Preparation And Characteirzation Of Novel Immobilized Protease Using Different Types Of Substrate Materials And Their Application In Proteomics Research

Proteomic research attracts great attention in recent years because of the urgent needs in biologicaland clinical research, such as biomarkers identification and drug targets discovery and verification.Currently, shut-gun based strategy which relies on the analysis of proteolytic peptides has beenwildly applied in qualitative and quantitative proteomic research. Compared with traditionalsolution based protease digestion, the application of immobilized protease offers largely reduceddigestion time and increased sample processing throughput.In this work, we prepared a few types of immobilized trypsin using different polymer modifiedsilica microparticals (PGMP) and magnetic nanoparticals (PGNP and PGMANP) synthesized byatom transfer radical polymerization (ATRP) as the substrate material. ATRP modification leads todensely packed polymer chains grafted on the microparticles and nanoparticles surface which notonly provide large amount of binding sites but also work as scaffolds to support three-dimensionaltrypsin immobilized. Therefore, increased loading amount and improved accessibility of theimmobilized trypsin is achieved. For standard protein (BSA),1minute digestion leads to completeprotein digestion into peptides and90%amino acid sequence coverage of the protein which isobviously higher that the results obtained by traditional solution digestion or using other types ofimmobilized trypsin reported in the literature. Next the effect of size and surface properties ofsubstrate materials on the digestion efficiency of immobilized enzyme is investigated usingcomplex proteome samples,1-3minutes digestion of Tengchong thermophiles and yeast using ourimmobilized trypsin results in the identification of7%more proteins than the number obtained by12hours solution digestion. Furthermore, we found that the digestion of PGMP-Trypsin is lowerthan PGNP-Trypsin and PGMANP-Trypsin, which can be attributed to larger specific surface areaof nanoparticles and increased trypsin loading amount. Comparison of the digestion results obtainedby PGNP-Trypsin (hydrophilic substrate) and PGMANP-Trypsin (hydrophobic substrate) leadsto an interesting finding that overlap of the identified protein and peptides is relatively low (58.43% and32.13%) indicating different affinity of substrate materials to proteins., The application ofmixture of PGNP-Trypsin and PGMANP-Trypsin obviously increased digestion comprehensivenessand completeness which was demonstrated by19.57%and55.24%increase in the number ofidentified proteins and peptides as well as remarkably reduced undigested proteins residuescompared with that obtained using conventional free trypsin digestion.