E. P. %A Wikoff, W. R. %A Shen, Z. %A O'Maille, G. %A Morita, H. %A Conrads, T. P. %A Nordstrom, A. %A Trauger, S. A. %A Uritboonthai, W. %A Lucas, D. A. %A Chan, K. C. %A Veenstra, T. D. %A Lewicki, H. %A Oldstone, M. B. %A Schneemann, A. %A Siuzdak, G. %K *Gene Expression Regulation %K Animals %K Cells, Cultured %K Drosophila melanogaster %K HeLa Cells %K Humans %K Mass Spectrometry/methods %K Measles virus/*metabolism %K Nodaviridae/*metabolism %K Oxygen Isotopes %K Proteins/*analysis %K Proteomics/*methods %K RNA Virus Infections/*metabolism %X Mass spectrometry analysis was used to target three different aspects of the viral infection process: the expression kinetics of viral proteins, changes in the expression levels of cellular proteins, and the changes in cellular metabolites in response to viral infection. The combination of these methods represents a new, more comprehensive approach to the study of viral infection revealing the complexity of these events within the infected cell. The proteins associated with measles virus (MV) infection of human HeLa cells were measured using a label-free approach. On the other hand, the regulation of cellular and Flock House Virus (FHV) proteins in response to FHV infection of Drosophila cells was monitored using stable isotope labeling. Three complementary techniques were used to monitor changes in viral protein expression in the cell and host protein expression. A total of 1500 host proteins was identified and quantified, of which over 200 proteins were either up- or down-regulated in response to viral infection, such as the up-regulation of the Drosophila apoptotic croquemort protein, and the down-regulation of proteins that inhibited cell death. These analyses also demonstrated the up-regulation of viral proteins functioning in replication, inhibition of RNA interference, viral assembly, and RNA encapsidation. Over 1000 unique metabolites were also observed with significant changes in over 30, such as the down-regulated cellular phospholipids possibly reflecting the initial events in cell death and viral release. Overall, the cellular transformation that occurs upon viral infection is a process involving hundreds of proteins and metabolites, many of which are structurally and functionally uncharacterized. %B J Proteome ResJ Proteome ResJ Proteome Res %7 2006/09/02 %V 5 %P 2405-16 %8 Sep %@ 1535-3893 (Print)1535-3893 (Linking) %G eng %9 Comparative StudyResearch Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, Non-P.H.S. %M 16944953 %2 2566936 %! Journal of proteome researchJournal of proteome research %0 Journal Article %J Anal Chem %D 2006 %T Solvent-dependent metabolite distribution, clustering, and protein extraction for serum profiling with mass spectrometry %A Want, E. J. %A O'Maille, G. %A Smith, C. A. %A Brandon, T. R. %A Uritboonthai, W. %A Qin, C. %A Trauger, S. A. %A Siuzdak, G. %XThe aim of metabolite profiling is to monitor all metabolites within a biological sample for applications in basic biochemical research as well as pharmacokinetic studies and biomarker discovery. Here, novel data analysis software, XCMS, was used to monitor all metabolite features detected from an array of serum extraction methods, with application to metabolite profiling using electrospray liquid chromatography/mass spectrometry (ESI-LC/MS). The XCMS software enabled the comparison of methods with regard to reproducibility, the number and type of metabolite features detected, and the similarity of these features between different extraction methods. Extraction efficiency with regard to metabolite feature hydrophobicity was examined through the generation of unique feature density distribution plots, displaying feature distribution along chromatographic time. Hierarchical clustering was performed to highlight similarities in the metabolite features observed between the extraction methods. Protein extraction efficiency was determined using the Bradford assay, and the residual proteins were identified using nano-LC/MS/MS. Additionally, the identification of four of the most intensely ionized serum metabolites using FTMS and tandem mass spectrometry was reported. The extraction methods, ranging from organic solvents and acids to heat denaturation, varied widely in both protein removal efficiency and the number of mass spectral features detected. Methanol protein precipitation followed by centrifugation was found to be the most effective, straightforward, and reproducible approach, resulting in serum extracts containing over 2000 detected metabolite features and less than 2% residual protein. Interestingly, the combination of all approaches produced over 10,000 unique metabolite features, a number that is indicative of the complexity of the human metabolome and the potential of metabolomics in biomarker discovery.
