@article {269896, title = {Regulation of astrocyte activation by glycolipids drives chronic CNS inflammation}, journal = {Nat MedNat MedNat Med}, year = {2014}, note = {Mayo, LiorTrauger, Sunia ABlain, ManonNadeau, MeghanPatel, BonnyAlvarez, Jorge IMascanfroni, Ivan DYeste, AdaKivisakk, PiaKallas, KeithEllezam, BenjaminBakshi, RohitPrat, AlexandreAntel, Jack PWeiner, Howard LQuintana, Francisco JNat Med. 2014 Sep 14. doi: 10.1038/nm.3681.}, month = {Sep 14}, edition = {2014/09/14}, abstract = {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.}, isbn = {1546-170X (Electronic)1078-8956 (Linking)}, author = {Mayo, L. and Trauger, S. A. and Blain, M. and Nadeau, M. and Patel, B. and Alvarez, J. I. and Mascanfroni, I. D. and Yeste, A. and Kivisakk, P. and Kallas, K. and Ellezam, B. and Bakshi, R. and Prat, A. and Antel, J. P. and Weiner, H. L. and Quintana, F. J.} } @article {269821, title = {In vivo performance of a drug-eluting contact lens to treat glaucoma for a month}, journal = {BiomaterialsBiomaterialsBiomaterials}, volume = {35}, year = {2014}, note = {Ciolino, Joseph BStefanescu, Cristina FRoss, Amy ESalvador-Culla, BorjaCortez, PriscilaFord, Eden MWymbs, Kate ASprague, Sarah LMascoop, Daniel RRudina, Shireen STrauger, Sunia ACade, FabianoKohane, Daniel S1K08EY019686-01/EY/NEI NIH HHS/GM073626/GM/NIGMS NIH HHS/K08 EY019686/EY/NEI NIH HHS/R01 GM073626/GM/NIGMS NIH HHS/NetherlandsBiomaterials. 2014 Jan;35(1):432-9. doi: 10.1016/j.biomaterials.2013.09.032. Epub 2013 Oct 4.}, month = {Jan}, pages = {432-9}, type = {Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov{\textquoteright}t}, edition = {2013/10/08}, abstract = {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.}, keywords = {*Contact Lenses, *Drug Delivery Systems, Animals, Drug Stability, Glaucoma/*drug therapy, Intraocular Pressure/drug effects, Prostaglandins F, Synthetic/*administration \& dosage/pharmacology/therapeutic use, Rabbits}, isbn = {1878-5905 (Electronic)0142-9612 (Linking)}, author = {Ciolino, J. B. and Stefanescu, C. F. and Ross, A. E. and Salvador-Culla, B. and Cortez, P. and Ford, E. M. and Wymbs, K. A. and Sprague, S. L. and Mascoop, D. R. and Rudina, S. S. and Trauger, S. A. and Cade, F. and Kohane, D. S.} } @article {269816, title = {The metabolite alpha-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR}, journal = {NatureNatureNature}, volume = {510}, year = {2014}, note = {Chin, Randall MFu, XudongPai, Melody YVergnes, LaurentHwang, HeejunDeng, GangDiep, SimonLomenick, BrettMeli, Vijaykumar SMonsalve, Gabriela CHu, EileenWhelan, Stephen AWang, Jennifer XJung, GwanghyunSolis, Gregory MFazlollahi, FarbodKaweeteerawat, ChitradaQuach, AustinNili, MahtaKrall, Abby SGodwin, Hilary AChang, Helena RFaull, Kym FGuo, FengJiang, MeishengTrauger, Sunia ASaghatelian, AlanBraas, DanielChristofk, Heather RClarke, Catherine FTeitell, Michael APetrascheck, MichaelReue, KarenJung, Michael EFrand, Alison RHuang, JingP40 OD010440/OD/NIH HHS/T32 CA009120/CA/NCI NIH HHS/T32 GM007104/GM/NIGMS NIH HHS/T32 GM007185/GM/NIGMS NIH HHS/T32 GM008496/GM/NIGMS NIH HHS/EnglandNature. 2014 Jun 19;510(7505):397-401. doi: 10.1038/nature13264. Epub 2014 May 14.}, month = {Jun 19}, pages = {397-401}, type = {Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov{\textquoteright}t}, edition = {2014/05/16}, abstract = {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.}, keywords = {Animals, Caenorhabditis elegans/*drug effects, Cell Line, Enzyme Activation/drug effects, Enzyme Inhibitors/pharmacology, Gene Knockdown Techniques, HEK293 Cells, Humans, Jurkat Cells, Ketoglutaric Acids/*pharmacology, Longevity/drug effects/genetics/*physiology, Mice, Mitochondrial Proton-Translocating ATPases/genetics/*metabolism, Protein Binding, TOR Serine-Threonine Kinases/*metabolism}, isbn = {1476-4687 (Electronic)0028-0836 (Linking)}, author = {Chin, R. M. and Fu, X and Pai, M. Y. and Vergnes, L. and Hwang, H. and Deng, G. and Diep, S. and Lomenick, B. and Meli, V. S. and Monsalve, G. C. and Hu, E. and Whelan, S. A. and Wang, J. X. and Jung, G. and Solis, G. M. and Fazlollahi, F. and Kaweeteerawat, C. and Quach, A. and Nili, M. and Krall, A. S. and Godwin, H. A. and Chang, H. R. and Faull, K. F. and Guo, F. and Jiang, M. and Trauger, S. A. and Saghatelian, A. and Braas, D. and Christofk, H. R. and Clarke, C. F. and Teitell, M. A. and Petrascheck, M. and Reue, K. and Jung, M. E. and Frand, A. R. and Huang, J.} } @article {269831, title = {Warfarin untargeted metabolomics study identifies novel procoagulant ethanolamide plasma lipids}, journal = {Br J HaematolBr J HaematolBr J Haematol}, volume = {165}, year = {2014}, note = {Deguchi, HiroshiElias, Darlene JTrauger, SuniaZhang, Hui-MinKalisiak, EwaSiuzdak, GaryGriffin, John HHL021544/HL/NHLBI NIH HHS/HL101034/HL/NHLBI NIH HHS/R01 HL021544/HL/NHLBI NIH HHS/RC1 HL101034/HL/NHLBI NIH HHS/UL1 RR025774/RR/NCRR NIH HHS/UL1RR025774/RR/NCRR NIH HHS/EnglandBr J Haematol. 2014 May;165(3):409-12. doi: 10.1111/bjh.12720. Epub 2014 Jan 23.}, month = {May}, pages = {409-12}, type = {LetterResearch Support, N.I.H., Extramural}, edition = {2014/01/24}, keywords = {Ethanolamine/blood, Humans, Lipids/*blood, Mass Spectrometry/methods, Metabolomics/methods, Warfarin/*blood/pharmacology}, isbn = {1365-2141 (Electronic)0007-1048 (Linking)}, author = {Deguchi, H. and Elias, D. J. and Trauger, S. and Zhang, H. M. and Kalisiak, E. and Siuzdak, G. and Griffin, J. H.} } @article {269781, title = {Regulation of astrocyte activation by glycolipids drives chronic CNS inflammation}, journal = {Nature Medicine}, volume = {Sep 14}, year = {2014}, abstract = {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.}, author = {Mayo L1, Trauger SA2, Blain M3, Nadeau M1, Patel B1, Alvarez JI4, Mascanfroni ID1, Yeste A1, Kivis{\"a}kk P1, Kallas K1, Ellezam B5,} } @article {269866, title = {Identification of collagen-based materials in cultural heritage}, journal = {AnalystAnalystAnalyst}, volume = {138}, year = {2013}, note = {Kirby, Daniel PBuckley, MichaelPromise, EllenTrauger, Sunia AHoldcraft, T RoseEnglandAnalyst. 2013 Sep 7;138(17):4849-58. doi: 10.1039/c3an00925d. Epub 2013 Jun 28.}, month = {Sep 7}, pages = {4849-58}, type = {Research Support, Non-U.S. Gov{\textquoteright}t}, edition = {2013/06/29}, abstract = {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.}, keywords = {*Culture, Chemistry Techniques, Analytical/*methods, Collagen/*analysis/chemistry/metabolism, Humans, Museums, Proteolysis, Skin/chemistry, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization}, isbn = {1364-5528 (Electronic)0003-2654 (Linking)}, author = {Kirby, D. P. and Buckley, M. and Promise, E. and Trauger, S. A. and Holdcraft, T. R.} } @article {269881, title = {Lysine addressability and mammalian cell interactions of bacteriophage lambda procapsids}, journal = {BiomacromoleculesBiomacromoleculesBiomacromolecules}, volume = {14}, year = {2013}, note = {Koudelka, Kristopher JIppoliti, ShannonMedina, ElizabethShriver, Leah PTrauger, Sunia ACatalano, Carlos EManchester, MarianneCA112075/CA/NCI NIH HHS/Biomacromolecules. 2013 Dec 9;14(12):4169-76. doi: 10.1021/bm401577f. Epub 2013 Nov 26.}, month = {Dec 9}, pages = {4169-76}, type = {Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov{\textquoteright}tResearch Support, U.S. Gov{\textquoteright}t, Non-P.H.S.}, edition = {2013/11/21}, abstract = {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.}, keywords = {Amino Acid Sequence, Bacteriophage lambda/*chemistry/metabolism, Capsid Proteins/chemistry, Capsid/*chemistry/metabolism, Drug Carriers/*chemistry/metabolism, HeLa Cells, Humans, Lysine/*chemistry, Molecular Sequence Data, Receptors, Transferrin/metabolism, Tandem Mass Spectrometry, Transferrin/chemistry/metabolism, Virus Internalization}, isbn = {1526-4602 (Electronic)1525-7797 (Linking)}, author = {Koudelka, K. J. and Ippoliti, S. and Medina, E. and Shriver, L. P. and Trauger, S. A. and Catalano, C. E. and Manchester, M.} } @article {269801, title = {ABHD12 controls brain lysophosphatidylserine pathways that are deregulated in a murine model of the neurodegenerative disease PHARC}, journal = {Proc Natl Acad Sci U S AProc Natl Acad Sci U S AProc Natl Acad Sci U S A}, volume = {110}, year = {2013}, note = {Blankman, Jacqueline LLong, Jonathan ZTrauger, Sunia ASiuzdak, GaryCravatt, Benjamin FDA009789/DA/NIDA NIH HHS/DA017259/DA/NIDA NIH HHS/P01 DA017259/DA/NIDA NIH HHS/P30 MH062261/MH/NIMH NIH HHS/Proc Natl Acad Sci U S A. 2013 Jan 22;110(4):1500-5. doi: 10.1073/pnas.1217121110. Epub 2013 Jan 7.}, month = {Jan 22}, pages = {1500-5}, type = {Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov{\textquoteright}t}, edition = {2013/01/09}, abstract = {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.}, keywords = {Animals, Ataxia/*genetics/*metabolism/pathology/physiopathology, Behavior, Animal/physiology, Brain/*metabolism/pathology, Cataract/*genetics/*metabolism/pathology/physiopathology, Disease Models, Animal, Humans, Lipid Metabolism, Lysophospholipids/*metabolism, Metabolic Networks and Pathways, Mice, Mice, Inbred C57BL, Mice, Knockout, Microglia/metabolism, Models, Neurological, Monoacylglycerol Lipases/deficiency/*genetics/*metabolism, Mutation, Phenotype, Polyneuropathies/*genetics/*metabolism/pathology/physiopathology, Retinitis Pigmentosa/*genetics/*metabolism/pathology/physiopathology}, isbn = {1091-6490 (Electronic)0027-8424 (Linking)}, author = {Blankman, J. L. and Long, J. Z. and Trauger, S. A. and Siuzdak, G. and Cravatt, B. F.