Toward a full characterization of the human 20S proteasome subunits and their isoforms by a combination of proteomic approaches.
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.
Uttenweiler-Joseph
Sandrine
S
Institut de Pharmacologie et de Biologie Structurale, UMR 5089, Centre National de la Recherche Scientifique/Université Paul Sabatier, Toulouse, France.
Claverol
Stéphane
S
Sylvius
Loïk
L
Bousquet-Dubouch
Marie-Pierre
MP
Burlet-Schiltz
Odile
O
Monsarrat
Bernard
B
D000595:Amino Acid Sequence; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004912:Erythrocytes / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry*; D020033:Protein Isoforms / Q000032:analysis* / Q000235:genetics; D021122:Protein Subunits / Q000032:analysis* / Q000235:genetics; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results
D020033:Protein Isoforms; D021122:Protein Subunits; D046988:Proteasome Endopeptidase Complex