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

名为“In-cell Automated Flash”的GenNext阶段II SBIR提交 氧化(IC-AUTOFOX™)蛋白质足迹系统 满足对用于高阶结构分析(HOS)的新的和改进的工具的需求 生物制药和膜蛋白靶标研究。一项新兴的居屋分析 技术是羟基自由基足迹(HRPF)。HRPF涉及不可逆转的 利用后续MS与羟基自由基反应对蛋白质表面进行标记 分析以确定蛋白质的外部部分。最广泛使用的方法是 产生OH自由基使用快速爆发的紫外光，被恰当地称为FAST 蛋白质的光化学氧化(FPOP)。我们已经开发了商业解决方案来 进行体外FPOP。应用体外结构研究成果的实践 真实的体内行为的实验已经受到了质疑。 细胞内的大分子拥挤限制了扩散，从而改变了反应动力学， 蛋白质的缔合率和蛋白质与DNA的相互作用。这些影响并不是 在进行体外研究时观察到的。由于体外培养的缺陷，有 最近一直希望以体内的方式将FPOP的使用扩展到整个细胞。 体内或细胞内FPOP(IC-FPOP)的实践是由Lisa博士开创的 马里兰大学的琼斯。同时表现出解决未实现的问题的巨大承诺 药物研究中的挑战，IC-FPOP的重复性受到以下挑战 细胞内背景清除的可变性，细胞间分离的不可重复性， 以及强制使用危险、复杂和重复性差的准分子 激光。与琼斯实验室合作，我们的工作将把我们的创新扩展到 体外无激光FPOP技术在体内的应用。GenNext Technologies是 只有一家商业化FPOP HOS分析产品的公司。我们的目标是将 IC-FPOP从学术研究实验到有价值的分析过程 工具。一旦被简化并转变为一种健壮的技术，我们设想IC-FPOP将 使基于细胞的分析能够：对位和表位单抗的相互作用 生物药物及其膜靶标；阐明铅结合的动力学 到正构或变构的膜靶标；揭示二级信使信号 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.