Hydrophilic immobilized trypsin reactor with magnetic graphene oxide as support for high efficient proteome digestion

In this paper, magnetic Fe₃O₄ nanoparticles modified graphene oxide nanocomposites (GO-CO-NH-Fe₃O₄) were prepared by covalent bonding, via the reaction between the amino groups of fuctionalized Fe₃O₄ and the carboxylic groups of GO, confirmed by Fourier-transform infrared spectra, Raman spectroscopy, and transmission electron microscopy. With GO-CO-NH-Fe₃O₄ as a novel substrate, trypsin was immobilized via π-π stacking and hydrogen bonding interaction, and the binding capacity of trypsin reached as high as 0.275 mg/mg. Since GO-CO-NH-Fe₃O₄ worked as not only support for enzyme immobilization, but also as an excellent microwave irradiation absorber, the digestion efficiency could be further improved with microwave assistance. By such an immobilized enzymatic reactor (IMER), standard proteins could be efficiently digested within 15 s, with sequence coverages comparable or better than those obtained by conventional in-solution digestion (12 h). Since trypsin was immobilized under mild conditions, the enzymatic activity of IMER preserved at least for a month. In addition, due to the good hydrophilicity of GO, no peptide residue was observed in the sequent digestion of bovine serum albumin and myoglobin. To further confirm the efficiency of such an IMER for proteome analysis, it was applied to digest proteins extracted from rat liver, followed by nanoRPLC-ESI-MS/MS analysis. With only 5 min microwave-assisted digestion, in 3 parallel runs, totally 456 protein groups were identified, comparable to that obtained by 12 h in-solution digestion, indicating the great potential of IMERs with GO-CO-NH-Fe₃O₄ as the support for high throughput proteome study.