In-cell Automated Flash Oxidation (IC-AutoFox™) Protein Footprinting System

GenNext Phase II SBIR提交了题为“单元内自动闪存 氧化（IC-AutoFox™）蛋白足迹系统”是响应于蛋白足迹的。 高阶结构分析（HOS）需要新的改进工具 生物制药和膜蛋白靶点研究的重要性。一个新兴的HOS分析 羟基自由基足迹法（HRPF）。HRPF涉及不可逆的 通过与羟基自由基反应标记蛋白质的外部，随后进行MS 分析以识别蛋白质的外部部分。的最广泛使用的方法 产生OH自由基采用紫外光的快速爆发，并且被恰当地称为快速 蛋白质光化学氧化（FPOP）。我们已经开发出商业解决方案， 进行体外FPOP。体外结构试验结果的应用实践 真实的体内行为的实验已经受到质疑。 细胞内的大分子拥挤限制了扩散，从而改变了反应动力学， 蛋白质的结合率和蛋白质-DNA相互作用。这些影响不是 在进行体外研究时观察到。由于体外培养的缺点， 最近希望以体内方式将FPOP的使用扩展到全细胞。 体内或细胞内FPOP（IC-FPOP）的实践由丽莎博士开创 马里兰州大学的琼斯。虽然在解决未实现的问题方面表现出很大的希望， 在制药研究中，IC-FPOP的重现性受到以下挑战： 细胞内背景清除的可变性，细胞间分离的不可再现性， 以及强制使用危险的、复杂的和可再现性差的准分子 激光与琼斯实验室合作，我们的工作将扩大我们的创新， 体外无激光FPOP技术在体内的应用。GenNext Technologies是 唯一将FPOP HOS分析产品商业化的公司。我们的目标是 IC-FPOP从一个学术研究实验变成了一个有价值分析 工具.一旦简化并转化为一种强大的技术，我们设想IC-FPOP 使基于细胞的测定能够：互补位和表位mAb的相互作用 生物药物及其膜靶点;阐明铅结合的动力学 正构或变构膜靶点;揭示第二信使信号 GPCR先导化合物的级联;并检测抗肿瘤药物对 靶点如激酶和生长因子。

The GenNext Phase II SBIR submssion entitled “In-cell Automated Flash Oxidation (IC-AutoFox™) Protein Footprinting System,” is responsive to the ackowledged need for new and improved tools for higher order structural analysis (HOS) of biopharmaceuticals and membrane protein target studies. An emerging HOS analysis technique is hydroxyl radical foot-printing (HRPF). HRPF involves the irreversible labeling of a protein’s exterior by reaction with hydroxyl radicals with subsequent MS analysis to identify the outer portions of the protein. The most widely used method for generating OH radicals employs a quick burst of UV light, and is appropriately called fast photochemical oxidation of proteins (FPOP).We have developed commercial solutions to perform in vitro FPOP. The practice of applying the results of in vitro structural experiments to authentic in vivo behavior has been brought into question. Macromolecular crowding within a cell limits diffusion, thus altering reaction kinetics, association rates of proteins, and protein-DNA interactions. These effects are not observed while performing in vitro studies. Because of in vitro shortcomings, there has been recent desire to extend the use of FPOP to whole cells in an in vivo manner. The practice of in vivo or in-cell FPOP (IC-FPOP) has been pioneered by Dr. Lisa Jones of the University of Maryland. While showing great promise to address unmet challenges in pharmaceutical research, reproducibility for IC-FPOP is challenged by variability of intracellular background scavenging, cell-to-cell isolation irreproducibility, and the mandatory use of hazardous, complicated, and poorly reproducible excimer lasers. Collaborating with the Jones laboratory, our work will extend our innovative in vitro laser-free FPOP technology to in vivo applications. GenNext Technologies is the only company commercializing products for FPOP HOS analysis. Our goal is to convert the IC-FPOP process from an academic research experiment into a valuable analytical tool. Once simplified and transformed into a robust technique, we envision IC-FPOP to enable cell-based assays to: paratope and epitope the interaction of mAb biopharmaceuticals with their membrane targets; elucidate the dynamics of lead binding to orthosteric or allosteric membrane targets; to reveal secondary messenger signaling cascades of GPCR lead compounds; and to detect the impact of anti-neoplastics upon targets such as kinases and growth factors.