%B Anal Chem %7 2006/02/02 %V 78 %P 743-52 %8 Feb 1 %@ 0003-2700 (Print)0003-2700 (Linking) %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/16448047 %M 16448047 %0 Journal Article %J J Proteome Res %D 2006 %T Mass spectrometry reveals specific and global molecular transformations during viral infection %A Go, E. P. %A Wikoff, W. R. %A Shen, Z. %A O'Maille, G. %A Morita, H. %A Conrads, T. P. %A Nordstrom, A. %A Trauger, S. A. %A Uritboonthai, W. %A Lucas, D. A. %A Chan, K. C. %A Veenstra, T. D. %A Lewicki, H. %A Oldstone, M. B. %A Schneemann, A. %A Siuzdak, G. %XMass spectrometry analysis was used to target three different aspects of the viral infection process: the expression kinetics of viral proteins, changes in the expression levels of cellular proteins, and the changes in cellular metabolites in response to viral infection. The combination of these methods represents a new, more comprehensive approach to the study of viral infection revealing the complexity of these events within the infected cell. The proteins associated with measles virus (MV) infection of human HeLa cells were measured using a label-free approach. On the other hand, the regulation of cellular and Flock House Virus (FHV) proteins in response to FHV infection of Drosophila cells was monitored using stable isotope labeling. Three complementary techniques were used to monitor changes in viral protein expression in the cell and host protein expression. A total of 1500 host proteins was identified and quantified, of which over 200 proteins were either up- or down-regulated in response to viral infection, such as the up-regulation of the Drosophila apoptotic croquemort protein, and the down-regulation of proteins that inhibited cell death. These analyses also demonstrated the up-regulation of viral proteins functioning in replication, inhibition of RNA interference, viral assembly, and RNA encapsidation. Over 1000 unique metabolites were also observed with significant changes in over 30, such as the down-regulated cellular phospholipids possibly reflecting the initial events in cell death and viral release. Overall, the cellular transformation that occurs upon viral infection is a process involving hundreds of proteins and metabolites, many of which are structurally and functionally uncharacterized.
%B J Proteome Res %7 2006/09/02 %V 5 %P 2405-16 %8 Sep %@ 1535-3893 (Print)1535-3893 (Linking) %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/16944953 %9 Comparative StudyResearch Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, Non-P.H.S. %M 16944953 %2 2566936 %0 Journal Article %J Ther Drug MonitTher Drug MonitTher Drug Monit %D 2005 %T METLIN: a metabolite mass spectral database %A Smith, C. A. %A O'Maille, G. %A Want, E. J. %A Qin, C. %A Trauger, S. A. %A Brandon, T. R. %A Custodio, D. E. %A Abagyan, R. %A Siuzdak, G. %K *Databases as Topic %K *Mass Spectrometry %K Biological Markers/analysis/chemistry/metabolism %K Chromatography, Liquid %K Humans %K Molecular Structure %K Pharmaceutical Preparations/analysis/chemistry/metabolism %K Spectroscopy, Fourier Transform Infrared %X Endogenous metabolites have gained increasing interest over the past 5 years largely for their implications in diagnostic and pharmaceutical biomarker discovery. METLIN (http://metlin.scripps.edu), a freely accessible web-based data repository, has been developed to assist in a broad array of metabolite research and to facilitate metabolite identification through mass analysis. METLINincludes an annotated list of known metabolite structural information that is easily cross-correlated with its catalogue of high-resolution Fourier transform mass spectrometry (FTMS) spectra, tandem mass spectrometry (MS/MS) spectra, and LC/MS data. %B Ther Drug MonitTher Drug MonitTher Drug Monit %7 2006/01/13 %V 27 %P 747-51 %8 Dec %@ 0163-4356 (Print)0163-4356 (Linking) %G eng %9 Research Support, N.I.H., Extramural %M 16404815 %! Therapeutic drug monitoringTherapeutic drug monitoring %0 Journal Article %J Ther Drug Monit %D 2005 %T METLIN: a metabolite mass spectral database %A Smith, C. A. %A O'Maille, G. %A Want, E. J. %A Qin, C. %A Trauger, S. A. %A Brandon, T. R. %A Custodio, D. E. %A Abagyan, R. %A Siuzdak, G. %XEndogenous metabolites have gained increasing interest over the past 5 years largely for their implications in diagnostic and pharmaceutical biomarker discovery. METLIN (http://metlin.scripps.edu), a freely accessible web-based data repository, has been developed to assist in a broad array of metabolite research and to facilitate metabolite identification through mass analysis. METLINincludes an annotated list of known metabolite structural information that is easily cross-correlated with its catalogue of high-resolution Fourier transform mass spectrometry (FTMS) spectra, tandem mass spectrometry (MS/MS) spectra, and LC/MS data.
