Dendrimer-grafted graphene oxide nanosheets as novel support for trypsin immobilization to achieve fast on-plate digestion of proteins

In this study, dendrimer grafted graphene oxide nanosheets (dGO) were prepared by covalent reaction. The successful synthesis of dGO was confirmed by Fourier-transform infrared spectra, Raman spectra, Thermo gravimetric analysis and Zeta potential. Taking advantages of large surface area, excellent biocompatibility and abundant functional groups, dGO provided an ideal substrate for trypsin immobilization. Trypsin-linked dGO was synthesized through covalent bonding using glutaraldehyde as coupling agents. The amount of trypsin immobilized on dGO nanosheets was calculated to be about 649±20 mg/g. The activity of immobilized trypsin could be maintained for over 10 days at 4 °C. On-plate proteolysis could be performed without removing trypsin-linked dGO, because dGO did not interfere with matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry analysis. By such an immobilized enzymatic reactor, standard proteins could be efficiently digested within 15 min, with sequence coverages comparable or better than those obtained by conventional over-night in-solution digestion. Furthermore, trypsin-linked dGO showed high sensitivity when applied to trace samples analysis. All these results demonstrated that the developed dGO based enzymatic reactor might provide a promising tool for high throughput proteome identification.