High-throughput fluorescence HPLC detection of low abundance metabolites and functional groups

现代生物化学的挑战之一是在复杂的生物混合物中检测微量化合物。这些化合物包括代谢中间体，这些中间体在性质上是短暂的或以不可检测的水平存在。带有荧光加合物的衍生化化合物大大提高了检测和量化它们的能力，并防止了它们的代谢周转。所要求的设备将支持三个研究项目，对检测生物样品中的微量化合物感兴趣。这包括检测a)蛋白质上的巯基；b)含有抗氧化剂（如谷胱甘肽、半胱氨酸和同型半胱氨酸）的低分子量巯基；c)低分子量巯基化合物和蛋白质之间的混合二硫化物。用N-（1-芘基）马来酰亚胺或单溴茂烷衍生化还原巯基后，可用高效液相色谱（HPLC）对游离巯基进行荧光检测。该技术允许检测游离硫醇的微摩尔（ppm）量。荧光高效液相色谱法也将用于检测脂肪酸的荧光代谢物。所要求的设备将建立在先前购买的带有电化学和光电二极管阵列探测器的瓦里安高效液相色谱系统的基础上。本次资助购买的设备将日常用于痕量化合物检测领域的本科生、研究生、博士后研究人员和访问科学家的研究和培训。该设备包括用于HPLC的荧光检测器以及用于高通量样品处理的自动进样器。这些添加到高效液相色谱将使我们能够确定低分子量和细胞内蛋白质硫醇的还原和氧化状态，以及生物合成途径的代谢中间体（脂质）。它还将用于复杂动植物样品中所含荧光化合物的跨学科研究。

One of the challenges of modern day biochemistry is the detection of trace amounts of compounds in complex biological mixtures. Such compounds include metabolic intermediates that are either transitory in nature or present in undetectable levels. Derivatization of compounds with fluorescent adducts greatly enhances the ability to detect and to quantify them as well as preventing their metabolic turnover. The requested equipment will support three research programs with an interest in detection of trace compounds in biological samples. These includes the detection of sulfhydryl groups present on a) proteins, b) low molecular weight sulfhydryl containing antioxidants such as glutathione, cysteine and homocysteine and c) mixed disulfides between low molecular weight sulfhydryl compounds and proteins. Fluorescence detection of free thiols can be performed by High Performance Liquid Chromatography (HPLC) after derivatizing reduced sulfhydryl groups with either N-(1-pyrenyl)maleimide or monobromobimane. This technique allows for detection of micromolar (ppm) amounts of free thiols. Fluorescence HPLC will also be utilized to detect fluorescent metabolites of fatty acids. The requested equipment will build upon a previously purchased Varian HPLC system with electrochemical and photodiode array detectors. The equipment purchased with this grant will be used on a daily basis in the research and training of undergraduates, graduate students, postdoctoral researchers and visiting scientists in the field trace compound detection. The equipment includes a fluorescence detector for the HPLC as well as an autosampler for high throughput processing of samples. These additions to the HPLC will enable us to determine the reduced vs. oxidized state of low molecular weight as well as protein thiols within the cell as well as metabolic intermediates of biosynthetic (lipids) pathways. It will also be used for cross-disciplinary studies of fluorescent compounds contained within complex animal and plant samples.