Live cell affinity receptor chromatography (LARC)

细胞表面受体是药物靶向治疗疾病的一类关键蛋白。细胞表面受体可以结合蛋白质或其他携带信息到细胞的靶分子。自上世纪中叶以来，人们一直在努力寻找一种直接分离和分析受体复合物中的蛋白质的方法，但基本上没有成功。我建议创造一种方法，通过将受体复合物的结合目标固定在微珠上，直接从活细胞表面捕获受体复合物。我会用血液中的一种蛋白质从活的白细胞表面捕获它的受体复合物。人类白细胞具有与细菌结合的受体，可以吞噬并消灭细菌。用于细菌识别的抗体可以附着在微珠上。这些小珠子将被用来捕获受体复合物，以从白细胞表面吞噬细菌。将收集携带受体珠的微珠，并通过质谱法鉴定捕获的受体蛋白。不同类型的盐和清洁剂以及酶将被用来去除常见的细胞物质，如DNA、RNA或核糖体，这些物质往往会粘在珠子上。将确定最佳的珠粒类型、珠粒大小、附着珠粒的化学方法以及收集珠粒的时间。将确定提取受体蛋白的最佳方法，并在鉴定前用色谱法对其进行分离。这些蛋白质可以通过酶将它们分解成碎片，然后通过质谱分析和使用经典数学的计算机搜索将它们分离出来来识别。从提取的受体复合物中鉴定的蛋白质将通过激光显微镜通过独立的方法（如使用特异性抗体和绿色荧光标记）进行确认。该方法将阐明受体复合体吞噬细菌所需的蛋白质。更重要的是，我们的成功将意味着加拿大将有一种直接从活细胞表面分离受体复合物的通用方法。这将允许识别和验证用于治疗许多疾病的新的和特定的药物靶点。

Cell surface receptors are a key group of proteins targeted by drugs for the treatment of disease. Cell surface receptors may bind proteins or other target molecules that carry messages to the cell. There has been a concerted, but largely unsuccessful, effort to find a way to directly isolate and analyze the proteins in receptor complexes since the middle of the last century. I propose to create a method to directly capture a receptor complex from the surface of live cells by fixing their binding target onto microscopic beads. I will use a protein from blood to capture its receptor complex from the surface of living white blood cells. Human white blood cells have receptors that bind to germs to engulf and destroy them. Antibodies for bacterial recognition may be attached to microscopic beads. The beads will be used to capture the receptor complex for engulfing germs from the surface of the white blood cells. The microbeads carrying the receptor beads will be collected and the receptor proteins captured will be identified by mass spectrometry. Different types of salts and detergents as well as enzymes will be used to remove common cellular material such as DNA, RNA or ribosomes that tend to stick to beads. The optimal type of bead, size of bead, chemical method of attachment to the bead, and time for collecting the beads will be determined. The best methods for extracting the receptor proteins and to fractionate them using chromatography prior to identification will be determined. The proteins can be identified by breaking them into pieces with enzymes, and separating them followed by mass spectrometry and computer searching using classical mathematics. Proteins identified from extracted receptor complexes will be confirmed with a laser microscope by independent methods such as the use of specific antibodies and green fluorescence tagging. The method will elucidate the proteins required by the receptor complex for engulfing germs. More importantly, our success would mean that Canada will have a general method for isolating receptor complexes directly from the surface of live cells. This will permit the identification and validation of new and specific drug targets for the treatment of many diseases.