Asymmetric flow FFF and Composition Gradient/Light Scattering System

Yale University, New Haven CT

Investigators

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Abstract

[unreadable] 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. [unreadable] [unreadable] [unreadable]