Application of HPLC to glycosyltransferase research

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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.