} } @article {269916, title = {Short communication: quantitative proteomic plasma profiling reveals activation of host defense to oxidative stress in chronic SIV and methamphetamine comorbidity}, journal = {AIDS Res Hum RetrovirusesAIDS Res Hum RetrovirusesAIDS Res Hum Retroviruses}, volume = {27}, year = {2011}, note = {Pendyala, GuruduttTrauger, Sunia ASiuzdak, GaryFox, Howard SP01 DA026146/DA/NIDA NIH HHS/P30 MH062261/MH/NIMH NIH HHS/R01 MH073490/MH/NIMH NIH HHS/R01 MH073490-01/MH/NIMH NIH HHS/R01 MH073490-02/MH/NIMH NIH HHS/R01 MH073490-03/MH/NIMH NIH HHS/R01 MH073490-04/MH/NIMH NIH HHS/R01 MH073490-05/MH/NIMH NIH HHS/R01 MH073490-06/MH/NIMH NIH HHS/AIDS Res Hum Retroviruses. 2011 Feb;27(2):179-82. doi: 10.1089/aid.2010.0090. Epub 2010 Oct 7.}, month = {Feb}, pages = {179-82}, type = {Research Support, N.I.H., Extramural}, edition = {2010/10/12}, abstract = {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.}, keywords = {*Oxidative Stress, *Proteomics, Animals, Blood Proteins/*metabolism, Chronic Disease, Humans, Methamphetamine/*administration \& dosage, Simian Acquired Immunodeficiency Syndrome/*blood}, isbn = {1931-8405 (Electronic)0889-2229 (Linking)}, author = {Pendyala, G. and Trauger, S. A. and Siuzdak, G. and Fox, H. S.} } @article {269921, title = {Type I signal peptidase and protein secretion in Staphylococcus epidermidis}, journal = {J BacteriolJ BacteriolJ Bacteriol}, volume = {193}, year = {2011}, note = {Powers, Michael ESmith, Peter ARoberts, Tucker CFowler, Bruce JKing, Charles CTrauger, Sunia ASiuzdak, GaryRomesberg, Floyd EAI081126/AI/NIAID NIH HHS/J Bacteriol. 2011 Jan;193(2):340-8. doi: 10.1128/JB.01052-10. Epub 2010 Nov 12.}, month = {Jan}, pages = {340-8}, type = {Research Support, N.I.H., ExtramuralResearch Support, U.S. Gov{\textquoteright}t, Non-P.H.S.}, edition = {2010/11/16}, abstract = {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.}, keywords = {Bacterial Proteins/*secretion, Chromatography, Liquid, Electrophoresis, Gel, Two-Dimensional, Enzyme Inhibitors/chemistry/pharmacology, Mass Spectrometry, Membrane Proteins/antagonists \& inhibitors/*metabolism, Molecular Structure, Oligopeptides/chemistry/pharmacology, Protein Sorting Signals/genetics, Serine Endopeptidases/*metabolism, Staphylococcus epidermidis/*metabolism}, isbn = {1098-5530 (Electronic)0021-9193 (Linking)}, author = {Powers, M. E. and Smith, P. A. and Roberts, T. C. and Fowler, B. J. and King, C. C. and Trauger, S. A. and Siuzdak, G. and Romesberg, F. E.} } @article {269886, title = {A computational framework for proteome-wide pursuit and prediction of metalloproteins using ICP-MS and MS/MS data}, journal = {BMC BioinformaticsBMC BioinformaticsBMC Bioinformatics}, volume = {12}, year = {2011}, note = {Lancaster, W AndrewPraissman, Jeremy LPoole, Farris L 2ndCvetkovic, AleksandarMenon, Angeli LalScott, Joseph WJenney, Francis E JrThorgersen, Michael PKalisiak, EwaApon, Junefredo VTrauger, Sunia ASiuzdak, GaryTainer, John AAdams, Michael W WEnglandBMC Bioinformatics. 2011 Feb 28;12:64. doi: 10.1186/1471-2105-12-64.}, pages = {64}, type = {Research Support, U.S. Gov{\textquoteright}t, Non-P.H.S.}, edition = {2011/03/02}, abstract = {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.}, keywords = {*Tandem Mass Spectrometry, Amino Acid Sequence, Automatic Data Processing/methods, Bacterial Proteins/analysis/chemistry/isolation \& purification, Computational Biology/*methods, Databases, Protein, Metalloproteins/*analysis/chemistry/isolation \& purification, Metals/analysis/chemistry/metabolism, Molybdenum/chemistry, Nickel/chemistry, Protein Interaction Domains and Motifs, Proteome/*analysis, Pyrococcus furiosus/metabolism}, isbn = {1471-2105 (Electronic)1471-2105 (Linking)}, author = {Lancaster, W. A. and Praissman, J. L. and Poole, F. L., 2nd and Cvetkovic, A. and Menon, A. L. and Scott, J. W. and Jenney, F. E., Jr. and Thorgersen, M. P. and Kalisiak, E. and Apon, J. V. and Trauger, S. A. and Siuzdak, G. and Tainer, J. A. and Adams, M. W.} } @article {269891, title = {Metabolomics annotates ABHD3 as a physiologic regulator of medium-chain phospholipids}, journal = {Nat Chem BiolNat Chem BiolNat Chem Biol}, volume = {7}, year = {2011}, note = {Long, Jonathan ZCisar, Justin SMilliken, DavidNiessen, SherryWang, ChuTrauger, Sunia ASiuzdak, GaryCravatt, Benjamin FCA132630/CA/NCI NIH HHS/R01 CA132630/CA/NCI NIH HHS/R01 CA132630-04/CA/NCI NIH HHS/Nat Chem Biol. 2011 Sep 18;7(11):763-5. doi: 10.1038/nchembio.659.}, month = {Nov}, pages = {763-5}, type = {Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov{\textquoteright}t}, edition = {2011/09/20}, abstract = {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.}, keywords = {Animals, Gene Expression Regulation, Enzymologic, Gene Expression Regulation/physiology, HEK293 Cells, Humans, Hydrolases/genetics/*metabolism, Membrane Proteins/genetics/*metabolism, Metabolomics/*methods, Mice, Mice, Knockout, Phospholipids/chemistry/*metabolism, Small Molecule Libraries, Substrate Specificity}, isbn = {1552-4469 (Electronic)1552-4450 (Linking)}, author = {Long, J. Z. and Cisar, J. S. and Milliken, D. and Niessen, S. and Wang, C. and Trauger, S. A. and Siuzdak, G. and Cravatt, B. F.} } @article {8106, title = {Short communication: quantitative proteomic plasma profiling reveals activation of host defense to oxidative stress in chronic SIV and methamphetamine comorbidity}, journal = {AIDS Res Hum Retroviruses}, volume = {27}, year = {2011}, note = {Pendyala, GuruduttTrauger, Sunia ASiuzdak, GaryFox, Howard SP01 DA026146/DA/NIDA NIH HHS/P01 DA026146-01/DA/NIDA NIH HHS/P01 DA026146-010002/DA/NIDA NIH HHS/P01 DA026146-019001/DA/NIDA NIH HHS/P01 DA026146-02/DA/NIDA NIH HHS/P01 DA026146-020002/DA/NIDA NIH HHS/P01 DA026146-029001/DA/NIDA NIH HHS/P01 DA026146-03/DA/NIDA NIH HHS/P30 MH062261/MH/NIMH NIH HHS/P30 MH062261-01/MH/NIMH NIH HHS/P30 MH062261-02/MH/NIMH NIH HHS/P30 MH062261-03/MH/NIMH NIH HHS/P30 MH062261-04/MH/NIMH NIH HHS/P30 MH062261-05/MH/NIMH NIH HHS/P30 MH062261-05S1/MH/NIMH NIH HHS/P30 MH062261-06A1/MH/NIMH NIH HHS/P30 MH062261-06A19008/MH/NIMH NIH HHS/P30 MH062261-079008/MH/NIMH NIH HHS/P30 MH062261-089008/MH/NIMH NIH HHS/P30 MH062261-097599/MH/NIMH NIH HHS/P30 MH062261-107599/MH/NIMH NIH HHS/R01 MH073490/MH/NIMH NIH HHS/R01 MH073490-01/MH/NIMH NIH HHS/R01 MH073490-02/MH/NIMH NIH HHS/R01 MH073490-03/MH/NIMH NIH HHS/R01 MH073490-04/MH/NIMH NIH HHS/R01 MH073490-05/MH/NIMH NIH HHS/R01 MH073490-06/MH/NIMH NIH HHS/AIDS Res Hum Retroviruses. 2011 Feb;27(2):179-82. Epub 2010 Oct 7. }, month = {Feb}, pages = {179-82}, type = {Research Support, N.I.H., Extramural}, edition = {2010/10/12}, abstract = {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. }, isbn = {1931-8405 (Electronic)0889-2229 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/20929344}, author = {Pendyala, G. and Trauger, S. A. and Siuzdak, G. and Fox, H. S.} } @article {8104, title = {A computational framework for proteome-wide pursuit and prediction of metalloproteins using ICP-MS and MS/MS data}, journal = {BMC Bioinformatics}, volume = {12}, year = {2011}, note = {Lancaster, W AndrewPraissman, Jeremy LPoole, Farris L 2ndCvetkovic, AleksandarMenon, Angeli LalScott, Joseph WJenney, Francis E JrThorgersen, Michael PKalisiak, EwaApon, Junefredo VTrauger, Sunia ASiuzdak, GaryTainer, John AAdams, Michael W WEnglandBMC Bioinformatics. 2011 Feb 28;12:64. }, pages = {64}, type = {Research Support, U.S. Gov{\textquoteright}t, Non-P.H.S.}, edition = {2011/03/02}, abstract = {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. }, isbn = {1471-2105 (Electronic)1471-2105 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/21356119}, author = {Lancaster, W. A. and Praissman, J. L. and Poole, F. L., 2nd and Cvetkovic, A. and Menon, A. L. and Scott, J. W. and Jenney, F. E., Jr. and Thorgersen, M. P. and Kalisiak, E. and Apon, J. V. and Trauger, S. A. and Siuzdak, G. and Tainer, J. A. and Adams, M. W.} } @article {8105, title = {Type I signal peptidase and protein secretion in Staphylococcus epidermidis}, journal = {J Bacteriol}, volume = {193}, year = {2011}, note = {Powers, Michael ESmith, Peter ARoberts, Tucker CFowler, Bruce JKing, Charles CTrauger, Sunia ASiuzdak, GaryRomesberg, Floyd EAI081126/AI/NIAID NIH HHS/J Bacteriol. 2011 Jan;193(2):340-8. Epub 2010 Nov 12. }, month = {Jan}, pages = {340-8}, type = {Research Support, N.I.H., ExtramuralResearch Support, U.S. Gov{\textquoteright}t, Non-P.H.S.}, edition = {2010/11/16}, abstract = {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. }, isbn = {1098-5530 (Electronic)0021-9193 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/21075926}, author = {Powers, M. E. and Smith, P. A. and Roberts, T. C. and Fowler, B. J. and King, C. C. and Trauger, S. A. and Siuzdak, G. and Romesberg, F. E.} } @article {8103, title = {Metabolomics annotates ABHD3 as a physiologic regulator of medium-chain phospholipids}, journal = {Nat Chem Biol}, volume = {7}, year = {2011}, note = {Long, Jonathan ZCisar, Justin SMilliken, DavidNiessen, SherryWang, ChuTrauger, Sunia ASiuzdak, GaryCravatt, Benjamin FCA132630/CA/NCI NIH HHS/R01 CA132630-04/CA/NCI NIH HHS/Nat Chem Biol. 2011 Sep 18;7(11):763-5. doi: 10.1038/nchembio.659. }, month = {Nov}, pages = {763-5}, type = {Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov{\textquoteright}t}, edition = {2011/09/20}, abstract = {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. }, isbn = {1552-4469 (Electronic)1552-4450 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/21926997}, author = {Long, J. Z. and Cisar, J. S. and Milliken, D. and Niessen, S. and Wang, C. and Trauger, S. A. and Siuzdak, G. and Cravatt, B. F.} } @article {269976, title = {Metabolic oxidation regulates embryonic stem cell differentiation}, journal = {Nat Chem BiolNat Chem BiolNat Chem Biol}, volume = {6}, year = {2010}, note = {Yanes, OscarClark, JulieWong, Diana MPatti, Gary JSanchez-Ruiz, AntonioBenton, H PaulTrauger, Sunia ADesponts, CarolineDing, ShengSiuzdak, GaryR24 EY017540/EY/NEI NIH HHS/R24 EY017540-03/EY/NEI NIH HHS/R24 EY017540-05/EY/NEI NIH HHS/Nat Chem Biol. 2010 Jun;6(6):411-7. doi: 10.1038/nchembio.364. Epub 2010 May 2.