Enzyme Immobilization Technique Based On Aptamer And Its Application In The Study Of Differential Proteomics

In the study of differential proteomics, protein enzymolysis and peptides labelling isthe key steps. So the preparation of immobilized enzyme with the properties that theimmobilized substrate would be reproducible, the large quantity of the enzyme fixingamount, and less enzymatic activity loss will be of great significance for the developmentof proteomics. In the paper, we grafted the aptamer on the silica gel particles using the atomtransfer radical polymerization (ATRP), and then used the grafted silica gel particles as theenzyme immobilization medium for the immobilization of trypsin. In addition, we made afurther investigation about the enzyme fixing amount and enzymatic activity. Moreover, weused the differential proteomics based on the18O labelled for the study of environmentalcontaminant hexabromocyclododecane (HBCD) exposure, and compared with the result ofconventional in-solution enzymolysis. The results are as follows:Firstly, the trypsin aptamer were grafted on the silica gel particles using the atomtransfer radical polymerization (ATRP). The immobilization of trypsin was realized basedon the high affinity and specificity between aptamer and trypsin. We measured the enzymefixing amount was10.6μg trypsin per1mg silica gel particles. Compared with theprevious method of glutaraldehyde crosslinked (the enzyme fixing amount was3.1μg/mg),the enzyme fixing amount by ATRP has been greatly improved. In addition, we studied theenzymolysis activity and the intraday and interday stability of the immobilized trypsinbased on ATRP using the standard protein bovine serum albumin (BSA) as the substrate.Then we compared it with the method of glutaraldehyde crosslinked and the free trypsin.The results indicated that enzymolysis activity of immobilized trypsin based the ATRPwas better than the method of glutaraldehyde crosslinked, and the number of enzymolysispeptides and sequence coverage rate are more than the latter and reached the level of freetrypsin. The number of enzymolysis peptides is about40, and sequence coverage rate isabout60%. Moreover, the immobilized trypsin had a better stability in the five timeenzymolysis activity of intra-day and inter-day. The relative standard deviation (RSD) ofenzymolysis peptides is28.82%and10.52%, respectively. The RSD of sequence coverage rate is20.13%and12.03%, respectively. Finally, we investigated the regenerability of theimmobilized trypsin based on ATRP. When the immobilized trypsin was eluted and thenimmobilized it again, the enzyme fixing amount was5.7μg/mg. Although the enzymefixing amount was less than before, but the number of enzymolysis peptides is also about40, sequence coverage rate is about56%. All those indicated that the immobilized trypsinbased on the aptamer has a good regenerability.In the rat model of environmental contaminant HBCD exposure, we found that therewas not shown statistical difference in the analysis of rat weight. The immobilized trypsinbased on ATRP was used for the analysis of the rat liver mitochondrial protein. We got the18O labeling efficiency was85.97%, the relative labeling efficiency reached91.26%. Thisis comparable to free enzyme (the labeling efficiency was90.30%, the relative labelingefficiency reached95.97%). After the analysis of SCX one-dimensional separation and highperformance liquid chromatography mass spectrometry,245proteins was identified, andthere was36differential proteins,8proteins were up-regulated, and28proteinsdown-regulated. But in the group of free enzyme,581proteins were identified and227proteins satisfied the requirement of quantitative analysis. And there was64differentialproteins,7proteins were up-regulated and57proteins down-regulated. Then we analyzedthe differential proteins using GO classification and Pathway, the results showed that theimmobilized and free enzyme have the similar proteomics results. The most of differentialproteins were related with metabolism, including the glycometabolism, lipid metabolismand amino acid metabolism. The results were shown in above indicated that theimmobilized trypsin based on ATRP can be used for the analysis of the differentialproteomics in the practical sample.