%B Ther Drug Monit %7 2006/01/13 %V 27 %P 747-51 %8 Dec %@ 0163-4356 (Print)0163-4356 (Linking) %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/16404815 %9 Research Support, N.I.H., Extramural %M 16404815 %0 Journal Article %J ChembiochemChembiochemChembiochem %D 2004 %T The identification of an adenovirus receptor by using affinity capture and mass spectrometry %A Trauger, S. A. %A Wu, E. %A Bark, S. J. %A Nemerow, G. R. %A Siuzdak, G. %K Antigens, CD/metabolism %K Chromatography, Affinity/*methods %K Electrophoresis, Polyacrylamide Gel %K Flow Cytometry %K Nanotechnology %K Receptors, Virus/*chemistry/metabolism %K Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/*methods %X A tandem mass spectrometry--based approach is demonstrated for detecting a receptor for Ad37, one of the causative agents for epidemic keratoconjunctivitis. Partial purification of membrane glycoproteins was performed by using lectin-affinity chromatography and SDS-PAGE. Gel bands that were shown to bind Ad37 by using Viral Overlay Protein Blot Assay (VOPBA) were excised, proteolyzed and analyzed by using nanoLC-MS/MS to identify putative receptors contained in a mixture of proteins. Four candidate receptors were identified among approximately 50 proteins based on a search against a protein database. Inhibition of gene delivery mediated by an Ad37 vector, with antibodies against the glycoproteins identified by tandem mass spectrometry, strongly indicated that Membrane Cofactor Protein (MCP), a member of the complement regulatory family of proteins, is the receptor. This rapid and sensitive MS/MS-based strategy is perceived to have wide potential applications for the detection of viral receptors. %B ChembiochemChembiochemChembiochem %7 2004/08/10 %V 5 %P 1095-9 %8 Aug 6 %@ 1439-4227 (Print)1439-4227 (Linking) %G eng %9 Research Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S. %M 15300833 %! Chembiochem : a European journal of chemical biologyChembiochem : a European journal of chemical biology %0 Journal Article %J J VirolJ VirolJ Virol %D 2004 %T Membrane cofactor protein is a receptor for adenoviruses associated with epidemic keratoconjunctivitis %A Wu, E. %A Trauger, S. A. %A Pache, L. %A Mullen, T. M. %A von Seggern, D. J. %A Siuzdak, G. %A Nemerow, G. R. %K Adenoviridae Infections/*etiology/virology %K Adenoviruses, Human/classification/genetics/*pathogenicity %K Amino Acid Sequence %K Animals %K Antigens, CD/chemistry/genetics/*physiology %K Antigens, CD46 %K Base Sequence %K Cell Line %K CHO Cells %K Cricetinae %K DNA, Viral/genetics %K HeLa Cells %K Humans %K Keratoconjunctivitis/*etiology/virology %K Membrane Glycoproteins/chemistry/genetics/*physiology %K Molecular Sequence Data %K Receptors, Virus/chemistry/genetics/*physiology %X Subgroup D adenovirus (Ad) types 8, 19, and 37 (Ad8, -19, and -37, respectively) are causative agents of epidemic keratoconjunctivitis and genital tract infections. Previous studies showed that Ad37 binds to a 50-kDa membrane glycoprotein expressed on human ocular (conjunctival) cells. To identify and characterize the role of the 50-kDa glycoprotein in Ad37 infection, we partially purified this molecule from solubilized Chang C conjunctival cell membranes by using lentil lectin chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Liquid chromatography coupled to nano-electrospray ionization-tandem mass spectrometry was subsequently used to identify four Ad37 receptor candidates: CD46, CD87, CD98, and CD147. Immunodepletion analyses demonstrated that the 50-kDa protein is identical to CD46 (also known as membrane cofactor protein). The Ad37, but not Ad5, fiber knob bound to the extracellular domain of CD46, demonstrating a direct interaction of an Ad37 capsid