}, month = {Jun}, pages = {411-7}, type = {Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov{\textquoteright}tResearch Support, U.S. Gov{\textquoteright}t, Non-P.H.S.}, edition = {2010/05/04}, abstract = {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.}, keywords = {Amino Acids/metabolism, Carboxylic Acids/metabolism, Carnitine/metabolism, Cell Differentiation, Eicosanoids/metabolism, Embryonic Stem Cells/*cytology/*metabolism, Gene Expression Regulation, Glutathione/metabolism, Humans, Oxidation-Reduction, Phenotype, Proteome/metabolism, Software, Stem Cells/cytology/metabolism}, isbn = {1552-4469 (Electronic)1552-4450 (Linking)}, author = {Yanes, O. and Clark, J. and Wong, D. M. and Patti, G. J. and Sanchez-Ruiz, A. and Benton, H. P. and Trauger, S. A. and Desponts, C. and Ding, S. and Siuzdak, G.} } @article {269911, title = {Quantitative plasma proteomic profiling identifies the vitamin E binding protein afamin as a potential pathogenic factor in SIV induced CNS disease}, journal = {J Proteome ResJ Proteome ResJ Proteome Res}, volume = {9}, year = {2010}, note = {Pendyala, GuruduttTrauger, Sunia ASiuzdak, GaryFox, Howard SP01 DA026146/DA/NIDA NIH HHS/P01 DA026146-02/DA/NIDA NIH HHS/P01DA026146/DA/NIDA NIH HHS/P30 MH062261/MH/NIMH NIH HHS/P30 MH062261-09/MH/NIMH NIH HHS/P30MH062261/MH/NIMH NIH HHS/R01 MH073490/MH/NIMH NIH HHS/R01 MH073490-06/MH/NIMH NIH HHS/J Proteome Res. 2010 Jan;9(1):352-8. doi: 10.1021/pr900685u.}, month = {Jan}, pages = {352-8}, type = {Research Support, N.I.H., Extramural}, edition = {2009/11/17}, abstract = {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.}, keywords = {Animals, Carrier Proteins/*blood, Encephalitis, Viral/*blood/virology, Glycoproteins/*blood, Macaca mulatta, Peptide Mapping, Proteome, Proteomics/*methods, Reproducibility of Results, Serum Albumin/*analysis, Simian Acquired Immunodeficiency Syndrome/*blood, Simian immunodeficiency virus, Tandem Mass Spectrometry, Vitamin E/metabolism}, isbn = {1535-3907 (Electronic)1535-3893 (Linking)}, author = {Pendyala, G. and Trauger, S. A. and Siuzdak, G. and Fox, H. S.} } @article {269871, title = {The glycerophospho metabolome and its influence on amino acid homeostasis revealed by brain metabolomics of GDE1(-/-) mice}, journal = {Chem BiolChem BiolChem Biol}, volume = {17}, year = {2010}, note = {Kopp, FlorianKomatsu, ToruNomura, Daniel KTrauger, Sunia AThomas, Jason RSiuzdak, GarySimon, Gabriel MCravatt, Benjamin FK99 DA030908/DA/NIDA NIH HHS/P01 DA017259/DA/NIDA NIH HHS/R01 CA132630/CA/NCI NIH HHS/R01 CA132630-04/CA/NCI NIH HHS/R01CA132630/CA/NCI NIH HHS/Chem Biol. 2010 Aug 27;17(8):831-40. doi: 10.1016/j.chembiol.2010.06.009.}, month = {Aug 27}, pages = {831-40}, type = {Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov{\textquoteright}t}, edition = {2010/08/28}, abstract = {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.}, keywords = {*Homeostasis, *Metabolome, Amino Acids/*metabolism, Animals, Brain/cytology/*metabolism, Inositol Phosphates/*metabolism, Male, Mass Spectrometry, Metabolomics/*methods, Mice, Phosphoric Diester Hydrolases/*deficiency/metabolism, Serine/metabolism, Solubility, Water/chemistry}, isbn = {1879-1301 (Electronic)1074-5521 (Linking)}, author = {Kopp, F. and Komatsu, T. and Nomura, D. K. and Trauger, S. A. and Thomas, J. R. and Siuzdak, G. and Simon, G. M. and Cravatt, B. F.} } @article {269841, title = {Large scale physiological readjustment during growth enables rapid, comprehensive and inexpensive systems analysis}, journal = {BMC Syst BiolBMC Syst BiolBMC Syst Biol}, volume = {4}, year = {2010}, note = {Facciotti, Marc TPang, Wyming LLo, Fang-yinWhitehead, KeniaKoide, TieMasumura, Ken-ichiPan, MinKaur, AmardeepLarsen, David JReiss, David JHoang, LinhKalisiak, EwaNorthen, TrentTrauger, Sunia ASiuzdak, GaryBaliga, Nitin S1R01GM077398-01A2/GM/NIGMS NIH HHS/P50 GM076547/GM/NIGMS NIH HHS/P50GM076547/GM/NIGMS NIH HHS/EnglandBMC Syst Biol. 2010 May 14;4:64. doi: 10.1186/1752-0509-4-64.}, pages = {64}, type = {Research Support, N.I.H., ExtramuralResearch Support, U.S. Gov{\textquoteright}t, Non-P.H.S.}, edition = {2010/05/18}, abstract = {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.}, keywords = {*Systems Analysis, *Systems Biology, Chromatography, Liquid/methods, Escherichia coli/metabolism, Gene Expression Profiling, Gene Regulatory Networks, Halobacterium salinarum/*genetics/metabolism, Mass Spectrometry/methods, Models, Biological, Models, Genetic, Oligonucleotide Array Sequence Analysis, Phenotype, RNA, Messenger/metabolism, Transcription, Genetic}, isbn = {1752-0509 (Electronic)1752-0509 (Linking)}, author = {Facciotti, M. T. and Pang, W. L. and Lo, F. Y. and Whitehead, K. and Koide, T. and Masumura, K. and Pan, M. and Kaur, A. and Larsen, D. J. and Reiss, D. J. and Hoang, L. and Kalisiak, E. and Northen, T. and Trauger, S. A. and Siuzdak, G. and Baliga, N. S.} } @article {269836, title = {Maintaining retinal astrocytes normalizes revascularization and prevents vascular pathology associated with oxygen-induced retinopathy}, journal = {GliaGliaGlia}, volume = {58}, year = {2010}, note = {Dorrell, Michael IAguilar, EdithJacobson, RuthTrauger, Sunia AFriedlander, JeffreySiuzdak, GaryFriedlander, MartinR01 EY011254/EY/NEI NIH HHS/R01 EY011254-12S1/EY/NEI NIH HHS/R01EY11254/EY/NEI NIH HHS/R24 EY017540/EY/NEI NIH HHS/R24 EY017540-05/EY/NEI NIH HHS/R24EY017540/EY/NEI NIH HHS/Glia. 2010 Jan 1;58(1):43-54. doi: 10.1002/glia.20900.}, month = {Jan 1}, pages = {43-54}, type = {Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov{\textquoteright}t}, edition = {2009/06/23}, abstract = {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.}, keywords = {Age Factors, Animals, Animals, Newborn, Antigens, CD11b/metabolism, Astrocytes/chemistry/pathology/*physiology, Bone Marrow Cells/physiology, Bone Marrow Transplantation/methods, Cells, Cultured, Cerebral Cortex/cytology, Culture Media, Conditioned/pharmacology, Disease Models, Animal, Fibroblast Growth Factors/therapeutic use, Glial Fibrillary Acidic Protein/metabolism, Humans, induced/*complications/*pathology/*prevention \& control, Infant, Newborn, Injections, Intraventricular/methods, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Myeloid Cells/physiology, Neovascularization, Physiologic/*physiology, Oxygen/adverse effects, Proteomics/methods, Retinal Neovascularization/*etiology, Retinopathy of Prematurity/chemically, Vascular Endothelial Growth Factor A/therapeutic use}, isbn = {1098-1136 (Electronic)0894-1491 (Linking)}, author = {Dorrell, M. I. and Aguilar, E. and Jacobson, R. and Trauger, S. A. and Friedlander, J. and Siuzdak, G. and Friedlander, M.} } @article {269826, title = {Microbial metalloproteomes are largely uncharacterized}, journal = {NatureNatureNature}, volume = {466}, year = {2010}, note = {Cvetkovic, AleksandarMenon, Angeli LalThorgersen, Michael PScott, Joseph WPoole, Farris L 2ndJenney, Francis E JrLancaster, W AndrewPraissman, Jeremy LShanmukh, SaratchandraVaccaro, Brian JTrauger, Sunia AKalisiak, EwaApon, Junefredo VSiuzdak, GaryYannone, Steven MTainer, John AAdams, Michael W WEnglandNature. 2010 Aug 5;466(7307):779-82. doi: 10.1038/nature09265. Epub 2010 Jul 18.}, month = {Aug 5}, pages = {779-82}, type = {Research Support, U.S. Gov{\textquoteright}t, Non-P.H.S.}, edition = {2010/07/20}, abstract = {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.}, keywords = {Bacterial Proteins/*analysis/chemistry, Chromatography, Liquid, Escherichia coli/chemistry, Metalloproteins/*analysis/*chemistry, Metals/*analysis/chemistry/metabolism, Proteome/*analysis/chemistry, Proteomics, Pyrococcus furiosus/*chemistry/metabolism, Sulfolobus solfataricus/chemistry, Tandem Mass Spectrometry}, isbn = {1476-4687 (Electronic)0028-0836 (Linking)}, author = {Cvetkovic, A. and Menon, A. L. and Thorgersen, M. P. and Scott, J. W. and Poole, F. L., 2nd and Jenney, F. E., Jr. and Lancaster, W. A. and Praissman, J. L. and Shanmukh, S. and Vaccaro, B. J. and Trauger, S. A. and Kalisiak, E. and Apon, J. V. and Siuzdak, G. and Yannone, S. M. and Tainer, J. A. and Adams, M. W.} } @article {8112, title = {Maintaining retinal astrocytes normalizes revascularization and prevents vascular pathology associated with oxygen-induced retinopathy}, journal = {Glia}, volume = {58}, year = {2010}, note = {Dorrell, Michael IAguilar, EdithJacobson, RuthTrauger, Sunia AFriedlander, JeffreySiuzdak, GaryFriedlander, MartinR01 EY011254-12S1/EY/NEI NIH HHS/R01EY11254/EY/NEI NIH HHS/R24 EY017540-05/EY/NEI NIH HHS/R24EY017540/EY/NEI NIH HHS/Glia. 2010 Jan 1;58(1):43-54. }, month = {Jan 1}, pages = {43-54}, type = {Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov{\textquoteright}t}, edition = {2009/06/23}, abstract = {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. }, isbn = {1098-1136 (Electronic)0894-1491 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19544395}, author = {Dorrell, M. I. and Aguilar, E. and Jacobson, R. and Trauger, S. A. and Friedlander, J. and Siuzdak, G. and Friedlander, M.} } @article {8109, title = {Large scale physiological readjustment during growth enables rapid, comprehensive and inexpensive systems analysis}, journal = {BMC Syst Biol}, volume = {4}, year = {2010}, note = {Facciotti, Marc TPang, Wyming LLo, Fang-yinWhitehead, KeniaKoide, TieMasumura, Ken-ichiPan, MinKaur, AmardeepLarsen, David JReiss, David JHoang, LinhKalisiak, EwaNorthen, TrentTrauger, Sunia ASiuzdak, GaryBaliga, Nitin S1R01GM077398-01A2/GM/NIGMS NIH HHS/P50GM076547/GM/NIGMS NIH HHS/EnglandBMC Syst Biol. 2010 May 14;4:64. }, pages = {64}, type = {Research Support, N.I.H., ExtramuralResearch Support, U.S. Gov{\textquoteright}t, Non-P.H.S.