Fluoresence detector for high performance liquid chromatography

这项申请是为了资助滑铁卢大学化学系斯科特·泰勒博士的实验室为高压液相色谱(HPLC)系统购买一个荧光检测器。这个检测器对于基于活性的硫酸酯酶蛋白质组研究是必不可少的，一类具有重要医学意义的酶。这项蛋白质组学研究的主要目标是量化硫酸酯酶表达水平的全球差异，并发现新的硫酸酯酶，最终目的是在正常和病理过程中将功能分配给硫酸盐酶网络。基于活性的蛋白质组学的核心是被称为基于活性的探针(ABP)的小分子，它们被设计为与酶的活性部位残基相互作用，从而形成稳定的共价键。我们已经构建了一系列化合物，这些化合物被设计为ABP的硫酸盐酶。这些都是基于机制的抑制剂将硫酸盐酶灭活并用荧光标签标记。为了确定硫酸盐酶中的哪些残基被荧光标记，灭活的酶将接受蛋白水解酶的消化，并使用配备荧光检测器的高效液相色谱分离荧光蛋白质片段。然后使用质谱学技术对荧光片段进行测序。我们目前的高效液相系统仅配备了紫外线检测器，我们无法随时访问任何配备荧光检测器的高效液相系统。我们必须能够访问这样的系统，因为它将使我们能够快速和经济地确定进行蛋白质组研究的最佳ABP和条件。

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.