Application of HPLC to glycosyltransferase research

碳水化合物修饰蛋白质的作用是复杂而多样的。在人类中，我们才刚刚开始了解其中的一些作用，但在细菌中，这些修饰的生物学原理在很大程度上是未知的。我们的研究项目正在寻求将生物功能与土壤细菌中简单而广泛的蛋白质修饰联系起来，这些细菌可以降解复杂的碳水化合物（植物细胞壁）和其他对环境重要的分子。我们正在描述这种碳水化合物修饰的蛋白质类型以及产生这种修饰的酶机制。我们的实验室已经在糖基转移酶研究中开发和使用荧光受体18年了，使用一种叫做毛细管电泳的技术作为检测技术。这些分子为我们提供了一种非常敏感的方法来追踪酶的反应，只用很少的材料，但这些只能用一种特定的检测分子标记，这限制了你可以做的分析的种类。维护毛细管电泳设备的局限性和高成本使得我们难以扩大可以分析的反应种类。由于这些原因，我们正在寻求一种更强大的替代方法，称为HPLC，以使我们能够将分析能力扩展到各种荧光团和更多的分离模式。使用更广泛的荧光仪和分离模式意味着我们不仅能够分析简单的糖缀合物，还可以扩展到使用毛细管电泳更难以分析的糖肽类/蛋白质和糖脂类。这种扩展的分析能力确保我们可以继续研究这些蛋白质修饰如何在这些生物体的生物学中发挥作用。

The role of carbohydrate modifications on proteins is complex and varied. In humans we are just beginning to understand some of these roles, but in bacteria the biology of these modifications is largely unknown. Our research project is seeking to link a biological function to a simple and widespread protein modification in soil bacteria that degrade complex carbohydrates (plant cell walls) and other environmentally important molecules. We are characterizing the types of proteins with this carbohydrate modification and also the enzymatic machinery that produces this modification. Our lab had previously developed and used fluorescent acceptors in glycosyltransferase research for 18 years using a technique called capillary electrophoresis as the assay technique. These molecules provide us with a very sensitive way to follow enzyme reactions using very little material but these could only be tagged with one specific detection molecule which limits the kinds of analysis you can do. The limitations and high cost of maintaining capillary electrophoresis equipment make it difficult to expand the kinds of reactions we can analyze. For these reasons we are seeking a more robust alternative called HPLC to allow us to expand our assay capability to a wide variety of fluorophores and more separation modes. Using an expanded range of flurophores and seperation modes means we will be able to analyze not only simple glycoconjugates, but expand to use glycopeptides/proteins and glycolipids which are more difficult to analyze by capillary electrophoresis. This expanded analysis capability ensures we can continue working out how these protein modifications play a role in the biology of these organisms.