Fluoresence detector for high performance liquid chromatography

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This application is for funds to purchase a fluorescence detector for a high pressure liquid chromatography (HPLC) system to be located in the laboratory of Dr. Scott Taylor in the department of chemistry at the University of Waterloo.  This detector is essential for an activity-based proteomic study of sulfatases, a class of medicinally significant enzymes.  The primary goals of this proteomic study is to quantify global differences in sulfatase expression levels and discover new sulfatases with the ultimate goal of assigning function to networks of sulfatases in normal and pathological processes.  At the heart of activity-based proteomics are small molecules called activity-based probes (ABP's) that are designed to interact with active site residues of enzymes resulting in the formation of a stable covalent bond.  We have constructed a series of compounds that are designed to act as ABP's of sulfatases.  These are mechanism-based inhibitors that inactivate and label the sulfatases with a fluorescent tag.  To determine which residues in the sulfatases are being fluorescently labeled the inactivated enzyme will be subjected to proteolytic digestion and the fluorescent protein fragments isolated using an HPLC equipped with a fluorescent detector.  The fluorescent fragments are then sequenced using mass spectrometry techniques.  Our current HPLC system is equipped with a UV detector only and we do not have ready access to any HPLC system equipped with a fluorescent detector.  It is essential that we have access to such as system as it will enable us to rapidly and economically determine the optimal ABP and conditions for our proteomic studies.