Toward a Full Characterization of the Human 20S Proteasome Subunits and Their Isoforms by a Combination of Proteomic Approaches
| Autor: | Loïk Sylvius, Stéphane Claverol, Marie-Pierre Bousquet-Dubouch, Sandrine Uttenweiler-Joseph, Bernard Monsarrat, Odile Burlet-Schiltz |
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| Přispěvatelé: | Institut de pharmacologie et de biologie structurale (IPBS), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Plateforme génomique fonctionnelle (PGFB), Université Bordeaux Segalen - Bordeaux 2, Plate-forme Protéomique CLIPP - Clinical and Innovation Proteomic Platform [Dijon] (CLIPP), Franche-Comté Électronique Mécanique, Thermique et Optique - Sciences et Technologies (UMR 6174) (FEMTO-ST), Université de Technologie de Belfort-Montbeliard (UTBM)-Ecole Nationale Supérieure de Mécanique et des Microtechniques (ENSMM)-Université de Franche-Comté (UFC), Université Bourgogne Franche-Comté [COMUE] (UBFC)-Université Bourgogne Franche-Comté [COMUE] (UBFC)-Centre National de la Recherche Scientifique (CNRS)-Université de Technologie de Belfort-Montbeliard (UTBM)-Ecole Nationale Supérieure de Mécanique et des Microtechniques (ENSMM)-Université de Franche-Comté (UFC), Université Bourgogne Franche-Comté [COMUE] (UBFC)-Université Bourgogne Franche-Comté [COMUE] (UBFC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie Moléculaire de l'Université de Bourgogne [Dijon] (ICMUB), Université de Bourgogne (UB)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Bourgogne (UB)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS) |
| Jazyk: | angličtina |
| Rok vydání: | 2008 |
| Předmět: |
Gene isoform
Gel electrophoresis 0303 health sciences Two-dimensional gel electrophoresis Molecular mass Chemistry Protein digestion Protein subunit 010401 analytical chemistry catalytic protein complex top-down and bottom-up proteomic approaches Mass spectrometry 2D gel electrophoresis 01 natural sciences protein modifications 0104 chemical sciences 03 medical and health sciences Biochemistry [SDV.BBM]Life Sciences [q-bio]/Biochemistry Molecular Biology Beta (finance) 030304 developmental biology protein gel elution mass spectrometry |
| Zdroj: | Functional Proteomics : Methods and Protocols Functional Proteomics : Methods and Protocols, pp.111-130, 2008, 978-1-59745-398-1. ⟨10.1007/978-1-59745-398-1_8⟩ Functional Proteomics ISBN: 9781588299710 |
| DOI: | 10.1007/978-1-59745-398-1_8⟩ |
| Popis: | International audience; The 20S proteasome is a multicatalytic protein complex, present in all eukaryotic cells, that plays a major role in intracellular protein degradation. In mammalian cells, this symmetrical cylindrical complex is composed of two copies each of seven different alpha and beta subunits arranged into four stacked rings (alpha(7)beta(7)beta(7)alpha(7)). Separation by two-dimensional (2D) gel electrophoresis of the human erythrocytes 20S proteasome subunits and mass spectrometry (MS) identification of all the observed spots reveal the presence of multiple isoforms for most of the subunits. These isoforms could correspond to protein variants and/or posttranslational modifications that may influence the 20S proteasome proteolytic activity. Their characterization is therefore important to establish the rules governing structure/activity relationships of the human 20S proteasome. This chapter describes the use of a combination of proteomic approaches to characterize the human 20S proteasome subunit isoforms separated by 2D gel electrophoresis. A "top-down" strategy was developed to determine by electrospray MS the molecular mass of the intact protein after its passive elution from the gel. Comparison of the experimental molecular mass to the theoretical one can reveal the presence of possible modifications. "Bottom-up" proteomic approaches are then performed and, after protein digestion, tandem MS analyses of the modified peptides allow the characterization and location of the modification. These methods are discussed for the study of the human erythrocytes 20S proteasome subunit isoforms. |
| Databáze: | OpenAIRE |
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