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Utilizing Untargeted Ion Mobility-Mass Spectrometry To Profile Changes in the Gut Metabolome Following Biliary Diversion Surgery.
Poland JC, Schrimpe-Rutledge AC, Sherrod SD, Flynn CR, McLean JA
(2019) Anal Chem 91: 14417-14423
MeSH Terms: Anastomosis, Surgical, Animals, Bile Acids and Salts, Bile Ducts, Cholesterol, Chromatography, Liquid, Duodenum, Fatty Acids, Volatile, Feces, Gastrointestinal Microbiome, Ileum, Jejunum, Male, Mass Spectrometry, Mice, Inbred C57BL
Show Abstract · Added December 17, 2019
Obesity and obesity-related disorders are a global epidemic affecting over 10% of the world's population. Treatment of these diseases has become increasingly challenging and expensive. The most effective and durable treatment for Class III obesity (body mass index ≥35 kg/m) is bariatric surgery, namely, Roux-en-Y gastric bypass (RYGB) and vertical sleeve gastrectomy. These procedures are associated with increased circulating bile acids, molecules that not only facilitate intestinal fat absorption but are also potent hormones regulating numerous metabolic pathways. We recently reported on a novel surgical procedure in mice, termed distal gallbladder bile diversion to the ileum (GB-IL), that emulates the altered bile flow after RYGB without other manipulations of gastrointestinal anatomy. GB-IL improves oral glucose tolerance in mice made obese with high-fat diet. This is accompanied by fat malabsorption and weight loss, which complicates studying the role of elevated circulating bile acids in metabolic control. A less aggressive surgery in which the gallbladder bile is diverted to the proximal ileum, termed GB-IL, also improves glucose control but is not accompanied by fat malabsorption. To better understand the differential effects achieved by these bile diversion procedures, an untargeted ultraperformance liquid chromatography-ion mobility-mass spectrometry (UPLC-IM-MS) method was optimized for fecal samples derived from mice that have undergone bile diversion surgery. Utilizing the UPLC-IM-MS method, we were able to identify dysregulation of bile acids, short-chain fatty acids, and cholesterol derivatives that contribute to the differential metabolism resulting from these surgeries.
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15 MeSH Terms
Picturing Polarized Myeloid Phagocytes and Regulatory Cells by Mass Cytometry.
Roussel M, Bartkowiak T, Irish JM
(2019) Methods Mol Biol 1989: 217-226
MeSH Terms: Cytokines, Dendritic Cells, Flow Cytometry, Humans, Mass Spectrometry, Monocytes, Myeloid-Derived Suppressor Cells, Phagocytes, Phenotype, Single-Cell Analysis
Show Abstract · Added May 13, 2019
The immune monocyte/phagocyte system (MPS) includes numerous cell subsets of the myeloid lineage including monocyte, macrophage, and dendritic cell (DC) populations that are heterogeneous both phenotypically and functionally. Previously, we characterized these diverse MPS phenotypes with multi-parametric mass cytometry (CyTOF). In order to expansively characterize monocytes, macrophages, and dendritic cells, a CyTOF panel was designed to measure 35 identity-, activation-, and polarization-markers. Here we provide a protocol to define a reference map for the myeloid compartment, including sample preparation, to produce reference cell subsets from the monocyte/phagocyte system. In particular, we focused on monocyte-derived macrophages that were further polarized in vitro with cytokine stimulation (i.e., M-CSF, GM-CSF, IL-4, IL-10, IFNγ, and LPS), as well as monocyte-derived DCs, and myeloid-derived suppressor cells (MDSCs), generated in vitro from human bone marrow and/or peripheral blood.
3 Communities
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10 MeSH Terms
Multi-metal Restriction by Calprotectin Impacts De Novo Flavin Biosynthesis in Acinetobacter baumannii.
