This project proposes unique column packings for the fast high-performance HPLC of biomacromolecules. Initial focus will be on developing techniques to modify the chemistry of silica surfaces using hydrothermal methods, so that biomolecular separations are optimized for efficiency, peak shape and compound yields. Second, three new particle types will be synthesized and tested, with two of the new particles optimized for the rapid separation of peptides and proteins, respectively. The third new particle type is for the high-speed separation of larger biomolecular weight components by size-exclusion (gel filtration) liquid chromatography. In Phase 1 grant, 1R43GM077688-01, we developed unique """"""""fused-core"""""""" silica microspheres using new nanoparticle technology. These particles are prepared by a proprietary multi- multilayering method that permits the fabrication of strong porous outer shells on solid silica cores. The outer shells can be made with different thicknesses and pore sizes, depending on intended application. Narrow-pore fused-core particles recently were commercialized to produce HaloTM columns. These columns demonstrate very fast separations of pharmaceutical and small biological compounds because of unusually high chromatographic efficiency and excellent mass transfer (kinetic) properties. Because of an extremely narrow particle size distribution and higher particle density, packed columns of fused-core particles demonstrate superior efficiency and unusual stability at high mobile phase flow rates and column inlet pressures. The proposed program will synthesize new wide-pore particles that have the pore size, particle size, outer shell thickness and surface chemistry optimized for the rapid separation of peptides, proteins, DNA fragments, and other higher molecular weight biomacromolecules. These new fused-core particles will be superior for proteomic and genetic studies, especially when multidimensional (""""""""2-D"""""""") separations are needed, and are highly suited for HPLC/MS bioscience applications. ? ? The new materials proposed in this project for rapid high-resolution liquid chromatographic separations will strongly enhance research efforts in proteomics, DNA-based studies and related bioscience applications. The increased capability for rapid characterizations and measurements will assist in the search for defining the cause and cure of disease-related problems, help in developing new drugs and support projects to improve the environment and general health. ? ? ?
| Johnson, Darryl; Boyes, Barry; Orlando, Ron (2013) The use of ammonium formate as a mobile-phase modifier for LC-MS/MS analysis of tryptic digests. J Biomol Tech 24:187-97 |
| Johnson, Darryl; Boyes, Barry; Fields, Taylor et al. (2013) Optimization of data-dependent acquisition parameters for coupling high-speed separations with LC-MS/MS for protein identifications. J Biomol Tech 24:62-72 |
| Schuster, Stephanie A; Boyes, Barry E; Wagner, Brian M et al. (2012) Fast high performance liquid chromatography separations for proteomic applications using Fused-Core® silica particles. J Chromatogr A 1228:232-41 |
