NEW TECHNIQUES FOR RAPID IDENTIFICATION OF GEL SEPARATED

凝胶分离快速鉴定新技术

• 批准号: 2771085
• 负责人: PETER WILLIAMS
• 金额: $ 12.51万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2771085
• 关键词: endopeptidases; enzyme activity; exopeptidase; gel electrophoresis; immobilized enzymes; mass spectrometry; method development; protein sequence

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.

描述：将开发极其快速（几个 分钟）未知蛋白质皮摩尔量的表征 最初通过一维或二维凝胶电泳分离。 这 将通过洗脱定位在凝胶上的蛋白质来完成 染色，到飞行时间质谱仪样品的表面 由一种或多种消化酶的共价连接激活的探针， 通常是内切蛋白酶。 这些活化的表面将用于催化 分析物的快速消化，随后可能是溶液相 外切蛋白酶或化学消化以产生阶梯状片段 来自一个或多个内切蛋白酶片段。 基质辅助激光 内切蛋白酶的解吸/电离飞行时间质谱分析 消化片段和外切蛋白酶消化产生的混合物 将产生内切蛋白酶图谱和内部序列的长度 初始蛋白质足以在数据库中识别它 搜索。 蛋白质定位和洗脱的方法以及化学 消化将得到优化，并且该技术的局限性 蛋白质大小和化学性质以及样品量将在 合作研究。