NEW TECHNIQUES FOR RAPID IDENTIFICATION OF GEL SEPARATED

描述：将开发非常快速的方法(几种 分钟)表征未知蛋白质的皮摩尔量 通过一维或二维凝胶电泳法进行初步分离。这 将通过洗脱蛋白质来完成，该蛋白质通过以下方式定位在凝胶上 在飞行时间质谱仪样品表面进行染色 通过一种或多种消化酶的共价连接而激活的探针， 典型的内切酶。这些活化的表面将被用来催化 分析物的快速消化，随后可能是溶液相 外蛋白水解酶或化学消化，以产生一系列的片段 从一个或多个内切酶片段中分离出来。基质辅助激光器 解吸/电离飞行时间质谱仪内切酶的研究 消化片段和外切酶消化产生的混合物 将产生内切酶图谱和一段内部序列 将足以在数据库中识别的初始蛋白质 搜索。蛋白质定位、洗脱方法及化学研究进展 消化将得到优化，技术在以下方面的限制 蛋白质的大小、化学成分和样本量将在 合作研究。

DESCRIPTION: Methods will be developed for extremely rapid (several minutes) characterization of picomole quantities of unknown proteins initially separated by one- or two-dimensional gel electrophoresis. This will be accomplished by elution of an protein, localized on a gel by staining, onto the surface of a time-of-flight mass spectrometer sample probe activated by covalent attachment of one or more digestive enzymes, typically endoproteases. These activated surfaces will be used to catalyze rapid digestion of the analyte, which may be followed by solution-phase exoprotease or chemical digestion to produce a ladder of fragments stemming from one or more of the endoprotease fragments. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of the endoprotease digest fragments and of the mixture resulting from exoprotease digestion will yield an endoprotease map and a length of internal sequence for the initial protein which will be sufficient to identify it in a database search. Methods of protein localization and elution and the chemistries of digestion will be optimized and the limits of the technology in terms of protein size and chemistry, and sample amount, will be explored in collaborative studies.