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  • mass spectrometry  (5)
  • 1
    Keywords: brain ; RECEPTOR ; CELLS ; Germany ; NETWORKS ; SYSTEM ; TOOL ; DISTINCT ; PROTEIN ; PROTEINS ; TRANSDUCTION ; COMPLEX ; MESSENGER-RNA ; RAT ; signal transduction ; MEMBRANE ; SIGNAL-TRANSDUCTION ; mass spectrometry ; MASS-SPECTROMETRY ; CHROMATOGRAPHY ; PROTEOMIC ANALYSIS ; glutathione-S-transferase ; BINDING PROTEIN ; signaling ; molecular ; NEURONS ; analysis ; cilia ; ENGLAND ; XENOBIOTIC-METABOLIZING ENZYMES ; affinity chromatography ; calcium-calmodulin ; CHEMOSENSORY CILIA ; NUCLEOTIDE-GATED CHANNEL ; olfaction ; olfactory receptor neurons ; PHOSPHOLIPID-BINDING ; SENSORY NEURONS
    Abstract: The olfactory neuroepithelium represents a unique interface between the brain and the external environment. Olfactory function comprises a distinct set of molecular tasks: sensory signal transduction, cytoprotection and adult neurogenesis. A multitude of biochemical studies has revealed the central role of Ca2+ signaling in the function of olfactory receptor neurons (ORNs). We set out to establish Ca2+-dependent signaling networks in ORN cilia by proteomic analysis. We subjected a ciliary membrane preparation to Ca2+/calmodulin-affinity chromatography using mild detergent conditions in order to maintain functional protein complexes involved in olfactory Ca2+ signaling. Thus, calmodulin serves as a valuable tool to gain access to novel Ca2+-regulated protein complexes. Tandem mass spectrometry (nanoscale liquid-chromatography-electrospray injection) identified 123 distinct proteins. Ninety-seven proteins (79%) could be assigned to specific olfactory functions, including 32 to sensory signal transduction and 40 to cytoprotection. We point out novel perspectives for research on the Ca2+-signaling networks in the olfactory system of the rat. (C) 2007 IBRO. Published by Elsevier Ltd. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 18155848
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  • 2
    Keywords: RECEPTOR ; EXPRESSION ; Germany ; INFORMATION ; SUPPORT ; TOOL ; SITE ; PROTEIN ; PROTEINS ; TRANSDUCTION ; MECHANISM ; primary ; RAT ; mechanisms ; signal transduction ; MEMBRANE ; SIGNAL-TRANSDUCTION ; mass spectrometry ; MASS-SPECTROMETRY ; LOCALIZATION ; RECEPTORS ; FOOD ; protein expression ; PROTEOMIC ANALYSIS ; POLYACRYLAMIDE GELS ; NEURONS ; analysis ; TECHNOLOGY ; EPITHELIUM ; ENGLAND ; enzymatic ; XENOBIOTIC-METABOLIZING ENZYMES ; CHEMOSENSORY CILIA ; NUCLEOTIDE-GATED CHANNEL ; olfactory receptor neurons ; ODORANT RECEPTORS ; SENSITIVE ADENYLATE-CYCLASE ; sensory cilia
    Abstract: The cilia of mammalian olfactory receptor neurons (ORNs) represent the sensory interface that is exposed to the air within the nasal cavity. The cilia are the site where odorants bind to specific receptors and initiate olfactory transduction that leads to excitation of the neuron. This process involves a multitude of ciliary proteins that mediate chemoelectrical transduction, amplification, and adaptation of the primary sensory signal. Many of these proteins were initially identified by their enzymatic activities using a membrane protein preparation from olfactory cilia. This so-called "calcium-shock" preparation is a versatile tool for the exploration of protein expression, enzyme kinetics, regulatory mechanisms, and ciliary development. To support such studies, we present a first proteomic analysis of this membrane preparation. We subjected the cilia preparation to liquid chromatography-electrospray ionisation (LC-ESI-MS/MS) tandem mass spectrometry and identified 268 proteins, of which 49% are membrane proteins. A detailed analysis of their cellular and subcellular localization showed that the cilia preparation obtained by calcium shock not only is highly enriched in ORN proteins but also contains a significant amount of nonciliary material. Although our proteomic study does not identify the entire set of ciliary and nonciliary proteins, it provides the first estimate of the purity of the calcium-shock preparation and provides valuable biochemical information for further research
    Type of Publication: Journal article published
    PubMed ID: 18032372
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  • 3
    Keywords: RECEPTOR ; Germany ; PATHWAY ; PROTEIN ; PROTEINS ; COMPLEX ; RAT ; signal transduction ; SIGNAL ; MOUSE ; MEMBRANE ; SIGNAL-TRANSDUCTION ; mass spectrometry ; PROTEOMIC ANALYSIS ; signaling ; NEURONS ; EPITHELIUM ; NUCLEOTIDE-GATED CHANNEL ; olfactory receptor neurons ; sensory cilia ; ADENYLYL-CYCLASE ; CHLORIDE ACCUMULATION ; MAMMALIAN ODORANT RECEPTORS ; ORGANELLAR PROTEOMICS
    Abstract: Olfactory sensory neurons expose to the inhaled air chemosensory cilia which bind odorants and operate as transduction organelles. Odorant receptors in the ciliary membrane activate a transduction cascade which uses cAMP and Ca2+ for sensory signaling in the ciliary lumen. Although the canonical transduction pathway is well established, molecular components for more complex aspects of sensory transduction, like adaptation, regulation, and termination of the receptor response have not been systematically identified. Moreover, open questions in olfactory physiology include how the cilia exchange solutes with the surrounding mucus, assemble their highly polarized set of proteins, and cope with noxious substances in the ambient air. A specific ciliary proteome would promote research efforts in all of these fields. We have improved a method to detach cilia from rat olfactory sensory neurons and have isolated a preparation specifically enriched in ciliary membrane proteins. Using LC-ESI-MS/MS analysis, we identified 377 proteins which constitute the olfactory cilia proteome. These proteins represent a comprehensive data set for olfactory research since more than 80% can be attributed to the characteristic functions of olfactory sensory neurons and their cilia: signal processing, protein targeting, neurogenesis, solute transport, and cytoprotection. Organellar proteomics thus yielded decisive information about the diverse physiological functions of a sensory organelle
