Preparation And Characterization Of Novel Immobilized Enzyme Using Thermoresponsive Magnetofluid Materials And Their Application In Proteomics Research

As the most successful method for large-scale protein identification and quantification, mass spectrometry based "shotgun" strategy has been widely applied in the field of proteomics research. In this strategy, the qualitative and quantitative information of proteins is obtained by analyzing their proteolytic peptides, which makes the highly efficient and complete digestion the key step. Compared with traditional in-solution digestion, immobilized enzyme for in situ proteolysis has attracted particular attentions due to its obvious advantages of rapidity, low costs and reusability. However, most of the previous studies focused on using solid materials as the immobilization matrix, the catalytic efficiency of which is limited by their heterogeneous reaction mechanism. Hence, this work proposed to develop a series of novel immobilized enzyme reagents to achieve "homogeneous catalysis and heterogeneous separation" using thermoresponsive copolymers and magnetofluids. In addition, by attaching different kinds of enzymes onto different thermoresponsive magnetic matrix, the reagents were successfully used in the study of proteomics or glycoproteomics research:(1) To prepare the thermoresponsive magnetofluid as the matrix for enzyme immobilization, the initiator of atom transfer radical polymerization (ATRP) was first anchored to the surface of Fe3O4 nanoparticle via ligand exchange. Then thermoresponsive copolymer chains were grafted by copolymerization of N-isopropylacrylamide (NIPAM) and 10-Undecenal (UnAl). Because of the hydrophicility and thermosensitivity of their copolymer shell, the grafted Fe3O4 nanoparticles were not only well dispersed in water and form aqueous magnetofluid, but also flocculated by temperature triggering. Next, Peptide-N-glycosidase F (PNGase F) was immobilized on this thermoresponsive magnetic matrix via Borch reduction between the aldehyde groups of the copolymer chains and the free amino groups of the enzyme. The properties of this immobilized PNGase F reagent were characterized and tested by deglycosylation of two standard glycoproteins, RNase B and asialofetuin (ASF) and compared with the result obtained using in-solution digestion.(2) The PNIPAM-based thermoresponsive magnetofluid immobilized enzyme reagents allow us to achieve homogeneous digestion at temperatures below the lower critical solution temperature (LCST) of the matrix polymer, and separate them heterogeneously by magnetic field after a simple heating procedure. This property indeed increases the reaction rate and efficiency significantly. However, if the substrate contains heat-sensitive proteins, processing flocculation-separation via heating may lead to protein denaturation. Hence, in the second part of this work, a novel magnetofluid immobilized enzyme reagent with an upper critical solution temperature (UCST) thermosensitivity was developed to address the above issue. By immobilization of trypsin on the UCST-type thermoresponsive copolymer poly(NAGA-co-UnAl) and combining with the citric acid coated Fe3O4 nanoparticles, a novel UCST-type immobilized trypsin reagent was successfully prepared. The properties of this reagent were characterized and tested by digestion of a standard protein BSA and compared with the result obtained using in-solution digestion. Finally, the reagent was successfully applied to the highly efficien digestion and identification of membrane proteins from macrophages.