Toward a Full Characterization of the Human 20S Proteasome Subunits and Their Isoforms by a Combination of Proteomic Approaches

  • Sandrine Uttenweiler-Joseph
  • Stéphane Claverol
  • Loïk Sylvius
  • Marie-Pierre Bousquet-Dubouch
  • Odile Burlet-Schiltz
  • Bernard Monsarrat
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 484)

Abstract

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 α and β subunits arranged into four stacked rings (α7β7β7α7). 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.

Key Words

2D gel electrophoresis protein gel elution top-down and bottom-up proteomic approaches mass spectrometry protein modifications catalytic protein complex 
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Copyright information

© Humana Press, Totowa, NJ 2008

Authors and Affiliations

  • Sandrine Uttenweiler-Joseph
    • 1
  • Stéphane Claverol
    • 2
  • Loïk Sylvius
    • 3
  • Marie-Pierre Bousquet-Dubouch
    • 1
  • Odile Burlet-Schiltz
    • 1
  • Bernard Monsarrat
    • 1
  1. 1.Institut de Pharmacologie et de Biologie Structurale, UMR 5089Centre National de la Recherche Scientifique/Université Paul SabatierToulouseFrance
  2. 2.Pole protéomique, Plateforme Génomique FonctionelleUniversité V. Ségalen BordeauxBordeauxFrance
  3. 3.Plate-forme protéomique IFR-100Etablissement Français du SangDijonFrance

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