Roesli Christoph, Neri Dario, Rybak Jascha-N
Institute of Pharmaceutical Sciences, ETH Zurich, 8093 Zurich, Switzerland.
Nat Protoc. 2006;1(1):192-9. doi: 10.1038/nprot.2006.29.
Targeted delivery of bioactive molecules to diseased organs or tissues by means of binding molecules specific to markers of diseases represents a promising area of pharmaceutical intervention. The availability of markers of pathology, ideally accessible from the vasculature, is crucial for such strategies. To this aim, here we present a protocol based on terminal perfusion of mice with a reactive ester derivate of biotin that enables the covalent modification of proteins readily accessible from the bloodstream. Biotinylated proteins from total organ or tissue extracts are (i) purified on streptavidin resin in the presence of strong detergents, (ii) digested on the resin and (iii) subjected to proteomic analysis. This technology is applicable to comparative proteomic investigations of differentially expressed, accessible proteins in numerous animal models having different physiological and pathological processes.
通过与疾病标志物特异性结合的分子将生物活性分子靶向递送至患病器官或组织，是药物干预的一个有前景的领域。病理标志物的可用性对于此类策略至关重要，理想情况下，这些标志物应可从脉管系统获取。为此，我们在此介绍一种基于用生物素的反应性酯衍生物对小鼠进行终末灌注的方案，该方案能够对易于从血液中获取的蛋白质进行共价修饰。来自全器官或组织提取物的生物素化蛋白质会（i）在强去污剂存在的情况下在链霉亲和素树脂上纯化，（ii）在树脂上进行消化，以及（iii）进行蛋白质组学分析。该技术适用于对具有不同生理和病理过程的众多动物模型中差异表达的可及蛋白质进行比较蛋白质组学研究。