protein with CD46. An antibody specific for the N-terminal 19 amino acids of CD46 also blocked Ad37 infection of human cervical carcinoma and conjunctival cells, indicating a requirement for CD46 in infection. Finally, expression of a 50-kDa isoform of human CD46 in a CD46-null cell line increased cell binding by wild-type Ad37 and gene delivery by an Ad vector pseudotyped with the Ad37 fiber, but not by a vector bearing the Ad5 fiber. Together, these studies demonstrate that CD46 serves as an attachment receptor for Ad37 and shed further light on the cell entry pathway of subgroup D Ads. %B J VirolJ VirolJ Virol %7 2004/03/30 %V 78 %P 3897-905 %8 Apr %@ 0022-538X (Print)0022-538X (Linking) %G eng %9 Research Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, Non-P.H.S.Research Support, U.S. Gov't, P.H.S. %M 15047806 %2 374279 %! Journal of virologyJournal of virology %0 Journal Article %J BiochemistryBiochemistryBiochemistry %D 2004 %T Assignment of endogenous substrates to enzymes by global metabolite profiling %A Saghatelian, A. %A Trauger, S. A. %A Want, E. J. %A Hawkins, E. G. %A Siuzdak, G. %A Cravatt, B. F. %K Amidohydrolases/chemistry/deficiency/*metabolism %K Animals %K Brain/enzymology/metabolism %K Chromatography, Liquid/methods/standards %K Ethanolamines/metabolism %K Fatty Acids/chemistry/*metabolism %K Hydrolysis %K Mass Spectrometry/methods/standards %K Mice %K Mice, Knockout %K Predictive Value of Tests %K Spinal Cord/enzymology/metabolism %K Substrate Specificity %K Taurine/metabolism %X Enzymes regulate biological processes through the conversion of specific substrates to products. Therefore, of fundamental interest for every enzyme is the elucidation of its natural substrates. Here, we describe a general strategy for identifying endogenous substrates of enzymes by untargeted liquid chromatography-mass spectrometry (LC-MS) analysis of tissue metabolomes from wild-type and enzyme-inactivated organisms. We use this method to discover several brain lipids regulated by the mammalian enzyme fatty acid amide hydrolase (FAAH) in vivo, including known signaling molecules (e.g., the endogenous cannabinoid anandamide) and a novel family of nervous system-enriched natural products, the taurine-conjugated fatty acids. Remarkably, the relative hydrolytic activity that FAAH exhibited for lipid metabolites in vitro was not predictive of the identity of specific FAAH substrates in vivo. Thus, global metabolite profiling establishes unanticipated connections between the proteome and metabolome that enable assignment of an enzyme's unique biochemical functions in vivo. %B BiochemistryBiochemistryBiochemistry %7 2004/11/10 %V 43 %P 14332-9 %8 Nov 16 %@ 0006-2960 (Print)0006-2960 (Linking) %G eng %9 Comparative StudyResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S. %M 15533037 %! BiochemistryBiochemistry %0 Journal Article %J Anal ChemAnal ChemAnal Chem %D 2004 %T High sensitivity and analyte capture with desorption/ionization mass spectrometry on silylated porous silicon %A Trauger, S. A. %A Go, E. P. %A Shen, Z. %A Apon, J. V. %A Compton, B. J. %A Bouvier, E. S. %A Finn, M. G. %A Siuzdak, G. %K Amino Acids/blood/isolation & purification %K Animals %K Cattle %K Hemoglobins/chemistry %K Indicators and Reagents %K Sensitivity and Specificity %K Serum Albumin, Bovine/chemistry %K Silicon Dioxide %K Silicon/*chemistry %K Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/*methods %X Silylation chemistry on porous silicon provides for ultrahigh sensitivity and analyte specificity with desorption/ionization on silicon mass spectrometry (DIOS-MS) analysis. Here, we report that the silylation of oxidized porous silicon offers a DIOS platform that is resistant to air oxidation and