Asymmetric flow FFF and Composition Gradient/Light Scattering System

描述(由申请人提供)：申请资助最先进的非对称流场流动分离(AFFF)和自动成分梯度(CG)注射器输送系统，该系统将共享光散射(LS)、折射率、吸光度和荧光检测器。当预期有超过一种大小的大分子复合体时，将使用AFFF，而CG将向相同的检测器提供作为各种组成的纯化蛋白质的溶液的样品，以便能够评估同种或异种缔合的平衡。AFFF是一种单相层析技术。在施加垂直力场的非常薄的流动中，可以实现按大小进行高分辨率分离。使用AFFF分离时，不需要柱介质与样品相互作用。整个分离过程温和、快速、无干扰--没有可能降解或改变样品的固定相。AFFF能够对大小从1纳米到20微米的样品进行分级。AFFF作为分离步骤和光散射(LS)作为检测方法的组合允许在一次实验中对样品进行分级并确定大小和摩尔质量：摩尔质量由静态LS测量确定，流体动力学半径由AFFF洗脱时间和动态LS测量确定。光散射测量与分级步骤的结合提供了最直接和最具成本效益的方法来确定各种不同的大分子的摩尔质量和低聚状态，包括天然或修饰的蛋白质及其络合物、核酸、结合蛋白质、脂质体和多糖。与超速离心法分级不同，AFFF/LS分级和分级可以方便地收集组分，以供进一步分析(例如，生物活性、电子显微镜和质谱分析)。所要求的AFFF/CG/LS系统是通用的，将支持23个项目，这些项目由13名耶鲁大学调查人员和来自其他8所机构的10名调查人员指导，包括哥伦比亚大学、哈佛大学、宾夕法尼亚州立大学、普渡大学和洛克菲勒大学。这23名研究人员建议使用AFFF/GC/LS推进各种生物医学研究，包括胰腺炎、沙门氏菌病(沙门氏菌病仍然是一个主要的全球健康问题，估计每年对美国造成30亿美元的经济影响)、与急性肾损伤或肠炎有关的金属蛋白酶、多巴胺转运体、催乳素受体、细胞间连接、局部控制区的重塑复合体、核孔复合体、A组链球菌感染，以及利用纳米和微颗粒开发先进的药物输送系统。这项研究的进展可能会促进我们对如何最好地理解、预防和治疗人类疾病的了解。如果这项拨款申请获得资助，AFFF/CG/LS系统将是耶鲁大学和邻近学术机构独一无二的，它肯定会为远远超出耶鲁大学的生物医学研究做出独特的贡献。

DESCRIPTION (provided by applicant): Funding is requested for state-of-the-art asymmetric flow Field-Flow Fractionation (AFFF) and automated composition gradient (CG) syringe delivery systems that would share light scattering (LS), refractive index, absorbance, and fluorescence detectors. AFFF would be used when more than one size macro- molecular complex is expected while CG would deliver to the same detectors samples that are solutions of purified proteins of various compositions to enable the evaluation of equilibria for homo- or hetero-associations. AFFF is a single phase chromatography technique. High-resolution separation by size is achieved within a very thin flow against which a perpendicular force field is applied. With AFFF separation there is no column medium to interact with the samples. The entire separation is gentle, rapid, and non-disruptive - without a stationary phase that may degrade, or other wise alter the sample. AFFF is capable of fractionating samples ranging in size from 1nm to >20 microns. The combination of AFFF as a fractionation step and light scattering (LS) as a detection method allows sample fractionation and determination of size and molar mass in a single experiment: the molar mass is determined from a static LS measurement and the hydrodynamic radius is determined from AFFF elution time and from dynamic LS measurements. The coupling of light scattering measurement to a fractionation step provides the most straightforward and cost effective method to determine the molar masses and oligomeric states of a wide range of diverse macromolecules including native or modified proteins and their complexes, nucleic acids, conjugated proteins, liposomes, and polysaccharides. In contrast to sizing by ultracentrifugation, fractionation and sizing on AFFF/LS allows the facile collection of fractions for further analyses (e.g., biological activities, electron microscopy, and mass spectrometry). The requested AFFF/CG/LS system is versatile and would support 23 projects directed by 13 Yale investigators and 10 investigators from 8 other institutions including Columbia, Harvard, Pennsylvania State, Purdue, and Rockefeller University. These 23 investigators propose to use AFFF/GC/LS to advance a diverse range of biomedical research on pancreatitis, Salmonellosis (which continues to be a major world-wide health concern and has an estimated annual economic impact on the U.S. of $3 billion), metalloproteases involved in acute renal injury or intestinal inflammation, dopamine transporters, prolactin receptor, intercellular junctions, remodeling complexes at locus control regions, nuclear pore complex, infection by group A streptococci, and the development of advanced drug delivery systems utilizing nano and microparticles. Progress in this research will likely advance our knowledge of how best to understand, prevent, and treat human diseases. If this grant application is funded, the AFFF/CG/LS system would be unique to Yale University and neighboring academic institutions, and it surely would make a unique contribution to biomedical research that would extend far beyond Yale University.