}, edition = {2010/05/18}, abstract = {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. }, isbn = {1752-0509 (Electronic)1752-0509 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/20470417}, author = {Facciotti, M. T. and Pang, W. L. and Lo, F. Y. and Whitehead, K. and Koide, T. and Masumura, K. and Pan, M. and Kaur, A. and Larsen, D. J. and Reiss, D. J. and Hoang, L. and Kalisiak, E. and Northen, T. and Trauger, S. A. and Siuzdak, G. and Baliga, N. S.} } @article {8110, title = {Metabolic oxidation regulates embryonic stem cell differentiation}, journal = {Nat Chem Biol}, volume = {6}, year = {2010}, note = {Yanes, OscarClark, JulieWong, Diana MPatti, Gary JSanchez-Ruiz, AntonioBenton, H PaulTrauger, Sunia ADesponts, CarolineDing, ShengSiuzdak, GaryR24 EY017540-03/EY/NEI NIH HHS/R24 EY017540-05/EY/NEI NIH HHS/Nat Chem Biol. 2010 Jun;6(6):411-7. Epub 2010 May 2. }, month = {Jun}, pages = {411-7}, type = {Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov{\textquoteright}tResearch Support, U.S. Gov{\textquoteright}t, Non-P.H.S.}, edition = {2010/05/04}, abstract = {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. }, isbn = {1552-4469 (Electronic)1552-4450 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/20436487}, author = {Yanes, O. and Clark, J. and Wong, D. M. and Patti, G. J. and Sanchez-Ruiz, A. and Benton, H. P. and Trauger, S. A. and Desponts, C. and Ding, S. and Siuzdak, G.} } @article {8111, title = {Quantitative plasma proteomic profiling identifies the vitamin E binding protein afamin as a potential pathogenic factor in SIV induced CNS disease}, journal = {J Proteome Res}, volume = {9}, year = {2010}, note = {Pendyala, GuruduttTrauger, Sunia ASiuzdak, GaryFox, Howard SP01 DA026146-02/DA/NIDA NIH HHS/P01DA026146/DA/NIDA NIH HHS/P30 MH062261-09/MH/NIMH NIH HHS/P30MH062261/MH/NIMH NIH HHS/R01 MH073490/MH/NIMH NIH HHS/R01 MH073490-06/MH/NIMH NIH HHS/J Proteome Res. 2010 Jan;9(1):352-8. }, month = {Jan}, pages = {352-8}, type = {Research Support, N.I.H., Extramural}, edition = {2009/11/17}, abstract = {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. }, isbn = {1535-3907 (Electronic)1535-3893 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19908921}, author = {Pendyala, G. and Trauger, S. A. and Siuzdak, G. and Fox, H. S.} } @article {8107, title = {The glycerophospho metabolome and its influence on amino acid homeostasis revealed by brain metabolomics of GDE1(-/-) mice}, journal = {Chem Biol}, volume = {17}, year = {2010}, note = {Kopp, FlorianKomatsu, ToruNomura, Daniel KTrauger, Sunia AThomas, Jason RSiuzdak, GarySimon, Gabriel MCravatt, Benjamin FK99 DA030908/DA/NIDA NIH HHS/P01 DA017259-08/DA/NIDA NIH HHS/R01 CA132630-04/CA/NCI NIH HHS/R01 CA132630-05/CA/NCI NIH HHS/R01CA132630/CA/NCI NIH HHS/Chem Biol. 2010 Aug 27;17(8):831-40. }, month = {Aug 27}, pages = {831-40}, type = {Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov{\textquoteright}t}, edition = {2010/08/28}, abstract = {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. }, isbn = {1879-1301 (Electronic)1074-5521 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/20797612}, author = {Kopp, F. and Komatsu, T. and Nomura, D. K. and Trauger, S. A. and Thomas, J. R. and Siuzdak, G. and Simon, G. M. and Cravatt, B. F.} } @article {8108, title = {Microbial metalloproteomes are largely uncharacterized}, journal = {Nature}, volume = {466}, year = {2010}, note = {Cvetkovic, AleksandarMenon, Angeli LalThorgersen, Michael PScott, Joseph WPoole, Farris L 2ndJenney, Francis E JrLancaster, W AndrewPraissman, Jeremy LShanmukh, SaratchandraVaccaro, Brian JTrauger, Sunia AKalisiak, EwaApon, Junefredo VSiuzdak, GaryYannone, Steven MTainer, John AAdams, Michael W WEnglandNature. 2010 Aug 5;466(7307):779-82. Epub 2010 Jul 18. }, month = {Aug 5}, pages = {779-82}, type = {Research Support, U.S. Gov{\textquoteright}t, Non-P.H.S.}, edition = {2010/07/20}, abstract = {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. }, isbn = {1476-4687 (Electronic)0028-0836 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/20639861}, author = {Cvetkovic, A. and Menon, A. L. and Thorgersen, M. P. and Scott, J. W. and Poole, F. L., 2nd and Jenney, F. E., Jr. and Lancaster, W. A. and Praissman, J. L. and Shanmukh, S. and Vaccaro, B. J. and Trauger, S. A. and Kalisiak, E. and Apon, J. V. and Siuzdak, G. and Yannone, S. M. and Tainer, J. A. and Adams, M. W.} } @article {269981, title = {Generation of induced pluripotent stem cells using recombinant proteins}, journal = {Cell Stem CellCell Stem CellCell Stem Cell}, volume = {4}, year = {2009}, note = {Zhou, HongyanWu, ShiliJoo, Jin YoungZhu, SaiyongHan, Dong WookLin, TongxiangTrauger, SuniaBien, GeofferyYao, SusanZhu, YongSiuzdak, GaryScholer, Hans RDuan, LingxunDing, ShengR01 HD064610/HD/NICHD NIH HHS/Cell Stem Cell. 2009 May 8;4(5):381-4. doi: 10.1016/j.stem.2009.04.005. Epub 2009 Apr 23.}, month = {May 8}, pages = {381-4}, type = {Research Support, Non-U.S. Gov{\textquoteright}t}, edition = {2009/04/29}, keywords = {Animals, Mice, Pluripotent Stem Cells/*cytology/metabolism, Recombinant Proteins/genetics/*metabolism, Transcription Factors/genetics/metabolism}, isbn = {1875-9777 (Electronic)}, author = {Zhou, H. and Wu, S. and Joo, J. Y. and Zhu, S. and Han, D. W. and Lin, T. and Trauger, S. and Bien, G. and Yao, S. and Zhu, Y. and Siuzdak, G. and Scholer, H. R. and Duan, L. and Ding, S.} } @article {269966, title = {Phosphonium labeling for increasing metabolomic coverage of neutral lipids using electrospray ionization mass spectrometry}, journal = {Rapid Commun Mass SpectromRapid Commun Mass SpectromRapid Commun Mass Spectrom}, volume = {23}, year = {2009}, note = {Woo, Hin-KoonGo, Eden PHoang, LinhTrauger, Sunia ABowen, BenjaminSiuzdak, GaryNorthen, Trent RMH062261/MH/NIMH NIH HHS/P30 MH062261/MH/NIMH NIH HHS/P30 MH062261-10/MH/NIMH NIH HHS/EnglandRapid Commun Mass Spectrom. 2009 Jun;23(12):1849-55. doi: 10.1002/rcm.4076.}, month = {Jun}, pages = {1849-55}, type = {Evaluation StudiesResearch Support, N.I.H., ExtramuralResearch Support, U.S. Gov{\textquoteright}t, Non-P.H.S.}, edition = {2009/05/19}, abstract = {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.}, keywords = {*Lipid Metabolism, *Metabolomics, Humans, Lipids/blood/*chemistry, Male, Organophosphorus Compounds/chemical synthesis/*chemistry, Sensitivity and Specificity, Spectrometry, Mass, Electrospray Ionization/*methods, Staining and Labeling}, isbn = {1097-0231 (Electronic)0951-4198 (Linking)}, author = {Woo, H. K. and Go, E. P. and Hoang, L. and Trauger, S. A. and Bowen, B. and Siuzdak, G. and Northen, T. R.} } @article {269961, title = {Response and recovery in the plasma metabolome tracks the acute LCMV-induced immune response}, journal = {J Proteome ResJ Proteome ResJ Proteome Res}, volume = {8}, year = {2009}, note = {Wikoff, William RKalisak, EwaTrauger, SuniaManchester, MarianneSiuzdak, GaryP30 MH062261/MH/NIMH NIH HHS/P30 MH062261-08/MH/NIMH NIH HHS/R01 MH073490/MH/NIMH NIH HHS/R01 MH073490-04/MH/NIMH NIH HHS/R01 MH073490-05/MH/NIMH NIH HHS/J Proteome Res. 2009 Jul;8(7):3578-87. doi: 10.1021/pr900275p.}, month = {Jul}, pages = {3578-87}, type = {Research Support, N.I.H., Extramural}, edition = {2009/06/06}, abstract = {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.}, keywords = {Animals, Blood Proteins/*chemistry, Immunity, Innate, Interferon-gamma/metabolism, Kinetics, Lymphocytic choriomeningitis virus/*metabolism, Male, Mass Spectrometry/methods, Metabolome, Metabolomics/*methods, Mice, Mice, Inbred C57BL, Models, Biological, Models, Chemical, Systems Biology/methods}, isbn = {1535-3893 (Print)1535-3893 (Linking)}, author = {Wikoff, W. R. and Kalisak, E. and Trauger, S. and Manchester, M. and Siuzdak, G.} } @article {269906, title = {Cerebrospinal fluid proteomics reveals potential pathogenic changes in the brains of SIV-infected monkeys}, journal = {J Proteome ResJ Proteome ResJ Proteome Res}, volume = {8}, year = {2009}, note = {Pendyala, GuruduttTrauger, Sunia AKalisiak, EwaEllis, Ronald JSiuzdak, GaryFox, Howard SDA026146/DA/NIDA NIH HHS/MH062261/MH/NIMH NIH HHS/MH062512/MH/NIMH NIH HHS/MH073490/MH/NIMH NIH HHS/P01 DA026146/DA/NIDA NIH HHS/P01 DA026146-010002/DA/NIDA NIH HHS/P30 MH062261/MH/NIMH NIH HHS/P30 MH062261-09/MH/NIMH NIH HHS/P30 MH062512/MH/NIMH NIH HHS/P30 MH062512-09/MH/NIMH NIH HHS/R01 MH073490/MH/NIMH NIH HHS/R01 MH073490-06/MH/NIMH NIH HHS/J Proteome Res. 2009 May;8(5):2253-60. doi: 10.1021/pr800854t.}, month = {May}, pages = {2253-60}, type = {Research Support, N.I.H., Extramural}, edition = {2009/03/14}, abstract = {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.}, keywords = {Animals, Brain Diseases/*cerebrospinal fluid/virology, Brain/metabolism/pathology/virology, Chromatography, Liquid, Complement C3/genetics/metabolism, Hemopexin/genetics/metabolism, Host-Pathogen Interactions, Humans, Immunoglobulin Heavy Chains/genetics/metabolism, Immunoglobulin M/genetics/metabolism, Immunohistochemistry, Macaca mulatta, Plasminogen/genetics/metabolism, Proteins/*analysis/genetics, Proteomics/*methods, Reverse Transcriptase Polymerase Chain Reaction, Simian Acquired Immunodeficiency Syndrome/*cerebrospinal fluid/virology, Simian immunodeficiency virus/physiology, Tandem Mass Spectrometry}, isbn = {1535-3893 (Print)1535-3893 (Linking)}, author = {Pendyala, G. and Trauger, S. A. and Kalisiak, E. and Ellis, R. J. and Siuzdak, G. and Fox, H. S.