Identification of enzymes and activity from two-dimensional gel electrophoresis
Identification of enzymes and activity from two-dimensional gel electrophoresis
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DOI：
10.1038/nprot.2007.317
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发表时间：
2007-01-01
期刊：
影响因子：
14.8
通讯作者：
Lubec, Gert
中科院分区：
文献类型：
--
作者：
Afjehi-Sadat, Leila;Lubec, Gert
用质谱（MS）鉴定具有酶活性的蛋白质，同时用二维凝胶电泳（2DE）筛选酶活性来测定蛋白质的功能，这是凝胶蛋白质组学的挑战之一。在这个方案中，从脊髓组织中提取蛋白质，然后用凝胶内消化的2DE和基质辅助激光解吸/电离鉴定。确定为可能感兴趣的酶的蛋白点被打孔，用含有sds的Tris缓冲液洗脱，并在还原条件下用缓冲液还原。酶活性用微量法测定。在大约4周的时间内，可以生成结构和功能图，并验证MS鉴定，补充免疫化学方法。2DE分离可视为纯化前步骤，因此允许从2DE凝胶点中提供的微量蛋白质进行活性测定；该方法可以替代耗时的重组酶技术。
Identification of proteins with enzymatic activity by mass spectrometry (MS) and concomitant determination of function by screening enzyme activity from two-dimensional gel electrophoresis (2DE) is one of the challenges of gel-based proteomics. In this protocol, proteins are extracted from spinal cord tissue followed by 2DE with in-gel digestion and identification by matrix-assisted laser desorption/ionization. Protein spots identified as possible enzyme of interest are punched, eluted by SDS-containing Tris buffer and renatured by buffers under reductive conditions. Enzyme activity is determined using micromethods. Within about 4 weeks, a structural and functional map can be generated and MS identification can be validated, complementing immunochemical methods. 2DE separation can be seen as a prepurification step and therefore allows activity assays from minute amounts of protein as provided in a 2DE gel spot; the method may be an alternative to the time-consuming use of recombinant enzyme techniques.