Wang J, Lonergan ZR, Gonzalez-Gutierrez G, Nairn BL, Maxwell CN, Zhang Y, Andreini C, Karty JA, Chazin WJ, Trinidad JC, Skaar EP, Giedroc DP
(2019) Cell Chem Biol 26: 745-755.e7
MeSH Terms: Acinetobacter baumannii, Bacterial Proteins, Chromatography, High Pressure Liquid, Flavins, Heat-Shock Proteins, Iron, Leukocyte L1 Antigen Complex, Metallochaperones, Proteome, Tandem Mass Spectrometry, Zinc
Show Abstract · Added March 26, 2019
Calprotectin (CP) inhibits bacterial viability through extracellular chelation of transition metals. However, how CP influences general metabolism remains largely unexplored. We show here that CP restricts bioavailable Zn and Fe to the pathogen Acinetobacter baumannii, inducing an extensive multi-metal perturbation of cellular physiology. Proteomics reveals severe metal starvation, and a strain lacking the candidate Zn metallochaperone ZigA possesses altered cellular abundance of multiple essential Zn-dependent enzymes and enzymes in de novo flavin biosynthesis. The ΔzigA strain exhibits decreased cellular flavin levels during metal starvation. Flavin mononucleotide provides regulation of this biosynthesis pathway, via a 3,4-dihydroxy-2-butanone 4-phosphate synthase (RibB) fusion protein, RibBX, and authentic RibB. We propose that RibBX ensures flavin sufficiency under CP-induced Fe limitation, allowing flavodoxins to substitute for Fe-ferredoxins as cell reductants. These studies elucidate adaptation to nutritional immunity and define an intersection between metallostasis and cellular metabolism in A. baumannii.
Copyright © 2019 Elsevier Ltd. All rights reserved.
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11 MeSH Terms
Isomeric and Conformational Analysis of Small Drug and Drug-Like Molecules by Ion Mobility-Mass Spectrometry (IM-MS).
Phillips ST, Dodds JN, May JC, McLean JA
(2019) Methods Mol Biol 1939: 161-178
MeSH Terms: Algorithms, Amino Acids, Carbohydrates, Ion Mobility Spectrometry, Isomerism, Mass Spectrometry, Molecular Conformation, Pharmaceutical Preparations, Small Molecule Libraries, Software
Show Abstract · Added August 7, 2019
This chapter provides a broad overview of ion mobility-mass spectrometry (IM-MS) and its applications in separation science, with a focus on pharmaceutical applications. A general overview of fundamental ion mobility (IM) theory is provided with descriptions of several contemporary instrument platforms which are available commercially (i.e., drift tube and traveling wave IM). Recent applications of IM-MS toward the evaluation of structural isomers are highlighted and placed in the context of both a separation and characterization perspective. We conclude this chapter with a guided reference protocol for obtaining routine IM-MS spectra on a commercially available uniform-field IM-MS.
1 Communities
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Ion mobility conformational lipid atlas for high confidence lipidomics.
Leaptrot KL, May JC, Dodds JN, McLean JA
(2019) Nat Commun 10: 985
MeSH Terms: Animals, Databases, Chemical, Lipids, Mass Spectrometry, Metabolomics, Molecular Conformation, Molecular Structure
Show Abstract · Added August 7, 2019
Lipids are highly structurally diverse molecules involved in a wide variety of biological processes. Here, we use high precision ion mobility-mass spectrometry to compile a structural database of 456 mass-resolved collision cross sections (CCS) of sphingolipid and glycerophospholipid species. Our CCS database comprises sphingomyelin, cerebroside, ceramide, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidic acid classes. Primary differences observed are between lipid categories, with sphingolipids exhibiting 2-6% larger CCSs than glycerophospholipids of similar mass, likely a result of the sphingosine backbone's restriction of the sn1 tail length, limiting gas-phase packing efficiency. Acyl tail length and degree of unsaturation are found to be the primary structural descriptors determining CCS magnitude, with degree of unsaturation being four times as influential per mass unit. The empirical CCS values and previously unmapped quantitative structural trends detailed in this work are expected to facilitate prediction of CCS in broadscale lipidomics research.
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Synthesis and Characterization of Site-Specific O -Alkylguanine DNA-Alkyl Transferase-Oligonucleotide Crosslinks.
