Trypsin-based monolithic bioreactor coupled on-line with LC/MS/MS system for protein digestion and variant identification in standard solutions and serum samples.

The applicability of a trypsin-based monolithic bioreactor coupled on-line with LC/MS/MS for rapid proteolytic digestion and protein identification is here described. Dilute samples are passed through the bioreactor for generation of proteolytic fragments in less than 10 min. After digestion and peptide separation, electrospray ionization tandem mass spectrometry is used to generate a peptide map and to identify proteolytic peptides by correlating their fragmentation spectra with amino acid sequences from a protein database. By digesting picomoles of proteins sufficient data from ESI and MS/MS were obtained to unambiguously identify proteins alone and in serum samples. This approach was also extended to locate mutation sites in beta-lactoglobulin A and B variants. Keywords: immobilized trypsin • in-column digestion • LC−MS/MS • variant identification