} } @article {269901, title = {Novel multiprotein complexes identified in the hyperthermophilic archaeon Pyrococcus furiosus by non-denaturing fractionation of the native proteome}, journal = {Mol Cell ProteomicsMol Cell ProteomicsMol Cell Proteomics}, volume = {8}, year = {2009}, note = {Menon, Angeli LalPoole, Farris L 2ndCvetkovic, AleksandarTrauger, Sunia AKalisiak, EwaScott, Joseph WShanmukh, SaratchandraPraissman, JeremyJenney, Francis E JrWikoff, William RApon, John VSiuzdak, GaryAdams, Michael W WMol Cell Proteomics. 2009 Apr;8(4):735-51. doi: 10.1074/mcp.M800246-MCP200. Epub 2008 Nov 28.}, month = {Apr}, pages = {735-51}, type = {Research Support, U.S. Gov{\textquoteright}t, Non-P.H.S.}, edition = {2008/12/02}, abstract = {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.}, keywords = {Amino Acids/metabolism, Archaeal Proteins/*analysis/isolation \& purification, Chemical Fractionation/*methods, Cytoplasm/metabolism, Multiprotein Complexes/*analysis, Protein Denaturation, Protein Multimerization, Proteome/*analysis, Pyrococcus furiosus/*metabolism}, isbn = {1535-9484 (Electronic)1535-9476 (Linking)}, author = {Menon, A. L. and Poole, F. L., 2nd and Cvetkovic, A. and Trauger, S. A. and Kalisiak, E. and Scott, J. W. and Shanmukh, S. and Praissman, J. and Jenney, F. E., Jr. and Wikoff, W. R. and Apon, J. V. and Siuzdak, G. and Adams, M. W.} } @article {269876, title = {Endothelial targeting of cowpea mosaic virus (CPMV) via surface vimentin}, journal = {PLoS PathogPLoS PathogPLoS Pathog}, volume = {5}, year = {2009}, note = {Koudelka, Kristopher JDestito, GiuseppePlummer, Emily MTrauger, Sunia ASiuzdak, GaryManchester, MarianneCA112075/CA/NCI NIH HHS/PLoS Pathog. 2009 May;5(5):e1000417. doi: 10.1371/journal.ppat.1000417. Epub 2009 May 1.}, month = {May}, pages = {e1000417}, type = {Research Support, N.I.H., Extramural}, edition = {2009/05/05}, abstract = {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.}, keywords = {Animals, Aorta/metabolism, Cell Line, Cell Membrane/metabolism, Chromatography, Liquid, Comovirus/*metabolism, Endothelium, Vascular/*metabolism/*virology, HeLa Cells, Humans, Male, Mice, Protein Binding, Proteomics, Rats, Rats, Sprague-Dawley, Tandem Mass Spectrometry, Vimentin/*metabolism, Virion/metabolism}, isbn = {1553-7374 (Electronic)1553-7366 (Linking)}, author = {Koudelka, K. J. and Destito, G. and Plummer, E. M. and Trauger, S. A. and Siuzdak, G. and Manchester, M.} } @article {269861, title = {Identification of a new endogenous metabolite and the characterization of its protein interactions through an immobilization approach}, journal = {J Am Chem SocJ Am Chem SocJ Am Chem Soc}, volume = {131}, year = {2009}, note = {Kalisiak, JaroslawTrauger, Sunia AKalisiak, EwaMorita, HirotoshiFokin, Valery VAdams, Mike W WSharpless, K BarrySiuzdak, GaryP30 MH062261/MH/NIMH NIH HHS/P30 MH062261-05S1/MH/NIMH NIH HHS/R24 EY017540/EY/NEI NIH HHS/R24 EY017540-02/EY/NEI NIH HHS/J Am Chem Soc. 2009 Jan 14;131(1):378-86. doi: 10.1021/ja808172n.}, month = {Jan 14}, pages = {378-86}, type = {Research Support, N.I.H., ExtramuralResearch Support, U.S. Gov{\textquoteright}t, Non-P.H.S.}, edition = {2008/12/06}, abstract = {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.}, keywords = {Metabolomics/*methods, Proteins/*analysis/metabolism, Pyrococcus furiosus/chemistry/metabolism, Spermidine/*analogs \& derivatives, Tandem Mass Spectrometry/*methods}, isbn = {1520-5126 (Electronic)0002-7863 (Linking)}, author = {Kalisiak, J. and Trauger, S. A. and Kalisiak, E. and Morita, H. and Fokin, V. V. and Adams, M. W. and Sharpless, K. B. and Siuzdak, G.} } @article {269856, title = {Increased enzymatic O-GlcNAcylation of mitochondrial proteins impairs mitochondrial function in cardiac myocytes exposed to high glucose}, journal = {J Biol ChemJ Biol ChemJ Biol Chem}, volume = {284}, year = {2009}, note = {Hu, YongSuarez, JorgeFricovsky, EduardoWang, HongScott, Brian TTrauger, Sunia AHan, WenlongHu, YingOyeleye, Mary ODillmann, Wolfgang HHL-66917/HL/NHLBI NIH HHS/P60 MD00220/MD/NIMHD NIH HHS/J Biol Chem. 2009 Jan 2;284(1):547-55. doi: 10.1074/jbc.M808518200. Epub 2008 Nov 12.}, month = {Jan 2}, pages = {547-55}, type = {Research Support, N.I.H., Extramural}, edition = {2008/11/14}, abstract = {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.}, keywords = {Acetylglucosamine/genetics/metabolism, Animals, Cyclooxygenase 1/metabolism, Diabetes Mellitus/*enzymology/pathology, Gene Expression Regulation, Enzymologic/drug effects, Glucose/*pharmacology, Glucosyltransferases/*biosynthesis, Glycosylation/drug effects, Membrane Proteins/metabolism, Mice, Mitochondria, Heart/*enzymology/pathology, Mitochondrial Proteins/*biosynthesis, Myocytes, Cardiac/*enzymology/pathology, NADH Dehydrogenase/metabolism, Protein Processing, Post-Translational/*drug effects, Rats, Sweetening Agents/*pharmacology}, isbn = {0021-9258 (Print)0021-9258 (Linking)}, author = {Hu, Y. and Suarez, J. and Fricovsky, E. and Wang, H. and Scott, B. T. and Trauger, S. A. and Han, W. and Oyeleye, M. O. and Dillmann, W. H.} } @article {8121, title = {Increased enzymatic O-GlcNAcylation of mitochondrial proteins impairs mitochondrial function in cardiac myocytes exposed to high glucose}, journal = {J Biol Chem}, volume = {284}, year = {2009}, note = {Hu, YongSuarez, JorgeFricovsky, EduardoWang, HongScott, Brian TTrauger, Sunia AHan, WenlongHu, YingOyeleye, Mary ODillmann, Wolfgang HHL-66917/HL/NHLBI NIH HHS/P60 MD00220/MD/NCMHD NIH HHS/J Biol Chem. 2009 Jan 2;284(1):547-55. Epub 2008 Nov 12. }, month = {Jan 2}, pages = {547-55}, type = {Research Support, N.I.H., Extramural}, edition = {2008/11/14}, abstract = {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. }, isbn = {0021-9258 (Print)0021-9258 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19004814}, author = {Hu, Y. and Suarez, J. and Fricovsky, E. and Wang, H. and Scott, B. T. and Trauger, S. A. and Han, W. and Oyeleye, M. O. and Dillmann, W. H.} } @article {8117, title = {Cerebrospinal fluid proteomics reveals potential pathogenic changes in the brains of SIV-infected monkeys}, journal = {J Proteome Res}, volume = {8}, year = {2009}, note = {Pendyala, GuruduttTrauger, Sunia AKalisiak, EwaEllis, Ronald JSiuzdak, GaryFox, Howard SDA026146/DA/NIDA NIH HHS/MH062261/MH/NIMH NIH HHS/MH062512/MH/NIMH NIH HHS/MH073490/MH/NIMH NIH HHS/P01 DA026146-010002/DA/NIDA NIH HHS/P30 MH062261-09/MH/NIMH NIH HHS/P30 MH062512-09/MH/NIMH NIH HHS/R01 MH073490-06/MH/NIMH NIH HHS/J Proteome Res. 2009 May;8(5):2253-60. }, month = {May}, pages = {2253-60}, type = {Research Support, N.I.H., Extramural}, edition = {2009/03/14}, abstract = {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. }, isbn = {1535-3893 (Print)1535-3893 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19281240}, author = {Pendyala, G. and Trauger, S. A. and Kalisiak, E. and Ellis, R. J. and Siuzdak, G. and Fox, H. S.} } @article {8118, title = {Identification of a new endogenous metabolite and the characterization of its protein interactions through an immobilization approach}, journal = {J Am Chem Soc}, volume = {131}, year = {2009}, note = {Kalisiak, JaroslawTrauger, Sunia AKalisiak, EwaMorita, HirotoshiFokin, Valery VAdams, Mike W WSharpless, K BarrySiuzdak, GaryP30 MH062261/MH/NIMH NIH HHS/P30 MH062261-05S1/MH/NIMH NIH HHS/R24 EY017540/EY/NEI NIH HHS/R24 EY017540-02/EY/NEI NIH HHS/J Am Chem Soc. 2009 Jan 14;131(1):378-86. }, month = {Jan 14}, pages = {378-86}, type = {Research Support, N.I.H., ExtramuralResearch Support, U.S. Gov{\textquoteright}t, Non-P.H.S.}, edition = {2008/12/06}, abstract = {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. }, isbn = {1520-5126 (Electronic)0002-7863 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19055353}, author = {Kalisiak, J. and Trauger, S. A. and Kalisiak, E. and Morita, H. and Fokin, V. V. and Adams, M. W. and Sharpless, K. B. and Siuzdak, G.} } @article {8119, title = {Novel multiprotein complexes identified in the hyperthermophilic archaeon Pyrococcus furiosus by non-denaturing fractionation of the native proteome}, journal = {Mol Cell Proteomics}, volume = {8}, year = {2009}, note = {Menon, Angeli LalPoole, Farris L 2ndCvetkovic, AleksandarTrauger, Sunia AKalisiak, EwaScott, Joseph WShanmukh, SaratchandraPraissman, JeremyJenney, Francis E JrWikoff, William RApon, John VSiuzdak, GaryAdams, Michael W WMol Cell Proteomics. 2009 Apr;8(4):735-51. Epub 2008 Nov 28. }, month = {Apr}, pages = {735-51}, type = {Research Support, U.S. Gov{\textquoteright}t, Non-P.H.S.}, edition = {2008/12/02}, abstract = {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. }, isbn = {1535-9484 (Electronic)1535-9476 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19043064}, author = {Menon, A. L. and Poole, F. L., 2nd and Cvetkovic, A. and Trauger, S. A. and Kalisiak, E. and Scott, J. W. and Shanmukh, S. and Praissman, J. and Jenney, F. E., Jr. and Wikoff, W. R. and Apon, J. V. and Siuzdak, G. and Adams, M. W.} } @article {8115, title = {Endothelial targeting of cowpea mosaic virus (CPMV) via surface vimentin}, journal = {PLoS Pathog}, volume = {5}, year = {2009}, note = {Koudelka, Kristopher JDestito, GiuseppePlummer, Emily MTrauger, Sunia ASiuzdak, GaryManchester, MarianneCA112075/CA/NCI NIH HHS/PLoS Pathog. 2009 May;5(5):e1000417. Epub 2009 May 1. }, month = {May}, pages = {e1000417}, type = {Research Support, N.I.H., Extramural}, edition = {2009/05/05}, abstract = {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. }, isbn = {1553-7374 (Electronic)1553-7366 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19412526}, author = {Koudelka, K. J. and Destito, G. and Plummer, E. M. and Trauger, S. A. and Siuzdak, G. and Manchester, M.