Functionalized magnetic micro‐ and nanoparticles: Optimization and application to μ‐chip tryptic digestion

Abstract

The preparation of an easily replaceable protease microreactor for micro-chip application is described. Magnetic particles coated with poly(N-isopropylacrylamide), polystyrene, poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate), poly(glycidyl methacrylate), [(2-amino-ethyl)hydroxymethylen]biphosphonic acid, or alginic acid with immobilized trypsin were utilized for heterogeneous digestion. The properties were optimized, with the constraint of allowing immobilization in a microchannel by a magnetic field gradient. To obtain the highest digestion efficiency, sub-micrometer spheres were organized by an inhomogeneous external magnetic field perpendicularly to the direction of the channel. Kinetic parameters of the enzyme reactor immobilized in micro-chip capillary (micro-chip immobilized magnetic enzyme reactor (IMER)) were determined. The capability of the proteolytic reactor was demonstrated by five model (glyco)proteins ranging in molecular mass from 4.3 to 150 kDa. Digestion efficiency of proteins in various conformations was investigated using SDS-PAGE, HPCE, RP-HPLC, and MS. The compatibility of the micro-chip IMER system with total and limited proteolysis of high-molecular-weight (glyco)proteins was confirmed. It opens the route to automated, high-throughput proteomic micro-chip devices.

Immobilization of Trypsin on Superparamagnetic Nanoparticles for Rapid and Effective Proteolysis

“…[18][19][20] Magnetic polymer microspheres, due to unique magnetic responsivity and high dispersibility, are versatile supports for the immobilization of enzyme. [21][22][23][24] Application of immobilized enzymes using magnetic polymer microspheres as carriers offers a distinct advantage over soluble enzymes, because they can be easily removed from the reaction mixture with the assistance of magnetic field, and can be used repeatedly. The storage properties and the pH stability of enzymes are often improved by immobilization.…”

High Throughput Bioassays Using Nanoparticles

“…Trypsin is widely used for protein digestion as it yields tryptic peptides of an appropriate size for mass spectrometry (MS) analysis, but the process requires several hours. The use of magnetic NPs with immobilized trypsin has been proposed for the reduction of the digestion time from hours to minutes or seconds [19][20][21]. Thus, a method involving microwave-assisted protein digestion has been described in which magnetic NPs are used as support for enzyme immobilization and, also, as absorbents of the microwave radiation, improving the efficiency of the process [19].…”

Preparation and properties of magnetic nano‐ and microsized particles for biological and environmental separations

“…A unique fingerprint for each protein called a "peptide map" can thus be obtained. Digestion in l-chip immobilized magnetic enzyme reactor (IMER) using magnetic nano-and microparticles is a preferable alternative to the conventional batch mode for numerous large globular and highly glycosylated proteins [267]. On-line connection with mass spectrometer opens the route to automated, high-throughput proteomic devices, which significantly simplifies proteomic analysis protocols without compromising the quality and specificity of analysis.…”