acid/base hydrolysis. Furthermore, surface modification with appropriate hydrophobic silanes allows analytes to absorb to the surface via hydrophobic interactions for direct analyte extraction from complex matrixes containing salts and other nonvolatile interferences present in the sample matrix. This enables rapid cleanup by simply spotting the sample onto the modified DIOS target and removing the liquid phase containing the interferences. This approach is demonstrated in the analysis of protein digests and metabolites in biofluids, as well as for the characterizing of inhibitors from their enzyme complex. An unprecedented detection limit of 480 molecules (800 ymol) for des-Arg(9)-bradykinin is reported on a pentafluorophenyl-functionalized DIOS chip. %B Anal ChemAnal ChemAnal Chem %7 2004/07/31 %V 76 %P 4484-9 %8 Aug 1 %@ 0003-2700 (Print)0003-2700 (Linking) %G eng %9 Research Support, N.I.H., Extramural %M 15283591 %! Analytical chemistryAnalytical chemistry %0 Journal Article %J Anal Chem %D 2004 %T High sensitivity and analyte capture with desorption/ionization mass spectrometry on silylated porous silicon %A Trauger, S. A. %A Go, E. P. %A Shen, Z. %A Apon, J. V. %A Compton, B. J. %A Bouvier, E. S. %A Finn, M. G. %A Siuzdak, G. %XSilylation chemistry on porous silicon provides for ultrahigh sensitivity and analyte specificity with desorption/ionization on silicon mass spectrometry (DIOS-MS) analysis. Here, we report that the silylation of oxidized porous silicon offers a DIOS platform that is resistant to air oxidation and acid/base hydrolysis. Furthermore, surface modification with appropriate hydrophobic silanes allows analytes to absorb to the surface via hydrophobic interactions for direct analyte extraction from complex matrixes containing salts and other nonvolatile interferences present in the sample matrix. This enables rapid cleanup by simply spotting the sample onto the modified DIOS target and removing the liquid phase containing the interferences. This approach is demonstrated in the analysis of protein digests and metabolites in biofluids, as well as for the characterizing of inhibitors from their enzyme complex. An unprecedented detection limit of 480 molecules (800 ymol) for des-Arg(9)-bradykinin is reported on a pentafluorophenyl-functionalized DIOS chip.
%B Anal Chem %7 2004/07/31 %V 76 %P 4484-9 %8 Aug 1 %@ 0003-2700 (Print)0003-2700 (Linking) %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/15283591 %9 Research Support, N.I.H., Extramural %M 15283591 %0 Journal Article %J Chembiochem %D 2004 %T The identification of an adenovirus receptor by using affinity capture and mass spectrometry %A Trauger, S. A. %A Wu, E. %A Bark, S. J. %A Nemerow, G. R. %A Siuzdak, G. %XA tandem mass spectrometry--based approach is demonstrated for detecting a receptor for Ad37, one of the causative agents for epidemic keratoconjunctivitis. Partial purification of membrane glycoproteins was performed by using lectin-affinity chromatography and SDS-PAGE. Gel bands that were shown to bind Ad37 by using Viral Overlay Protein Blot Assay (VOPBA) were excised, proteolyzed and analyzed by using nanoLC-MS/MS to identify putative receptors contained in a mixture of proteins. Four candidate receptors were identified among approximately 50 proteins based on a search against a protein database. Inhibition of gene delivery mediated by an Ad37 vector, with antibodies against the glycoproteins identified by tandem mass spectrometry, strongly indicated that Membrane Cofactor Protein (MCP), a member of the complement regulatory family of proteins, is the receptor. This rapid and sensitive MS/MS-based strategy is perceived to have wide potential applications for the detection of viral receptors.
