Detection of activity and mass spectrometric identification of mouse liver carboxylesterase and aldehyde dehydrogenase separated by non-denaturing two-dimensional electrophoresis after extraction with detergents.
Detection of activity and mass spectrometric identification of mouse liver carboxylesterase and aldehyde dehydrogenase separated by non-denaturing two-dimensional electrophoresis after extraction with detergents.
复制标题
DOI：
10.1016/j.bbapap.2005.02.011
复制
发表时间：
2005-05
期刊：
影响因子：
--
通讯作者：
Y. Shimazaki;T. Manabe
中科院分区：
文献类型：
--
作者：
Y. Shimazaki;T. Manabe
为了检测与细胞器如微粒体和线粒体相关的酶的活性和特性，使用非离子去污剂Nonidet P-40（NP-40）、聚氧乙烯脱水山梨糖醇单油酸酯（PEG）和聚氧乙烯脱水山梨糖醇单油酸酯（PEG）从小鼠肝脏中提取蛋白质。（吐温80），聚氧乙烯异辛基苯基酯（Triton X）、正辛基β-d-葡糖苷（辛基糖苷）或阴离子去污剂十二烷基硫酸钠（SDS）。用非变性双向电泳（2-DE）分离洗涤剂提取的蛋白。非变性双向电泳结果表明，经各种非离子去污剂处理后，酯酶和乙醛脱氢酶的活性基本保持不变，但当SDS浓度超过0.5%时，酶活性降低或丧失。为了对小鼠肝脏中的细胞器相关蛋白进行蛋白质组学分析，通过非变性2-DE分离蛋白质，并在用辛基糖苷、NP-40和0.1%SDS溶解蛋白质后，使用电喷雾电离串联质谱（ESI-MS/MS）鉴定蛋白质。鉴定了羧酸酯酶、乙醛脱氢酶、葡萄糖调节蛋白和热休克蛋白60等细胞器相关蛋白。这些结果表明，活性和身份的洗涤剂可溶性酶可以检查这种非变性2-DE和质谱。
To examine the activities and identity of enzymes associated with organelles such as microsomes and mitochondria, proteins from mouse liver were extracted using the non-ionic detergents Nonidet P-40 (NP-40), polyoxyethylene sorbitan monooleate (Tween 80), polyoxyethylene isooctylphenyl ester (Triton X), n-octyl β-d-glucoside (octyl glycoside) or anionic detergent sodium dodecylsulfate (SDS) after the removal of cytosolic proteins. The proteins extracted by detergents were separated by non-denaturing two-dimensional electrophoresis (2-DE). The activities of esterase and aldehyde dehydrogenase were retained by non-denaturing 2-DE after treatment with each non-ionic detergent, but the activities were reduced or lost when the proteins were extracted with more than 0.5% SDS. For proteomic analysis of the organelle-associated proteins in mouse liver, proteins were separated by non-denaturing 2-DE and were identified using electrospray ionization tandem mass spectrometry (ESI-MS/MS) after the proteins were solubilized by octyl glycoside, NP-40 and 0.1% SDS. Several organelle-associated proteins such as carboxylesterase, aldehyde dehydrogenase, glucose regulated protein and HSP60 were identified. These results indicate that the activities and identity of detergent-soluble enzymes can be examined by this non-denaturing 2-DE and mass spectrometry.