Scaled-down purification protocol to access proteomic analysis of 20S proteasome from human tissue samples: comparison of normal and tumor colorectal cells.
The proteasome is a proteolytic complex that constitutes the main pathway for degradation of intracellular proteins in eukaryotic cells. It regulates many physiological processes and its dysfunction can lead to several pathologies like cancer. To study the 20S proteasome structure/activity relationship in cells that derive from human biopsy samples, we optimized an immuno-purification protocol for the analysis of samples containing a small number of cells using magnetic beads. This scaled-down protocol was used to purify the cytoplasmic 20S proteasome of adjacent normal and tumor colorectal cells arising from tissue samples of several patients. Proteomic analyses based on two-dimensional gel electrophoresis (2DE) and mass spectrometry showed that the subunit composition of 20S proteasomes from these normal and tumor cells were not significantly different. The proteasome activity was also assessed in the cytoplasmic extracts and was similar or higher in tumor colorectal than in the corresponding normal cells. The scaled-down 20S proteasome purification protocol developed here can be applied to any human clinical tissue samples and is compatible with further proteomic analyses.
Universite de Toulouse, Institute of Pharmacology and Structural Biology, IPBS, UPS, 205 route de Narbonne, 31077, Toulouse, cedex 4, France.
Manuelle
M
Ducoux-Petit
Sandrine
S
Uttenweiler-Joseph
Franck
F
Brichory
Marie-Pierre
MP
Bousquet-Dubouch
Odile
O
Burlet-Schiltz
Jean-François
JF
Haeuw
Bernard
B
Monsarrat
D000368:Aged; D000369:Aged, 80 and over; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000737:chemistry* / Q000473:pathology; D005260:Female; D006801:Humans; D047468:Immunoprecipitation; D008280:Magnetics; D008297:Male; D008875:Middle Aged; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000302:isolation & purification*; D021122:Protein Subunits / Q000737:chemistry / Q000302:isolation & purification; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry
D021122:Protein Subunits; D046988:Proteasome Endopeptidase Complex