} } @article {8116, title = {Generation of induced pluripotent stem cells using recombinant proteins}, journal = {Cell Stem Cell}, volume = {4}, year = {2009}, note = {Zhou, HongyanWu, ShiliJoo, Jin YoungZhu, SaiyongHan, Dong WookLin, TongxiangTrauger, SuniaBien, GeofferyYao, SusanZhu, YongSiuzdak, GaryScholer, Hans RDuan, LingxunDing, ShengCell Stem Cell. 2009 May 8;4(5):381-4. Epub 2009 Apr 23. }, month = {May 8}, pages = {381-4}, type = {Research Support, Non-U.S. Gov{\textquoteright}t}, edition = {2009/04/29}, abstract = {n/a}, isbn = {1875-9777 (Electronic)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19398399}, author = {Zhou, H. and Wu, S. and Joo, J. Y. and Zhu, S. and Han, D. W. and Lin, T. and Trauger, S. and Bien, G. and Yao, S. and Zhu, Y. and Siuzdak, G. and Scholer, H. R. and Duan, L. and Ding, S.} } @article {8114, title = {Phosphonium labeling for increasing metabolomic coverage of neutral lipids using electrospray ionization mass spectrometry}, journal = {Rapid Commun Mass Spectrom}, volume = {23}, year = {2009}, note = {Woo, Hin-KoonGo, Eden PHoang, LinhTrauger, Sunia ABowen, BenjaminSiuzdak, GaryNorthen, Trent RMH062261/MH/NIMH NIH HHS/P30 MH062261-10/MH/NIMH NIH HHS/EnglandRapid Commun Mass Spectrom. 2009 Jun;23(12):1849-55. }, month = {Jun}, pages = {1849-55}, type = {Evaluation StudiesResearch Support, N.I.H., ExtramuralResearch Support, U.S. Gov{\textquoteright}t, Non-P.H.S.}, edition = {2009/05/19}, abstract = {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. }, isbn = {1097-0231 (Electronic)0951-4198 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19449318}, author = {Woo, H. K. and Go, E. P. and Hoang, L. and Trauger, S. A. and Bowen, B. and Siuzdak, G. and Northen, T. R.} } @article {8113, title = {Response and recovery in the plasma metabolome tracks the acute LCMV-induced immune response}, journal = {J Proteome Res}, volume = {8}, year = {2009}, note = {Wikoff, William RKalisak, EwaTrauger, SuniaManchester, MarianneSiuzdak, GaryP30 MH062261-08/MH/NIMH NIH HHS/R01 MH073490-04/MH/NIMH NIH HHS/R01 MH073490-05/MH/NIMH NIH HHS/J Proteome Res. 2009 Jul;8(7):3578-87. }, month = {Jul}, pages = {3578-87}, type = {Research Support, N.I.H., Extramural}, edition = {2009/06/06}, abstract = {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. }, isbn = {1535-3893 (Print)1535-3893 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19496611}, author = {Wikoff, W. R. and Kalisak, E. and Trauger, S. and Manchester, M. and Siuzdak, G.} } @article {269941, title = {Correlating the transcriptome, proteome, and metabolome in the environmental adaptation of a hyperthermophile}, journal = {J Proteome ResJ Proteome ResJ Proteome Res}, volume = {7}, year = {2008}, note = {Trauger, Sunia AKalisak, EwaKalisiak, JaroslawMorita, HirotoshiWeinberg, Michael VMenon, Angeli LalPoole, Farris L 2ndAdams, Michael W WSiuzdak, GaryJ Proteome Res. 2008 Mar;7(3):1027-35. doi: 10.1021/pr700609j. Epub 2008 Feb 2.}, month = {Mar}, pages = {1027-35}, type = {Research Support, U.S. Gov{\textquoteright}t, Non-P.H.S.}, edition = {2008/02/06}, abstract = {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.}, keywords = {*Adaptation, Physiological, *Proteome, Bacterial Proteins/chemistry/genetics/*metabolism, Oligonucleotide Array Sequence Analysis, Peptides/isolation \& purification, RNA, Messenger/*genetics, Spectrometry, Mass, Electrospray Ionization, Thermus thermophilus/genetics/*metabolism/physiology}, isbn = {1535-3893 (Print)1535-3893 (Linking)}, author = {Trauger, S. A. and Kalisak, E. and Kalisiak, J. and Morita, H. and Weinberg, M. V. and Menon, A. L. and Poole, F. L., 2nd and Adams, M. W. and Siuzdak, G.} } @article {269811, title = {Quantitative ESI-TOF analysis of macromolecular assembly kinetics}, journal = {Anal ChemAnal ChemAnal Chem}, volume = {80}, year = {2008}, note = {Bunner, Anne ETrauger, Sunia ASiuzdak, GaryWilliamson, James RR37 GM053757-13/GM/NIGMS NIH HHS/R37-GM53757/GM/NIGMS NIH HHS/Anal Chem. 2008 Dec 15;80(24):9379-86. doi: 10.1021/ac8020505.}, month = {Dec 15}, pages = {9379-86}, type = {Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov{\textquoteright}t}, edition = {2008/11/15}, abstract = {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{\textquoteright} 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.}, keywords = {*Spectrometry, Mass, Electrospray Ionization, *Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Escherichia coli/genetics/metabolism, Kinetics, Macromolecular Substances/chemistry/*metabolism, Protein Subunits, Ribosomal Proteins/genetics/*metabolism, Ribosomes/*chemistry/genetics/*metabolism, RNA, Ribosomal, 16S/genetics/metabolism}, isbn = {1520-6882 (Electronic)0003-2700 (Linking)}, author = {Bunner, A. E. and Trauger, S. A. and Siuzdak, G. and Williamson, J. R.} } @article {269796, title = {XCMS2: processing tandem mass spectrometry data for metabolite identification and structural characterization}, journal = {Anal ChemAnal ChemAnal Chem}, volume = {80}, year = {2008}, note = {Benton, H PWong, D MTrauger, S ASiuzdak, GP30 MH062261/MH/NIMH NIH HHS/P30 MH062261-05S1/MH/NIMH NIH HHS/R24 EY017540/EY/NEI NIH HHS/R24 EY017540-02/EY/NEI NIH HHS/R24EY017540/EY/NEI NIH HHS/Anal Chem. 2008 Aug 15;80(16):6382-9. doi: 10.1021/ac800795f. Epub 2008 Jul 16.}, month = {Aug 15}, pages = {6382-9}, type = {Research Support, N.I.H., ExtramuralResearch Support, U.S. Gov{\textquoteright}t, Non-P.H.S.}, edition = {2008/07/17}, abstract = {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.}, keywords = {*Algorithms, *Databases as Topic, *Software, *Tandem Mass Spectrometry, Biological Markers/analysis/*chemistry, Chromatography, Liquid, Computational Biology/*methods, Databases, Protein, Humans, Molecular Structure, Peptide Mapping/methods, Sequence Analysis, Protein/methods, Serum/*chemistry}, isbn = {1520-6882 (Electronic)0003-2700 (Linking)}, author = {Benton, H. P. and Wong, D. M. and Trauger, S. A. and Siuzdak, G.} } @article {8123, title = {Correlating the transcriptome, proteome, and metabolome in the environmental adaptation of a hyperthermophile}, journal = {J Proteome Res}, volume = {7}, year = {2008}, note = {Trauger, Sunia AKalisak, EwaKalisiak, JaroslawMorita, HirotoshiWeinberg, Michael VMenon, Angeli LalPoole, Farris L 2ndAdams, Michael W WSiuzdak, GaryJ Proteome Res. 2008 Mar;7(3):1027-35. Epub 2008 Feb 2. }, month = {Mar}, pages = {1027-35}, type = {Research Support, U.S. Gov{\textquoteright}t, Non-P.H.S.}, edition = {2008/02/06}, abstract = {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. }, isbn = {1535-3893 (Print)1535-3893 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/18247545}, author = {Trauger, S. A. and Kalisak, E. and Kalisiak, J. and Morita, H. and Weinberg, M. V. and Menon, A. L. and Poole, F. L., 2nd and Adams, M. W. and Siuzdak, G.} } @article {8120, title = {Quantitative ESI-TOF analysis of macromolecular assembly kinetics}, journal = {Anal Chem}, volume = {80}, year = {2008}, note = {Bunner, Anne ETrauger, Sunia ASiuzdak, GaryWilliamson, James RR37 GM053757-13/GM/NIGMS NIH HHS/R37-GM53757/GM/NIGMS NIH HHS/Anal Chem. 2008 Dec 15;80(24):9379-86. }, month = {Dec 15}, pages = {9379-86}, type = {Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov{\textquoteright}t}, edition = {2008/11/15}, abstract = {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{\textquoteright} 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. }, isbn = {1520-6882 (Electronic)0003-2700 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19007188}, author = {Bunner, A. E. and Trauger, S. A. and Siuzdak, G. and Williamson, J. R.} } @article {8122, title = {XCMS2: processing tandem mass spectrometry data for metabolite identification and structural characterization}, journal = {Anal Chem}, volume = {80}, year = {2008}, note = {Benton, H PWong, D MTrauger, S ASiuzdak, GP30 MH062261/MH/NIMH NIH HHS/P30 MH062261-05S1/MH/NIMH NIH HHS/R24 EY017540-02/EY/NEI NIH HHS/R24EY017540/EY/NEI NIH HHS/Anal Chem. 2008 Aug 15;80(16):6382-9. Epub 2008 Jul 16. }, month = {Aug 15}, pages = {6382-9}, type = {Research Support, N.I.H., ExtramuralResearch Support, U.S. Gov{\textquoteright}t, Non-P.H.S.}, edition = {2008/07/17}, abstract = {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. }, isbn = {1520-6882 (Electronic)0003-2700 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/18627180}, author = {Benton, H. P. and Wong, D. M. and Trauger, S. A. and Siuzdak, G.} } @article {269951, title = {A metalloantibody that irreversibly binds a protein antigen}, journal = {J Biol ChemJ Biol ChemJ Biol Chem}, volume = {282}, year = {2007}, note = {Trisler, KirkLooger, Loren LSharma, VikramBaker, MartinBenson, David ETrauger, SuniaSchultz, Peter GSmider, Vaughn VJ Biol Chem. 2007 Sep 7;282(36):26344-53. Epub 2007 Jul 6.}, month = {Sep 7}, pages = {26344-53}, edition = {2007/07/10}, abstract = {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.}, keywords = {*Antibody Affinity/genetics, Antibodies, Monoclonal/*chemistry/diagnostic use/genetics/metabolism/therapeutic, Antigens/*chemistry, Humans, Kinetics, Metals/*chemistry/metabolism, Protein Binding/genetics, Tumor Necrosis Factor-alpha/*chemistry/metabolism, use}, isbn = {0021-9258 (Print)0021-9258 (Linking)}, author = {Trisler, K. and Looger, L. L. and Sharma, V. and Baker, M. and Benson, D. E. and Trauger, S. and Schultz, P. G. and Smider, V. V.} } @article {269846, title = {Selective metabolite and peptide capture/mass detection using fluorous affinity tags}, journal = {J Proteome ResJ Proteome ResJ Proteome Res}, volume = {6}, year = {2007}, note = {Go, Eden PUritboonthai, WilasineeApon, Junefredo VTrauger, Sunia ANordstrom, AndersO{\textquoteright}Maille, GraceBrittain, Scott MPeters, Eric CSiuzdak, GaryMH06226/MH/NIMH NIH HHS/P30 MH062261/MH/NIMH NIH HHS/P30 MH062261-04/MH/NIMH NIH HHS/J Proteome Res. 2007 Apr;6(4):1492-9. Epub 2007 Mar 8.