OPTIMIZATION OF ONLINE PEPTIDE-MAPPING BY CAPILLARY ZONE ELECTROPHORESIS

A variety of enzyme-catalyzed reactions can be performed in a enzyme-m odified fused-silica capillary microreactor which has been integrated with a CZE separation capillary via an open fluid junction, In this re port, we describe the utilization of two proteases (trypsin and pepsin ) and a peptidase, carboxypeptidase-Y, in the coupled enzyme-modified microreactor-CZE system to perform on-line analysis of protein hydroly sates. Several different proteins, including insulin and a heavily gly cosylated protein, alpha(1)-acid glycoprotein, were digested in the mi croreactor. Electrophoretic fluid transfer of an aliquot of the total microreactor sample (approximately 50 pmol of denatured glycoprotein) to the separation capillary minimizes loss of the hydrolysate sample a nd conserves the remainder for iterative injections. Carboxypeptidase- Y mediated hydrolysis of the carboxyl termini of the insulin B-chain w as accomplished with on-line CZE analysis of the truncated peptides. P eptide bond cleavages within bovine insulin B-chain were rapidly analy zed in the pepsin microreactor-CZE capillary coupled system, and the e lectropherogram is compared with a CZE analysis of the free solution p epsin digestion products of the insulin B-chain.