Development, Modification and Applications of Polymeric Substances for Biomolecules in Separation Science

Abstract

The availability of stationary phases for crude and specific separations is an important task achieved by a separation chemist. This necessity becomes vital when the complex samples like biofluids are to be dealt with proteome science. The emergence of new synthetic polymers has a massive and continuing effect on the direction and capabilities of modern analytical science. The derivatization of these polymers has made them an efficient class of substrate, having unique properties and the selectivity tailored surface chemistries for target molecules. The deeper and detailed characterization of complex sample types has become feasible due to the enhanced selectivity and sensitivity offered by these polymer materials. The work presented here involves the synthesis and application of a terpolymeric sorbent and two bi-functional polymers with different functionalizations adapted for the selective enrichment of biomolecules of interest from biological fluids. Also a polymeric monolith is prepared and modified by controlled growth of porous coordination network to enable it for the selective enrichment for biologically relevant phosphopeptides. Each of bifunctional monomers, glycidyl propargyl ether (GPE) and allylglycidyl ether (AGE) is thermally polymerized with divinylbenzene (DVB) to form poly(GPE/DVB) and poly(AGE/DVB). Synthesis of terpolymer is carried out by the radical polymerization of monomers; methyl acrylate, acrylic acid and vinyl acetate with diethylene glycol dimethacrylate as cross-linking agent benzoyl peroxide as initiator. Synthesized polymers are characterized by scanning electron microscopy (SEM), energy dispersive X-Ray spectroscopy (EDX) and Fourier transform infrared spectroscopy (FTIR). The polymers are further derivatized to IMAC (immobilized metal ion affinity chromatography) and are investigated by loading different metal ions (Fe3+, Ti4+, Zr4+ and La3+). The trypsin digested products of phosphoproteins, such as casein, non-fat milk, egg yolk, HeLa cell extract and human blood serum, are used to explore phosphopeptide enrichment ability of the modified polymers from complex samples. Serum profiling of healthy and diseased samples demonstrates the potential of new polymer to impart in the disease diagnosis. The identification with their sequence coverage is made using mascot and Phosphosite Plus. Poly(AGE/DVB) has high selectivity of 1:2000 with BSA background. Hydrophobicity is introduced to the polymers through octadecyl amine (ODA) which provides compatible results to commercially available reverse phase materials for the desalting of complex mixtures of all caseins. In another approach, an iron-benzenetricarboxylate (FeBTC) coordination network has been confined within the pores of a polystyrene-divinyl benzene-methacrylic acid polymer monolith by means of a step-by-step in-situ growth mechanism. The enhanced amount of active metal sites due to the gradual incorporation of Fe(III) through coordination with trimesic acid enables the selective enrichment of biologically relevant phosphopeptides. The FeBTC-porous polymer monolith preparation is adapted to a capillary column format, obtaining for the first time, a flow- through miniaturized device containing a porous coordination polymer thin layer, which is implementable for the efficient purification of biological samples.