Assays of proteasome-dependent cleavage products.
The degradation of misfolded, aged, or no longer needed cytosolic proteins depends largely on the ubiquitin-proteasome system. Proteasomes degrade their substrates into fragments of 3-20 amino acids. Human 20S proteasomes can be purified from human erythrocytes by batch adsorption to DEAE-cellulose, ammonium sulfate precipitation, anion-exchange fast protein liquid chromatography (FPLC), and glycerol density gradient ultracentrifugation. 20S proteasomes purified by this method are suitable for the in vitro digestion of synthetic peptides as well as full-length proteins. The degradation products produced by proteasomes are separated by reversed-phase HPLC using an acetonitrile gradient. The obtained fractions are further analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and Edman degradation, which allows a quantitative analysis of the digestion products.
Tenzer
Stefan
S
Institute of Immunology, University of Mainz, Germany.
Schild
Hansjörg
H
D002851:Chromatography, High Pressure Liquid; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D010455:Peptides / Q000737:chemistry*; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry* / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D025801:Ubiquitin / Q000737:chemistry*
D010455:Peptides; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex