Asymmetric flow FFF and Composition Gradient/Light Scattering System

非对称流 FFF 和成分梯度/光散射系统

• 批准号: 7213206
• 负责人: Ewa Joanna Folta-Stogniew
• 金额: $ 27.08万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7213206
• 关键词: Acute; Advanced Development; Applications Grants; Arts; Biological; Biomedical Research; Chromatography; Collection; Complex; Coupling; Detection; Drug Delivery Systems; Economics; Electron Microscopy; Equilibrium; Evaluation; Field Flow Fractionation; Fluorescence; Fractionation; Funding; Funding Applicant; Health; Homo; Infection; Inflammatory disease of the intestine; Injury; Institution; Intercellular Junctions; Kidney; Knowledge; Liposomes; Locus Control Region; Mass Spectrum Analysis; Metalloproteases; Methods; Molecular; Nuclear Pore Complex; Nucleic Acids; Pancreatitis; Pennsylvania; Phase; Polysaccharides; Prolactin Receptor; Proteins; Range; Refractive Indices; Research; Research Personnel; Resolution; Salmonella infections; Sampling; Solutions; Streptococcus pyogenes; Syringes; System; Techniques; Time; Ultracentrifugation; Universities; cost effective; detector; dopamine transporter; human disease; light scattering; macromolecule; nanoparticle; nephelometry; prevent; radius bone structure; research study; size

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.

描述（由申请人提供）：要求为最先进的非对称流场流分级（AFFF）和自动成分梯度（CG）注射器输送系统提供资金，这些系统将共享光散射（LS）、折射率、吸光度和荧光检测器。当预期超过一种大小的大分子复合物时，将使用AFFF，而CG将向相同的检测器递送样品，所述样品是各种组成的纯化蛋白质的溶液，以能够评价同源或异源缔合的平衡。AFFF是一种单相色谱技术。在施加垂直力场的非常薄的流动中实现了按尺寸的高分辨率分离。使用AFFF分离，没有柱介质与样品相互作用。整个分离过程温和、快速、无破坏性-没有可能降解或改变样品的固定相。AFFF能够分离尺寸从1 nm到>20微米的样品。AFFF作为分级分离步骤和光散射（LS）作为检测方法的组合允许在单个实验中进行样品分级分离和尺寸和摩尔质量的测定：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。光散射测量与分级分离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大分子的摩尔质量和低聚状态，所述大分子包括天然或修饰的蛋白质及其复合物、核酸、缀合蛋白质、脂质体和多糖。与通过超离心分级相比，在AFFF/LS上分级和分级允许容易地收集级分用于进一步分析（例如，生物活性、电子显微镜和质谱法）。所要求的AFFF/CG/LS系统是通用的，将支持由13名耶鲁大学研究人员和10名来自其他8个机构，包括哥伦比亚，哈佛，宾夕法尼亚州立大学，普渡大学和洛克菲勒大学的研究人员指导的23个项目。这23名研究人员建议使用AFFF/GC/LS来推进胰腺炎、沙门氏菌病、结核（这仍然是一个主要的世界范围的健康问题，估计每年对美国的经济影响为30亿美元），涉及急性肾损伤或肠道炎症的金属蛋白酶，多巴胺转运蛋白，催乳素受体，细胞间连接，基因座控制区的重塑复合物、核孔复合物、A组链球菌感染以及利用纳米和微粒的先进药物递送系统的开发。这项研究的进展可能会提高我们对如何最好地理解，预防和治疗人类疾病的认识。如果这项拨款申请得到资助，AFFF/CG/LS系统将是耶鲁大学和邻近学术机构所独有的，它肯定会对生物医学研究做出独特的贡献，远远超出耶鲁大学的范围。

项目成果

General Solution for Stabilizing Triple Helical Collagen.

• DOI: 10.1021/jacs.6b03823
• 发表时间: 2016-07
• 期刊: Journal of the American Chemical Society
• 影响因子: 15
• 作者: Yitao Zhang;Madison R Herling;D. Chenoweth

Molecular weight-gyration radius relation of globular proteins: a comparison of light scattering, small-angle X-ray scattering and structure-based data.

球状蛋白的分子量-回转半径关系：光散射、小角 X 射线散射和基于结构的数据的比较。

• DOI: 10.1107/s1600576715015551
• 发表时间: 2015
• 期刊: Journal of applied crystallography
• 影响因子: 6.1
• 作者: Smilgies,Detlef-M;Folta-Stogniew,Ewa
• 通讯作者: Folta-Stogniew,Ewa

Unraveling the mechanism of recognition of the 3' splice site of the adenovirus major late promoter intron by the alternative splicing factor PUF60.

• DOI: 10.1371/journal.pone.0242725
• 发表时间: 2020
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Hsiao HT;Crichlow GV;Murphy JW;Folta-Stogniew EJ;Lolis EJ;Braddock DT
• 通讯作者: Braddock DT

The structure of DcrB, a lipoprotein from Salmonella enterica, reveals flexibility in the N-terminal segment of the Mog1p/PsbP-like fold.

• DOI: 10.1016/j.jsb.2018.10.005
• 发表时间: 2018-12
• 期刊: JOURNAL OF STRUCTURAL BIOLOGY
• 影响因子: 3
• 作者: Rasmussen, Damien M.;Soens, Ross W.;Davie, Timothy J.;Vaneerd, Cody K.;Bhattacharyya, Basudeb;May, John F.
• 通讯作者: May, John F.

Oligomerization of the polycystin-2 C-terminal tail and effects on its Ca2+-binding properties.

• DOI: 10.1074/jbc.m115.641803
• 发表时间: 2015-04-17
• 期刊: The Journal of biological chemistry
• 作者: Yang Y;Keeler C;Kuo IY;Lolis EJ;Ehrlich BE;Hodsdon ME
• 通讯作者: Hodsdon ME