Ghodke PP, Albertolle ME, Johnson KM, Guengerich FP
(2019) Curr Protoc Nucleic Acid Chem 76: e74
MeSH Terms: Catalysis, Catalytic Domain, Chromatography, Liquid, Copper, Cross-Linking Reagents, Escherichia coli, O(6)-Methylguanine-DNA Methyltransferase, Oligonucleotides, Polymerization, Tandem Mass Spectrometry, Templates, Genetic, Trypsin
Show Abstract · Added March 3, 2020
O -Alkylguanine DNA-alkyltransferase (AGT), a DNA repair protein, can form crosslinks with DNA. The AGT-DNA crosslinks are known to be mutagenic when AGT is heterologously expressed in Escherichia coli, as well as in mammalian cells. To understand the biological consequences, reliable access to AGT-oligonucleotide crosslinks is needed. This article describes the synthesis and characterization of site-specific AGT-oligonucleotide crosslinks at the N2-position of deoxyguanosine and N6-position of deoxyadenosine. We developed a post-oligomerization strategy for the synthesis of propargyl-modified oligonucleotides. Copper-catalyzed azide-alkyne cycloaddition was used as a key step to obtain the iodoacetamide-linked oligonucleotides, which serve as good electrophiles for the crosslinking reaction with cysteine-145 of the active site of AGT. Trypsinization of AGT and hydrolysis of oligonucleotides, combined with analysis by liquid chromatography-tandem mass spectrometry, was utilized to confirm the nucleobase-adducted peptides. This method provides a useful strategy for the synthesis and characterization of site-specific DNA-protein crosslinks, which can be further used to understand proteolytic degradation-coupled DNA repair mechanisms. © 2019 by John Wiley & Sons, Inc.
© 2019 John Wiley & Sons, Inc.
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Quantitative Proteomic Analysis of Small and Large Extracellular Vesicles (EVs) Reveals Enrichment of Adhesion Proteins in Small EVs.
Jimenez L, Yu H, McKenzie AJ, Franklin JL, Patton JG, Liu Q, Weaver AM
(2019) J Proteome Res 18: 947-959
MeSH Terms: Cell Adhesion, Cell Adhesion Molecules, Cell Line, Tumor, Chromatography, Liquid, Exosomes, Extracellular Vesicles, Humans, Particle Size, Proteomics, Tandem Mass Spectrometry
Show Abstract · Added April 2, 2019
Extracellular vesicles (EVs) are important mediators of cell-cell communication due to their cargo content of proteins, lipids, and RNAs. We previously reported that small EVs (SEVs) called exosomes promote directed and random cell motility, invasion, and serum-independent growth. In contrast, larger EVs (LEVs) were not active in those assays, but might have unique functional properties. In order to identify protein cargos that may contribute to different functions of SEVs and LEVs, we used isobaric tags for relative and absolute quantitation (iTRAQ)-liquid chromatography (LC) tandem mass spectrometry (MS) on EVs isolated from a colon cancer cell line. Bioinformatics analyses revealed that SEVs are enriched in proteins associated with cell-cell junctions, cell-matrix adhesion, exosome biogenesis machinery, and various signaling pathways. In contrast, LEVs are enriched in proteins associated with ribosome and RNA biogenesis, processing, and metabolism. Western blot analysis of EVs purified from two different cancer cell types confirmed the enrichment of cell-matrix and cell-cell adhesion proteins in SEVs. Consistent with those data, we found that cells exhibit enhanced adhesion to surfaces coated with SEVs compared to an equal protein concentration of LEVs. These data suggest that a major function of SEVs is to promote cellular adhesion.
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10 MeSH Terms
Beyond the message: advantages of snapshot proteomics with single-cell mass cytometry in solid tumors.
Mistry AM, Greenplate AR, Ihrie RA, Irish JM
(2019) FEBS J 286: 1523-1539
MeSH Terms: Humans, Image Cytometry, Mass Spectrometry, Neoplasms, Precision Medicine, Proteins, Proteomics, Sensitivity and Specificity, Signal Transduction, Single-Cell Analysis, Tumor Microenvironment
Show Abstract · Added December 16, 2018
Single-cell technologies that can quantify features of individual cells within a tumor are critical for treatment strategies aiming to target cancer cells while sparing or activating beneficial cells. Given that key players in protein networks are often the primary targets of precision oncology strategies, it is imperative to transcend the nucleic acid message and read cellular actions in human solid tumors. Here, we review the advantages of multiplex, single-cell mass cytometry in tissue and solid tumor investigations. Mass cytometry can quantitatively probe nearly any cellular feature or target. In discussing the ability of mass cytometry to reveal and characterize a broad spectrum of cell types, identify rare cells, and study functional behavior through protein signaling networks in millions of individual cells from a tumor, this review surveys publications of scientific advances in solid tumor biology made with the aid of mass cytometry. Advances discussed include functional identification of rare tumor and tumor-infiltrating immune cells and dissection of cellular mechanisms of immunotherapy in solid tumors and the periphery. The review concludes by highlighting ways to incorporate single-cell mass cytometry in solid tumor precision oncology efforts and rapidly developing cytometry techniques for quantifying cell location and sequenced nucleic acids.