}, month = {Apr}, pages = {1492-9}, type = {Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov{\textquoteright}tResearch Support, U.S. Gov{\textquoteright}t, Non-P.H.S.}, edition = {2007/03/09}, abstract = {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.}, keywords = {Affinity Labels/*chemistry, Amino Acid Sequence, Amino Acids/*blood, Humans, Hydrocarbons, Fluorinated/*chemistry, Mass Spectrometry/*methods, Molecular Sequence Data, Peptides/*analysis, Silicon/chemistry, Surface Properties}, isbn = {1535-3893 (Print)1535-3893 (Linking)}, author = {Go, E. P. and Uritboonthai, W. and Apon, J. V. and Trauger, S. A. and Nordstrom, A. and O{\textquoteright}Maille, G. and Brittain, S. M. and Peters, E. C. and Siuzdak, G.} } @article {8124, title = {A metalloantibody that irreversibly binds a protein antigen}, journal = {J Biol Chem}, volume = {282}, year = {2007}, note = {Trisler, KirkLooger, Loren LSharma, VikramBaker, MartinBenson, David ETrauger, SuniaSchultz, Peter GSmider, Vaughn VJ Biol Chem. 2007 Sep 7;282(36):26344-53. Epub 2007 Jul 6. }, month = {Sep 7}, pages = {26344-53}, edition = {2007/07/10}, abstract = {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. }, isbn = {0021-9258 (Print)0021-9258 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/17617633}, author = {Trisler, K. and Looger, L. L. and Sharma, V. and Baker, M. and Benson, D. E. and Trauger, S. and Schultz, P. G. and Smider, V. V.} } @article {8125, title = {Selective metabolite and peptide capture/mass detection using fluorous affinity tags}, journal = {J Proteome Res}, volume = {6}, year = {2007}, note = {Go, Eden PUritboonthai, WilasineeApon, Junefredo VTrauger, Sunia ANordstrom, AndersO{\textquoteright}Maille, GraceBrittain, Scott MPeters, Eric CSiuzdak, GaryMH06226/MH/NIMH NIH HHS/P30 MH062261-04/MH/NIMH NIH HHS/J Proteome Res. 2007 Apr;6(4):1492-9. Epub 2007 Mar 8. }, month = {Apr}, pages = {1492-9}, type = {Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov{\textquoteright}tResearch Support, U.S. Gov{\textquoteright}t, Non-P.H.S.}, edition = {2007/03/09}, abstract = {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. }, isbn = {1535-3893 (Print)1535-3893 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/17343404}, author = {Go, E. P. and Uritboonthai, W. and Apon, J. V. and Trauger, S. A. and Nordstrom, A. and O{\textquoteright}Maille, G. and Brittain, S. M. and Peters, E. C. and Siuzdak, G.} } @article {269956, title = {Solvent-dependent metabolite distribution, clustering, and protein extraction for serum profiling with mass spectrometry}, journal = {Anal ChemAnal ChemAnal Chem}, volume = {78}, year = {2006}, note = {Want, Elizabeth JO{\textquoteright}Maille, GraceSmith, Colin ABrandon, Theodore RUritboonthai, WilasineeQin, ChuanTrauger, Sunia ASiuzdak, GaryAnal Chem. 2006 Feb 1;78(3):743-52.}, month = {Feb 1}, pages = {743-52}, edition = {2006/02/02}, abstract = {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.}, keywords = {Blood Proteins/*analysis/isolation \& purification, Chromatography, Liquid/methods, Humans, Male, Sensitivity and Specificity, Serum/*chemistry, Solvents/chemistry, Spectrometry, Mass, Electrospray Ionization/*methods}, isbn = {0003-2700 (Print)0003-2700 (Linking)}, author = {Want, E. J. and O{\textquoteright}Maille, G. and Smith, C. A. and Brandon, T. R. and Uritboonthai, W. and Qin, C. and Trauger, S. A. and Siuzdak, G.} } @article {269851, title = {Mass spectrometry reveals specific and global molecular transformations during viral infection}, journal = {J Proteome ResJ Proteome ResJ Proteome Res}, volume = {5}, year = {2006}, note = {Go, Eden PWikoff, William RShen, ZhouxinO{\textquoteright}Maille, GraceMorita, HirotoshiConrads, Thomas PNordstrom, AndersTrauger, Sunia AUritboonthai, WilasineeLucas, David AChan, King CVeenstra, Timothy DLewicki, HannaOldstone, Michael BSchneemann, AnetteSiuzdak, GaryAI036222/AI/NIAID NIH HHS/GM55775/GM/NIGMS NIH HHS/N01-CO-12400/CO/NCI NIH HHS/R01 GM055775/GM/NIGMS NIH HHS/R01 GM055775-02/GM/NIGMS NIH HHS/J Proteome Res. 2006 Sep;5(9):2405-16.}, month = {Sep}, pages = {2405-16}, type = {Comparative StudyResearch Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov{\textquoteright}tResearch Support, U.S. Gov{\textquoteright}t, Non-P.H.S.}, edition = {2006/09/02}, abstract = {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.}, keywords = {*Gene Expression Regulation, Animals, Cells, Cultured, Drosophila melanogaster, HeLa Cells, Humans, Mass Spectrometry/methods, Measles virus/*metabolism, Nodaviridae/*metabolism, Oxygen Isotopes, Proteins/*analysis, Proteomics/*methods, RNA Virus Infections/*metabolism}, isbn = {1535-3893 (Print)1535-3893 (Linking)}, author = {Go, E. P. and Wikoff, W. R. and Shen, Z. and O{\textquoteright}Maille, G. and Morita, H. and Conrads, T. P. and Nordstrom, A. and Trauger, S. A. and Uritboonthai, W. and Lucas, D. A. and Chan, K. C. and Veenstra, T. D. and Lewicki, H. and Oldstone, M. B. and Schneemann, A. and Siuzdak, G.} } @article {8127, title = {Solvent-dependent metabolite distribution, clustering, and protein extraction for serum profiling with mass spectrometry}, journal = {Anal Chem}, volume = {78}, year = {2006}, note = {Want, Elizabeth JO{\textquoteright}Maille, GraceSmith, Colin ABrandon, Theodore RUritboonthai, WilasineeQin, ChuanTrauger, Sunia ASiuzdak, GaryAnal Chem. 2006 Feb 1;78(3):743-52. }, month = {Feb 1}, pages = {743-52}, edition = {2006/02/02}, abstract = {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. }, isbn = {0003-2700 (Print)0003-2700 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/16448047}, author = {Want, E. J. and O{\textquoteright}Maille, G. and Smith, C. A. and Brandon, T. R. and Uritboonthai, W. and Qin, C. and Trauger, S. A. and Siuzdak, G.} } @article {8126, title = {Mass spectrometry reveals specific and global molecular transformations during viral infection}, journal = {J Proteome Res}, volume = {5}, year = {2006}, note = {Go, Eden PWikoff, William RShen, ZhouxinO{\textquoteright}Maille, GraceMorita, HirotoshiConrads, Thomas PNordstrom, AndersTrauger, Sunia AUritboonthai, WilasineeLucas, David AChan, King CVeenstra, Timothy DLewicki, HannaOldstone, Michael BSchneemann, AnetteSiuzdak, GaryAI036222/AI/NIAID NIH HHS/GM55775/GM/NIGMS NIH HHS/N01-CO-12400/CO/NCI NIH HHS/R01 GM055775-02/GM/NIGMS NIH HHS/J Proteome Res. 2006 Sep;5(9):2405-16. }, month = {Sep}, pages = {2405-16}, type = {Comparative StudyResearch Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov{\textquoteright}tResearch Support, U.S. Gov{\textquoteright}t, Non-P.H.S.}, edition = {2006/09/02}, abstract = {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. }, isbn = {1535-3893 (Print)1535-3893 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/16944953}, author = {Go, E. P. and Wikoff, W. R. and Shen, Z. and O{\textquoteright}Maille, G. and Morita, H. and Conrads, T. P. and Nordstrom, A. and Trauger, S. A. and Uritboonthai, W. and Lucas, D. A. and Chan, K. C. and Veenstra, T. D. and Lewicki, H. and Oldstone, M. B. and Schneemann, A. and Siuzdak, G.} } @article {269931, title = {METLIN: a metabolite mass spectral database}, journal = {Ther Drug MonitTher Drug MonitTher Drug Monit}, volume = {27}, year = {2005}, note = {Smith, Colin AO{\textquoteright}Maille, GraceWant, Elizabeth JQin, ChuanTrauger, Sunia ABrandon, Theodore RCustodio, Darlene EAbagyan, RubenSiuzdak, Gary5P30 EY012598-04/EY/NEI NIH HHS/5R24EY01474-04/EY/NEI NIH HHS/Ther Drug Monit. 2005 Dec;27(6):747-51.}, month = {Dec}, pages = {747-51}, type = {Research Support, N.I.H., Extramural}, edition = {2006/01/13}, abstract = {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.}, keywords = {*Databases as Topic, *Mass Spectrometry, Biological Markers/analysis/chemistry/metabolism, Chromatography, Liquid, Humans, Molecular Structure, Pharmaceutical Preparations/analysis/chemistry/metabolism, Spectroscopy, Fourier Transform Infrared}, isbn = {0163-4356 (Print)0163-4356 (Linking)}, author = {Smith, C. A. and O{\textquoteright}Maille, G. and Want, E. J. and Qin, C. and Trauger, S. A. and Brandon, T. R. and Custodio, D. E. and Abagyan, R. and Siuzdak, G.} } @article {8128, title = {METLIN: a metabolite mass spectral database}, journal = {Ther Drug Monit}, volume = {27}, year = {2005}, note = {Smith, Colin AO{\textquoteright}Maille, GraceWant, Elizabeth JQin, ChuanTrauger, Sunia ABrandon, Theodore RCustodio, Darlene EAbagyan, RubenSiuzdak, Gary5P30 EY012598-04/EY/NEI NIH HHS/5R24EY01474-04/EY/NEI NIH HHS/Ther Drug Monit. 2005 Dec;27(6):747-51. }, month = {Dec}, pages = {747-51}, type = {Research Support, N.I.H., Extramural}, edition = {2006/01/13}, abstract = {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. }, isbn = {0163-4356 (Print)0163-4356 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/16404815}, author = {Smith, C. A. and O{\textquoteright}Maille, G. and Want, E. J. and Qin, C. and Trauger, S. A. and Brandon, T. R. and Custodio, D. E. and Abagyan, R. and Siuzdak, G.} } @article {269946, title = {The identification of an adenovirus receptor by using affinity capture and mass spectrometry}, journal = {ChembiochemChembiochemChembiochem}, volume = {5}, year = {2004}, note = {Trauger, Sunia AWu, EugeneBark, Steve JNemerow, Glen RSiuzdak, GaryEY11431/EY/NEI NIH HHS/R01 GM 5-71094/GM/NIGMS NIH HHS/GermanyChembiochem. 2004 Aug 6;5(8):1095-9.}, month = {Aug 6}, pages = {1095-9}, type = {Research Support, Non-U.S. Gov{\textquoteright}tResearch Support, U.S. Gov{\textquoteright}t, P.H.S.}, edition = {2004/08/10}, abstract = {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.