Tenzer Stefan, Schild Hansjörg
Institute of Immunology, University of Mainz, Germany.
Methods Mol Biol. 2005;301:97-115. doi: 10.1385/1-59259-895-1:097.
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.
错误折叠、老化或不再需要的胞质蛋白的降解很大程度上依赖于泛素-蛋白酶体系统。蛋白酶体将其底物降解为3至20个氨基酸的片段。人20S蛋白酶体可通过分批吸附到DEAE-纤维素上、硫酸铵沉淀、阴离子交换快速蛋白质液相色谱（FPLC）和甘油密度梯度超速离心从人红细胞中纯化出来。用这种方法纯化的20S蛋白酶体适用于合成肽以及全长蛋白的体外消化。蛋白酶体产生的降解产物通过使用乙腈梯度的反相高效液相色谱进行分离。所得馏分通过基质辅助激光解吸电离质谱（MALDI-MS）和埃德曼降解进一步分析，这使得能够对消化产物进行定量分析。
