CHARGED PEPTIDE DERIVATIVES IN GEL MEMBRANE PRESERVE ELECTROPHORETIC SEPARATION

凝胶膜保护中的带电肽衍生物电泳分离

• 批准号: 6258778
• 负责人: JOHN ALLISON
• 金额: $ 0.29万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-06-01 至 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6258778
• 关键词: biological products; biomedical equipment development; biomedical resource; biotechnology; proteins; technology /technique development

When 2-D gel electrophoresis is used to separate a mixture of proteins, a high degree of separation can be achieved. The generation of charged derivatives offers substantial advantages when the analytes are subsequently prepared for MALDI analysis. However, the introduction of solutions to introduce reagents results in diffusion of analytes, degrading the separation. We are pursuing the gas phase introduction of reagents, for the derivatization of peptides and proteins on membranes. Membranes containing peptides can either be exposed to gas phase reagents, or the gaseous reagents can be perfused through the membrane, by using a vacuum on one side and a source of reagent on the other. When a peptide on a membrane is exposed to chloroacetyl chloride (g), this reacts with the N-terminus, eliminating HCI. The remaining CI can be displaced by a variety of bases such as trimethyyl amine (g) to make the N-terminal charged derivative. MALDI matrix can then be introduced for direct desorption of the derivatized analyte(s) from the membrane surface. Conditions are being optimized for such reactions, and bases are being investigated which will form charged-derivatives that yield structural information in the PSD experiment.

当2-D凝胶电泳用于分离 蛋白质，可以实现高度分离。 这一代 分析物时，带电的衍生工具具有很大的优势 随后准备进行MALDI分析。 但是， 引入引入试剂的解决方案导致扩散 分析物，降低分离。 我们正在追求汽油阶段 引入试剂，用于肽的衍生化和 膜上的蛋白质。 含有肽的膜可以是 暴露于气相试剂或气态试剂可以灌注 通过膜，一侧使用真空和 另一个试剂。 当膜上的肽暴露于 氯乙酰基氯化物（G），这与N末端反应 消除HCI。 其余的CI可以由多种 诸如三甲基胺（G）之类的碱基以使N端充电 衍生物。 然后可以引入MALDI矩阵以进行直接解吸 来自膜表面的衍生分析物。 状况 正在针对此类反应进行优化，并且基地正在 调查将形成带电的衍生物，产生结构性 PSD实验中的信息。