    Type of Publication: Journal article published
    PubMed ID: 19086097
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  • 4
    Keywords: EXPRESSION ; Germany ; MODEL ; INFORMATION ; SYSTEM ; GENE ; GENE-EXPRESSION ; GENOME ; PROTEIN ; PROTEINS ; RESOLUTION ; MECHANISM ; FAMILY ; DOMAIN ; mechanisms ; TOLERANCE ; CYCLE ; SEQUENCE ; IDENTIFICATION ; gene expression ; HEAT-SHOCK ; mass spectrometry ; SPECTROMETRY ; DATABASE ; MASS-SPECTROMETRY ; PROJECT ; PROTEOMICS ; PROTEIN IDENTIFICATION ; ARABIDOPSIS-THALIANA ; HIGH-RESOLUTION ; ANNOTATION ; SCIENCE ; LIFE ; MOLECULAR-MECHANISMS ; GLUTATHIONE S-TRANSFERASES ; Genetic ; protein extraction ; MILNESIUM-TARDIGRADUM ; RICHTERSIUS-CORONIFER ; ARTEMIA-FRANCISCANA ; DESICCATION TOLERANCE ; EST ; Sequence information ; Molecular mechanisms ; BRINE SHRIMP ; TREHALOSE
    Abstract: Background: Tardigrades are small, multicellular invertebrates which are able to survive times of unfavourable environmental conditions using their well-known capability to undergo cryptobiosis at any stage of their life cycle. Milnesium tardigradum has become a powerful model system for the analysis of cryptobiosis. While some genetic information is already available for Milnesium tardigradum the proteome is still to be discovered. Principal Findings: Here we present to the best of our knowledge the first comprehensive study of Milnesium tardigradum on the protein level. To establish a proteome reference map we developed optimized protocols for protein extraction from tardigrades in the active state and for separation of proteins by high resolution two-dimensional gel electrophoresis. Since only limited sequence information of M. tardigradum on the genome and gene expression level is available to date in public databases we initiated in parallel a tardigrade EST sequencing project to allow for protein identification by electrospray ionization tandem mass spectrometry. 271 out of 606 analyzed protein spots could be identified by searching against the publicly available NCBInr database as well as our newly established tardigrade protein database corresponding to 144 unique proteins. Another 150 spots could be identified in the tardigrade clustered EST database corresponding to 36 unique contigs and ESTs. Proteins with annotated function were further categorized in more detail by their molecular function, biological process and cellular component. For the proteins of unknown function more information could be obtained by performing a protein domain annotation analysis. Our results include proteins like protein member of different heat shock protein families and LEA group 3, which might play important roles in surviving extreme conditions. Conclusions: The proteome reference map of Milnesium tardigradum provides the basis for further studies in order to identify and characterize the biochemical mechanisms of tolerance to extreme desiccation. The optimized proteomics workflow will enable application of sensitive quantification techniques to detect differences in protein expression, which are characteristic of the active and anhydrobiotic states of tardigrades
    Type of Publication: Journal article published
    PubMed ID: 20224743
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  • 5
    Keywords: APOPTOSIS ; mass spectrometry ; DISC ; CD95 ; protein quantification ; DED ; AQUA technique ; protein platforms
    Abstract: Contemporary quantitative mass spectrometry provides fascinating opportunities in defining the stoichiometry of high-molecular weight complexes or multiprotein platforms. The composition stoichiometry of multiprotein platforms is a key to understand the regulation of complex signaling pathways and provides a basis for constructing models in systems biology. Here we present an improved AQUA technique workflow that we adapted for the quantitative mass spectrometry analysis of the stoichiometry of the CD95 (Fas/APO-1) death inducing signaling complex (DISC). The DISC is a high-molecular weight platform essential for the initiation of CD95-mediated apoptotic and non-apoptotic responses. For protein quantification, CD95 DISCs were immunoprecipitated and proteins in the immunoprecipitations were separated by one-dimensional gel electrophoresis, followed by protein quantification using the AQUA technique. We will discuss in detail AQUA analysis of the CD95 DISC focusing on the key issues of this methodology, i.e., selection and validation of AQUA peptides. The application of this powerful method allowed getting new insights into mechanisms of procaspase-8 activation at the DISC and apoptosis initiation. Here we discuss the AQUA methodology adapted by us for the analysis of the CD95 DISC in more detail. This approach paves the way for the successful quantification of multiprotein complexes and thereby delineating the intrinsic details of molecular interactions.
    Type of Publication: Journal article published
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