%B Chembiochem %7 2004/08/10 %V 5 %P 1095-9 %8 Aug 6 %@ 1439-4227 (Print)1439-4227 (Linking) %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/15300833 %9 Research Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S. %M 15300833 %0 Journal Article %J J Virol %D 2004 %T Membrane cofactor protein is a receptor for adenoviruses associated with epidemic keratoconjunctivitis %A Wu, E. %A Trauger, S. A. %A Pache, L. %A Mullen, T. M. %A von Seggern, D. J. %A Siuzdak, G. %A Nemerow, G. R. %XSubgroup D adenovirus (Ad) types 8, 19, and 37 (Ad8, -19, and -37, respectively) are causative agents of epidemic keratoconjunctivitis and genital tract infections. Previous studies showed that Ad37 binds to a 50-kDa membrane glycoprotein expressed on human ocular (conjunctival) cells. To identify and characterize the role of the 50-kDa glycoprotein in Ad37 infection, we partially purified this molecule from solubilized Chang C conjunctival cell membranes by using lentil lectin chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Liquid chromatography coupled to nano-electrospray ionization-tandem mass spectrometry was subsequently used to identify four Ad37 receptor candidates: CD46, CD87, CD98, and CD147. Immunodepletion analyses demonstrated that the 50-kDa protein is identical to CD46 (also known as membrane cofactor protein). The Ad37, but not Ad5, fiber knob bound to the extracellular domain of CD46, demonstrating a direct interaction of an Ad37 capsid protein with CD46. An antibody specific for the N-terminal 19 amino acids of CD46 also blocked Ad37 infection of human cervical carcinoma and conjunctival cells, indicating a requirement for CD46 in infection. Finally, expression of a 50-kDa isoform of human CD46 in a CD46-null cell line increased cell binding by wild-type Ad37 and gene delivery by an Ad vector pseudotyped with the Ad37 fiber, but not by a vector bearing the Ad5 fiber. Together, these studies demonstrate that CD46 serves as an attachment receptor for Ad37 and shed further light on the cell entry pathway of subgroup D Ads.
%B J Virol %7 2004/03/30 %V 78 %P 3897-905 %8 Apr %@ 0022-538X (Print)0022-538X (Linking) %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/15047806 %9 Research Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, Non-P.H.S.Research Support, U.S. Gov't, P.H.S. %M 15047806 %2 374279 %0 Journal Article %J Biochemistry %D 2004 %T Assignment of endogenous substrates to enzymes by global metabolite profiling %A Saghatelian, A. %A Trauger, S. A. %A Want, E. J. %A Hawkins, E. G. %A Siuzdak, G. %A Cravatt, B. F. %XEnzymes regulate biological processes through the conversion of specific substrates to products. Therefore, of fundamental interest for every enzyme is the elucidation of its natural substrates. Here, we describe a general strategy for identifying endogenous substrates of enzymes by untargeted liquid chromatography-mass spectrometry (LC-MS) analysis of tissue metabolomes from wild-type and enzyme-inactivated organisms. We use this method to discover several brain lipids regulated by the mammalian enzyme fatty acid amide hydrolase (FAAH) in vivo, including known signaling molecules (e.g., the endogenous cannabinoid anandamide) and a novel family of nervous system-enriched natural products, the taurine-conjugated fatty acids. Remarkably, the relative hydrolytic activity that FAAH exhibited for lipid metabolites in vitro was not predictive of the identity of specific FAAH substrates in vivo. Thus, global metabolite profiling establishes unanticipated connections between the proteome and metabolome that enable assignment of an enzyme's unique biochemical functions in vivo.
%B Biochemistry %7 2004/11/10 %V 43 %P 14332-9 %8 Nov 16 %@ 0006-2960 (Print)0006-2960 (Linking) %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/15533037 %9 Comparative StudyResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S. %M 15533037 %0 Journal Article %J Environ Toxicol ChemEnviron Toxicol ChemEnviron Toxicol Chem %D 2002 %T Metabonomic assessment of toxicity of 4-fluoroaniline, 3,5-difluoroaniline and 2-fluoro-4-methylaniline to the earthworm Eisenia veneta (Rosa): identification of new endogenous biomarkers %A Bundy, J. G. %A Lenz, E. M. %A Bailey, N. J. %A Gavaghan, C. L. %A Svendsen, C. %A Spurgeon, D. %A Hankard, P. K. %A Osborn, D. %A Weeks, J. M. %A Trauger, S. A. %A Speir, P. %A Sanders, I. %A Lindon, J. C. %A Nicholson, J. K. %A Tang, H. %K *Oligochaeta %K Aniline Compounds/*toxicity %K Animals %K Biological Markers/*analysis %K Chromatography, High