Live cell affinity receptor chromatography (LARC)

细胞表面受体是治疗疾病的药物靶向的一组关键蛋白质。细胞表面受体可以结合蛋白质或其他将信息传递到细胞的靶分子。自上世纪中叶以来，人们一直在共同努力，试图找到一种直接分离和分析受体复合体中蛋白质的方法，但基本上没有成功。我建议创造一种方法，直接从活细胞表面捕获受体复合体，方法是将它们的结合靶标固定在微珠上。我将使用血液中的一种蛋白质，从活的白细胞表面捕获其受体复合体。人类白血球有与细菌结合的受体，可以吞噬并摧毁细菌。细菌识别的抗体可以附着在显微镜下的珠子上。这些珠子将被用来捕获从白细胞表面吞噬细菌的受体复合体。将收集携带受体微珠的微球，并通过质谱仪对捕获的受体蛋白进行鉴定。不同类型的盐、洗涤剂以及酶将被用来去除常见的细胞物质，如DNA、RNA或倾向于粘在珠子上的核糖体。将确定最佳的珠子类型、珠子的大小、附着在珠子上的化学方法以及收集珠子的时间。将确定提取受体蛋白并在鉴定前使用层析对其进行分级的最佳方法。这些蛋白质可以通过用酶将其分解成碎片，然后进行质谱分析和使用经典数学进行计算机搜索来识别。从提取的受体复合体中鉴定出的蛋白质将通过使用特定抗体和绿色荧光标记等独立方法在激光显微镜下得到确认。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拥有一种直接从活细胞表面分离受体复合体的通用方法。这将允许确定和确认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.