Development of an on-line immobilized-enzyme reversed-phase HPLC method for protein digestion and peptide separation

This paper describes use of a novel glass bead-based immobilized-enzyme micro column for simple and swift on-line protein digestion then peptide separation by reversed-phase HPLC. The inexpensive and easily made immobilized-enzyme micro column was prepared from aminopropyl controlled-pore glass that was reacted first with glutaraldehyde then with trypsin in the presence of phosphate buffer. Tryptic digestion of bovine serum albumin (BSA) was achieved simply by passing pretreated protein solution through the laboratory made immobilized-trypsin column; the tryptic fragments were then separated by reversed-phase HPLC. The peptide separation was found to be identical to separation of a sample which had undergone conventional enzymatic protein digestion in solution. Digestion of BSA by the immobilized-trypsin column decreased with increasing flow rate of the solution through the column, and 1.0 μL min⁻¹ was found to be the optimum flow rate for on-line protein digestion with our system. It was also found that the sample required pretreatment with urea before injection, because of a change in the properties of the protein in the presence of urea, and the immobilized-trypsin column lost its function in the presence of acetonitrile. This on-line proteomics system enables simple and rapid protein digestion and was successfully applied to partially micro two-dimensional (2D) chromatographic separation of proteins.     

Zinc(II) complex immobilized on amine functionalized silica gel: a novel,highly efficient and recyclable catalyst for multicomponent click synthesis of 1,4-disubstituted 1,2,3-triazoles

We report a highly efficient and recyclable heterogeneous zinc catalytic system via covalent immobilization of 2-hydroxyacetophenone (2-HAP) onto an amine functionalized silica gel followed by metallation with zinc chloride and its catalytic application in three component click synthesis of 1,4-disubstituted 1,2,3-triazoles. The structure of the synthesized organic–inorganic hybrid material (SiO₂@APTES@2HAP-Zn) has been confirmed by various physicochemical characterization techniques, such as solid-state ¹³C CPMAS NMR spectroscopy, Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction analysis (XRD), Brunauer–Emmett–Teller (BET) surface area analysis, scanning electron microscopy (SEM), atomic absorption spectroscopy (AAS), energy-dispersive X-ray fluorescence spectroscopy (ED-XRF), and elemental analysis. The newly designed catalyst works under mild reaction conditions and also exhibits excellent performance in terms of good product yield and high turnover number (TON). One of the most important attributes of the present methodology is that the catalyst can be recycled several times without appreciable loss in its activity as proved by FTIR spectroscopy and SEM analysis. Besides, the heterogeneity test also confirms that no leaching of active catalytic species occurs from the silica supported zinc catalyst which confirms its remarkable structural stability under the reaction conditions.