Pressure Liquid %K Inosine Monophosphate/analysis %K Magnetic Resonance Spectroscopy %K Maltose/analysis %K Xenobiotics/*toxicity %X High-resolution 1H nuclear magnetic resonance (NMR) spectroscopy can be used to produce a biochemical fingerprint of low-molecular-weight metabolites from complex biological mixtures such as tissue extracts and biofluids. Changes in such fingerprint profiles can be used to characterize the effects of toxic insult in in vivo systems. The technique is nonselective and requires little sample preparation or derivatization. In the present study, earthworms (Eisenia veneta) were exposed to three different model xenobiotics by a standard filter paper contact test, and toxicant-induced biochemical changes were then investigated by characterizing the changes in endogenous metabolites visible in 600-MHz 1H NMR spectra of tissue extracts. The NMR spectral intensities were converted to discrete numerical values and tabulated in order to provide data matrices suitable for multivariate analysis. Principal component analysis showed that changes had occurred in the biochemical profiles relative to the undosed controls. The 2-fluoro-4-methylaniline-treated worms showed a decrease in a resonance from a compound identified as 2-hexyl-5-ethyl-3-furansulfonate using a combination of high-performance liquid chromatography (HPLC)-Fourier transform mass spectrometry (IonSpec, Lake Forest, CA, USA) and 1H and 13C NMR spectroscopy. An increase in inosine monophosphate was also observed. The 4-fluoroaniline-treated worms showed a decrease in maltose concentrations, and 3,5-difluoroaniline exerted the same effect as 2-fluoro-4-methylaniline but to a lesser extent. These changes could potentially be used as novel biomarkers of xenobiotic toxicity and could be used to determine the mechanism of action of other toxic chemicals. %B Environ Toxicol ChemEnviron Toxicol ChemEnviron Toxicol Chem %7 2002/09/11 %V 21 %P 1966-72 %8 Sep %@ 0730-7268 (Print)0730-7268 (Linking) %G eng %9 Research Support, Non-U.S. Gov't %M 12206438 %! Environmental toxicology and chemistry / SETACEnvironmental toxicology and chemistry / SETAC %0 Journal Article %J Environ Toxicol Chem %D 2002 %T Metabonomic assessment of toxicity of 4-fluoroaniline, 3,5-difluoroaniline and 2-fluoro-4-methylaniline to the earthworm Eisenia veneta (Rosa): identification of new endogenous biomarkers %A Bundy, J. G. %A Lenz, E. M. %A Bailey, N. J. %A Gavaghan, C. L. %A Svendsen, C. %A Spurgeon, D. %A Hankard, P. K. %A Osborn, D. %A Weeks, J. M. %A Trauger, S. A. %A Speir, P. %A Sanders, I. %A Lindon, J. C. %A Nicholson, J. K. %A Tang, H. %XHigh-resolution 1H nuclear magnetic resonance (NMR) spectroscopy can be used to produce a biochemical fingerprint of low-molecular-weight metabolites from complex biological mixtures such as tissue extracts and biofluids. Changes in such fingerprint profiles can be used to characterize the effects of toxic insult in in vivo systems. The technique is nonselective and requires little sample preparation or derivatization. In the present study, earthworms (Eisenia veneta) were exposed to three different model xenobiotics by a standard filter paper contact test, and toxicant-induced biochemical changes were then investigated by characterizing the changes in endogenous metabolites visible in 600-MHz 1H NMR spectra of tissue extracts. The NMR spectral intensities were converted to discrete numerical values and tabulated in order to provide data matrices suitable for multivariate analysis. Principal component analysis showed that changes had occurred in the biochemical profiles relative to the undosed controls. The 2-fluoro-4-methylaniline-treated worms showed a decrease in a resonance from a compound identified as 2-hexyl-5-ethyl-3-furansulfonate using a combination of high-performance liquid chromatography (HPLC)-Fourier transform mass spectrometry (IonSpec, Lake Forest, CA, USA) and 1H and 13C NMR spectroscopy. An increase in inosine monophosphate was also observed. The 4-fluoroaniline-treated worms showed a decrease in maltose concentrations, and 3,5-difluoroaniline exerted the same effect as 2-fluoro-4-methylaniline but to a lesser extent. These changes could potentially be used as novel biomarkers of xenobiotic toxicity and could be used to determine the mechanism of action of other toxic chemicals.
%B Environ Toxicol Chem %7 2002/09/11 %V 21 %P 1966-72 %8 Sep %@ 0730-7268 (Print)0730-7268 (Linking) %G eng %U http://www.ncbi.nlm.nih.gov/pubmed/12206438 %9 Research Support, Non-U.S. Gov't %M 12206438