Separation of complex mixtures from biological sources has been highly dependent on advances in microcolumn (capillary) liquid chromatography (LC). When used in combination with biomolecular mass spectrometry (MS), capillary LC columns have been at the heart of key omics technological approaches including glycoproteomic and glycomic profiling of biological fluids and tissues in search for disease biomarkers. While typical columns used in the analytical practice utilize 5 m-sized particles packed inside capillary tubes, more kinetically progressive column types can now be tightly packed with particles within 1.0- 1.5 m range. At the expense of higher inlet pressures and the needed time for optimizing column packing technologies, the capillaries packed with such small particles can clearly outperform the more conventional columns used in today's practice of proteomics and metabolomics (as it has been already shown with reversed-phase separations). However, the separation problems of the glycoscience need different column materials effectively retaining relatively hydrophilic carbohydrates. Highly promising new column materials have been recently developed at Indiana University and preliminarily tested to meet such needs: (1) macroporous silica microspheres, which are prepared by ultrasonic spray pyrolysis (USP) using inorganic salts as a removable pore template; and (2) macroporous carbon microspheres, employing organic carboxylate salts, whose USP yields morphologically well-defined entities. This proposal deals with appropriate modifications of the particle surfaces for the benefits of adsorption chromatography and hydrophilic interaction chromatography (HILIC) developments and toward providing thus far unachieved total resolution of numerous glycan isomers. This is to be achieved through the optimum combination of column selectivity and kinetic performance. The proposal involves a combination of expertise by the P.I., a bioanalytical chemist and a separation science specialist, and the co-I. specializing in materials chemistry. The study will also be significantly aided by a distinguished collaborator at the University of North Carolina and two other collaborators with significant expertise in carbohydrate synthesis.
Aim 1 concerns modification of macroporous silica microparticles for retention of polar glycoconjugate solutes in the HILIC separation mode. The USP-derived macroporous silica particles will be packed into capillaries of different lengths and diameters and evaluated under different regimes of ultrahigh pressure and different mobile phases. Special consideration will be given to biologically important fucosylated and sialylated isomeric structures. After packing silica materials, the column will be treated in situ to generate polar surface structures.
Aim 2 will deal with packing different types of particles, specifically carbonaceous materials. They will be subjected to simila evaluations as in Aim 1. The proposed research will address one of the urgent needs of contemporary glycobiology and biomedical field.
Quantitatively altered glycosylation of proteins is a common attribute of human diseases including various types and stages of cancer. Among the difficult measurements needed to assess these clinically important situations are the determinations of differently glycosylated and sialylated glycan isomers. To address the glycan isomerism issues, highly resolving chromatographic columns can now be constructed from newly developed materials featuring macro porous geometries and unique chemistry.
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