On-line multi-enzymatic approach for improved sequence coverage in protein analysis.

The development of a new mixed bioreactor for proteomic studies based on trypsin and chymotrypsin is described. Trypsin and chymotrypsin were simultaneously bonded to an epoxy monolithic silica column (100 mmx4.6 mm id) in a one-step reaction via epoxy-groups. In order to compare the catalytic properties of the two enzymes in the isolated and in the multi-enzymatic approach, two other single enzyme bioreactors based on trypsin and chymotrypsin were prepared following the same immobilization protocol. The kinetic parameters of the multi-enzymatic bioreactor were derived and it was demonstrated that it retains the individual catalytic activity of the two enzymes. To prove the power of this experimental approach the new mixed bioreactor was integrated in an LC-ESI-MS/MS system for digestion, enrichment, separation and identification of the test protein insulin-like growth factor binding-protein 1 (IGFBP-1). The peptide map and protein sequence coverage obtained with the three bioreactors were compared. The results clearly indicate that the proposed multi-enzyme approach can reduce both digestion and analysis time, accelerate data interpretation and increase the confidence degree in protein identification.