Dual-function monolithic enzyme reactor based on dopamine/graphene oxide coating for simultaneous protein enzymatic hydrolysis and glycopeptide enrichment

A new dual-function enzyme reactor was prepared based on a dopamine/graphene oxide coated boron affinity monolithic column, which can be used for simultaneous protein enzymatic hydrolysis and glycopeptide enrichment. Firstly, a boron affinity monolithic column was prepared as the carrier for enzyme reactor. Secondly, the monolithic column was coated with dopamine/graphene oxide to provide higher specific surface area for the increase in the amount of trypsin bound. Then, dopamine can self-polymerize under alkaline conditions to produce multiple reaction sites. By the Schiff base reaction and Michael addition reaction with amino, sulfhydryl groups to trypsin, enzyme were immobilized on the boron affinity monolithic carrier. The enzyme activity was characterized by kinetic parameters maximum rate (Vmax) of the enzyme reaction and Michaelis constant (Km). Km of the dual-function enzyme reactors doped with PDA/GO and without PDA/GO were 34.37 and 120.93 mM, Vmax were 1.35 and 3.35 mM/min, respectively. The performance of the dual-function enzyme reactor was evaluated by protein extraction of mouse liver. After digested by the dual-function enzyme reactor, the number of peptides was 4,801, which was 960 more than the number of peptides in the solution digestion. At the same time, the dual-function enzyme reactor displayed the ability to capture cis-dihydroxy compounds specificly. A total of 55 glycopeptides were enriched in the dual-functional enzyme reactor, corresponding to 33 glycoproteins. The dual-function enzyme reactor provided repeatable performance and robust with long-term storage.