© 2018 Federation of European Biochemical Societies.
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11 MeSH Terms
Human mAbs to Staphylococcus aureus IsdA Provide Protection Through Both Heme-Blocking and Fc-Mediated Mechanisms.
Bennett MR, Bombardi RG, Kose N, Parrish EH, Nagel MB, Petit RA, Read TD, Schey KL, Thomsen IP, Skaar EP, Crowe JE
(2019) J Infect Dis 219: 1264-1273
MeSH Terms: Animals, Antibodies, Monoclonal, Antigens, Bacterial, Bacterial Proteins, Disease Models, Animal, Female, Hemeproteins, Humans, Hydrogen Deuterium Exchange-Mass Spectrometry, Mice, Mice, Inbred BALB C, Receptors, Cell Surface, Staphylococcal Infections, Staphylococcus aureus
Show Abstract · Added March 31, 2019
The nutrient metal iron plays a key role in the survival of microorganisms. The iron-regulated surface determinant (Isd) system scavenges heme-iron from the human host, enabling acquisition of iron in iron-deplete conditions in Staphylococcus aureus during infection. The cell surface receptors IsdB and IsdH bind hemoproteins and transfer heme to IsdA, the final surface protein before heme-iron is transported through the peptidoglycan. To define the human B-cell response to IsdA, we isolated human monoclonal antibodies (mAbs) specific to the surface Isd proteins and determined their mechanism of action. We describe the first isolation of fully human IsdA and IsdH mAbs, as well as cross-reactive Isd mAbs. Two of the identified IsdA mAbs worked in a murine septic model of infection to reduce bacterial burden during staphylococcal infection. Their protection was a result of both heme-blocking and Fc-mediated effector functions, underscoring the importance of targeting S. aureus using diverse mechanisms.
© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.
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14 MeSH Terms
Proteomic Analysis of S-Palmitoylated Proteins in Ocular Lens Reveals Palmitoylation of AQP5 and MP20.
Wang Z, Schey KL
(2018) Invest Ophthalmol Vis Sci 59: 5648-5658
MeSH Terms: Animals, Aquaporin 5, Blotting, Western, Cattle, Chromatography, Liquid, Electrophoresis, Polyacrylamide Gel, Eye Proteins, Immunoblotting, Lens, Crystalline, Lipoylation, Membrane Proteins, Palmitates, Proteomics, Tandem Mass Spectrometry
Show Abstract · Added April 4, 2019
Purpose - The purpose of this study was to characterize the palmitoyl-proteome in lens fiber cells. S-palmitoylation is the most common form of protein S-acylation and the reversible nature of this modification functions as a molecular switch to regulate many biological processes. This modification could play important roles in regulating protein functions and protein-protein interactions in the lens.
Methods - The palmitoyl-proteome of bovine lens fiber cells was investigated by combining acyl-biotin exchange (ABE) chemistry and mass-spectrometry analysis. Due to the possibility of false-positive results from ABE experiment, a method was also developed for direct detection of palmitoylated peptides by mass spectrometry for validating palmitoylation of lens proteins MP20 and AQP5. Palmitoylation levels on AQP5 in different regions of the lens were quantified after iodoacetamide (IAA)-palmitate exchange.
Results - The ABE experiment identified 174 potential palmitoylated proteins. These proteins include 39 well-characterized palmitoylated proteins, 92 previously reported palmitoylated proteins in other tissues, and 43 newly identified potential palmitoylated proteins including two important transmembrane proteins in the lens, AQP5 and MP20. Further analysis by direct detection of palmitoylated peptides confirmed palmitoylation of AQP5 on C6 and palmitoylation of MP20 on C159. Palmitoylation of AQP5 was found to only occur in a narrow region of the inner lens cortex and does not occur in the lens epithelium, in the lens outer cortex, or in the lens nucleus.
Conclusions - AQP5 and MP20 are among 174 palmitoylated proteins found in bovine lens fiber cells. This modification to AQP5 and MP20 may play a role in their translocation from the cytoplasm to cell membranes during fiber cell differentiation.
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14 MeSH Terms