}, keywords = {Antigens, CD/metabolism, Chromatography, Affinity/*methods, Electrophoresis, Polyacrylamide Gel, Flow Cytometry, Nanotechnology, Receptors, Virus/*chemistry/metabolism, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/*methods}, isbn = {1439-4227 (Print)1439-4227 (Linking)}, author = {Trauger, S. A. and Wu, E. and Bark, S. J. and Nemerow, G. R. and Siuzdak, G.} } @article {269971, title = {Membrane cofactor protein is a receptor for adenoviruses associated with epidemic keratoconjunctivitis}, journal = {J VirolJ VirolJ Virol}, volume = {78}, year = {2004}, note = {Wu, EugeneTrauger, Sunia APache, LarsMullen, Tina-Marievon Seggern, Daniel JSiuzdak, GaryNemerow, Glen REY11431/EY/NEI NIH HHS/HL54352/HL/NHLBI NIH HHS/R01GM5-71094/GM/NIGMS NIH HHS/J Virol. 2004 Apr;78(8):3897-905.}, month = {Apr}, pages = {3897-905}, type = {Research Support, Non-U.S. Gov{\textquoteright}tResearch Support, U.S. Gov{\textquoteright}t, Non-P.H.S.Research Support, U.S. Gov{\textquoteright}t, P.H.S.}, edition = {2004/03/30}, abstract = {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.}, keywords = {Adenoviridae Infections/*etiology/virology, Adenoviruses, Human/classification/genetics/*pathogenicity, Amino Acid Sequence, Animals, Antigens, CD/chemistry/genetics/*physiology, Antigens, CD46, Base Sequence, Cell Line, CHO Cells, Cricetinae, DNA, Viral/genetics, HeLa Cells, Humans, Keratoconjunctivitis/*etiology/virology, Membrane Glycoproteins/chemistry/genetics/*physiology, Molecular Sequence Data, Receptors, Virus/chemistry/genetics/*physiology}, isbn = {0022-538X (Print)0022-538X (Linking)}, author = {Wu, E. and Trauger, S. A. and Pache, L. and Mullen, T. M. and von Seggern, D. J. and Siuzdak, G. and Nemerow, G. R.} } @article {269926, title = {Assignment of endogenous substrates to enzymes by global metabolite profiling}, journal = {BiochemistryBiochemistryBiochemistry}, volume = {43}, year = {2004}, note = {Saghatelian, AlanTrauger, Sunia AWant, Elizabeth JHawkins, Edward GSiuzdak, GaryCravatt, Benjamin FDA015197/DA/NIDA NIH HHS/DA017259/DA/NIDA NIH HHS/P01 DA017259/DA/NIDA NIH HHS/P01 DA017259-01/DA/NIDA NIH HHS/R01 DA015197/DA/NIDA NIH HHS/R01 DA015197-02/DA/NIDA NIH HHS/Biochemistry. 2004 Nov 16;43(45):14332-9.}, month = {Nov 16}, pages = {14332-9}, type = {Comparative StudyResearch Support, Non-U.S. Gov{\textquoteright}tResearch Support, U.S. Gov{\textquoteright}t, P.H.S.}, edition = {2004/11/10}, abstract = {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{\textquoteright}s unique biochemical functions in vivo.}, keywords = {Amidohydrolases/chemistry/deficiency/*metabolism, Animals, Brain/enzymology/metabolism, Chromatography, Liquid/methods/standards, Ethanolamines/metabolism, Fatty Acids/chemistry/*metabolism, Hydrolysis, Mass Spectrometry/methods/standards, Mice, Mice, Knockout, Predictive Value of Tests, Spinal Cord/enzymology/metabolism, Substrate Specificity, Taurine/metabolism}, isbn = {0006-2960 (Print)0006-2960 (Linking)}, author = {Saghatelian, A. and Trauger, S. A. and Want, E. J. and Hawkins, E. G. and Siuzdak, G. and Cravatt, B. F.} } @article {269936, title = {High sensitivity and analyte capture with desorption/ionization mass spectrometry on silylated porous silicon}, journal = {Anal ChemAnal ChemAnal Chem}, volume = {76}, year = {2004}, note = {Trauger, Sunia AGo, Eden PShen, ZhouxinApon, Junefredo VCompton, Bruce JBouvier, Edouard S PFinn, M GSiuzdak, Gary15066/PHS HHS/Anal Chem. 2004 Aug 1;76(15):4484-9.}, month = {Aug 1}, pages = {4484-9}, type = {Research Support, N.I.H., Extramural}, edition = {2004/07/31}, abstract = {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.}, keywords = {Amino Acids/blood/isolation \& purification, Animals, Cattle, Hemoglobins/chemistry, Indicators and Reagents, Sensitivity and Specificity, Serum Albumin, Bovine/chemistry, Silicon Dioxide, Silicon/*chemistry, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/*methods}, isbn = {0003-2700 (Print)0003-2700 (Linking)}, author = {Trauger, S. A. and Go, E. P. and Shen, Z. and Apon, J. V. and Compton, B. J. and Bouvier, E. S. and Finn, M. G. and Siuzdak, G.} } @article {8131, title = {High sensitivity and analyte capture with desorption/ionization mass spectrometry on silylated porous silicon}, journal = {Anal Chem}, volume = {76}, year = {2004}, note = {Trauger, Sunia AGo, Eden PShen, ZhouxinApon, Junefredo VCompton, Bruce JBouvier, Edouard S PFinn, M GSiuzdak, Gary15066/PHS HHS/Anal Chem. 2004 Aug 1;76(15):4484-9. }, month = {Aug 1}, pages = {4484-9}, type = {Research Support, N.I.H., Extramural}, edition = {2004/07/31}, abstract = {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. }, isbn = {0003-2700 (Print)0003-2700 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/15283591}, author = {Trauger, S. A. and Go, E. P. and Shen, Z. and Apon, J. V. and Compton, B. J. and Bouvier, E. S. and Finn, M. G. and Siuzdak, G.} } @article {8130, title = {The identification of an adenovirus receptor by using affinity capture and mass spectrometry}, journal = {Chembiochem}, volume = {5}, year = {2004}, note = {Trauger, Sunia AWu, EugeneBark, Steve JNemerow, Glen RSiuzdak, GaryEY11431/EY/NEI NIH HHS/R01 GM 5-71094/GM/NIGMS NIH HHS/GermanyChembiochem. 2004 Aug 6;5(8):1095-9. }, month = {Aug 6}, pages = {1095-9}, type = {Research Support, Non-U.S. Gov{\textquoteright}tResearch Support, U.S. Gov{\textquoteright}t, P.H.S.}, edition = {2004/08/10}, abstract = {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. }, isbn = {1439-4227 (Print)1439-4227 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/15300833}, author = {Trauger, S. A. and Wu, E. and Bark, S. J. and Nemerow, G. R. and Siuzdak, G.} } @article {8132, title = {Membrane cofactor protein is a receptor for adenoviruses associated with epidemic keratoconjunctivitis}, journal = {J Virol}, volume = {78}, year = {2004}, note = {Wu, EugeneTrauger, Sunia APache, LarsMullen, Tina-Marievon Seggern, Daniel JSiuzdak, GaryNemerow, Glen REY11431/EY/NEI NIH HHS/HL54352/HL/NHLBI NIH HHS/R01GM5-71094/GM/NIGMS NIH HHS/J Virol. 2004 Apr;78(8):3897-905. }, month = {Apr}, pages = {3897-905}, type = {Research Support, Non-U.S. Gov{\textquoteright}tResearch Support, U.S. Gov{\textquoteright}t, Non-P.H.S.Research Support, U.S. Gov{\textquoteright}t, P.H.S.}, edition = {2004/03/30}, abstract = {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. }, isbn = {0022-538X (Print)0022-538X (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/15047806}, author = {Wu, E. and Trauger, S. A. and Pache, L. and Mullen, T. M. and von Seggern, D. J. and Siuzdak, G. and Nemerow, G. R.} } @article {8129, title = {Assignment of endogenous substrates to enzymes by global metabolite profiling}, journal = {Biochemistry}, volume = {43}, year = {2004}, note = {Saghatelian, AlanTrauger, Sunia AWant, Elizabeth JHawkins, Edward GSiuzdak, GaryCravatt, Benjamin FDA015197/DA/NIDA NIH HHS/DA017259/DA/NIDA NIH HHS/P01 DA017259-01/DA/NIDA NIH HHS/R01 DA015197-02/DA/NIDA NIH HHS/Biochemistry. 2004 Nov 16;43(45):14332-9. }, month = {Nov 16}, pages = {14332-9}, type = {Comparative StudyResearch Support, Non-U.S. Gov{\textquoteright}tResearch Support, U.S. Gov{\textquoteright}t, P.H.S.}, edition = {2004/11/10}, abstract = {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{\textquoteright}s unique biochemical functions in vivo. }, isbn = {0006-2960 (Print)0006-2960 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/15533037}, author = {Saghatelian, A. and Trauger, S. A. and Want, E. J. and Hawkins, E. G. and Siuzdak, G. and Cravatt, B. F.} } @article {269806, title = {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}, journal = {Environ Toxicol ChemEnviron Toxicol ChemEnviron Toxicol Chem}, volume = {21}, year = {2002}, note = {Bundy, Jacob GLenz, Eva MBailey, Nigel JGavaghan, Claire LSvendsen, ClausSpurgeon, DavidHankard, Peter KOsborn, DanielWeeks, Jason MTrauger, Sunia ASpeir, PaulSanders, IanLindon, John CNicholson, Jeremy KTang, HuiruEnviron Toxicol Chem. 2002 Sep;21(9):1966-72.}, month = {Sep}, pages = {1966-72}, type = {Research Support, Non-U.S. Gov{\textquoteright}t}, edition = {2002/09/11}, abstract = {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.}, keywords = {*Oligochaeta, Aniline Compounds/*toxicity, Animals, Biological Markers/*analysis, Chromatography, High Pressure Liquid, Inosine Monophosphate/analysis, Magnetic Resonance Spectroscopy, Maltose/analysis, Xenobiotics/*toxicity}, isbn = {0730-7268 (Print)0730-7268 (Linking)}, author = {Bundy, J. G. and Lenz, E. M. and Bailey, N. J. and Gavaghan, C. L. and Svendsen, C. and Spurgeon, D. and Hankard, P. K. and Osborn, D. and Weeks, J. M. and Trauger, S. A. and Speir, P. and Sanders, I. and Lindon, J. C. and Nicholson, J. K. and Tang, H.} } @article {8133, title = {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}, journal = {Environ Toxicol Chem}, volume = {21}, year = {2002}, note = {Bundy, Jacob GLenz, Eva MBailey, Nigel JGavaghan, Claire LSvendsen, ClausSpurgeon, DavidHankard, Peter KOsborn, DanielWeeks, Jason MTrauger, Sunia ASpeir, PaulSanders, IanLindon, John CNicholson, Jeremy KTang, HuiruEnviron Toxicol Chem. 2002 Sep;21(9):1966-72. }, month = {Sep}, pages = {1966-72}, type = {Research Support, Non-U.S. Gov{\textquoteright}t}, edition = {2002/09/11}, abstract = {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. }, isbn = {0730-7268 (Print)0730-7268 (Linking)}, url = {http://www.ncbi.nlm.nih.gov/pubmed/12206438}, author = {Bundy, J. G. and Lenz, E. M. and Bailey, N. J. and Gavaghan, C. L. and Svendsen, C. and Spurgeon, D. and Hankard, P. K. and Osborn, D. and Weeks, J. M. and Trauger, S. A. and Speir, P. and Sanders, I. and Lindon, J. C. and Nicholson, J. K. and Tang, H.} }