Immobilize Con A On Magnetic Materials For Glycoprotein Enrichment Through Chelating Interaction

Glycosylation of proteins is the most ubiquitous post-translational modification observed in both eukaryotes and prokaryotes, plays crucial roles in various physiological processes such as cell adhesion, signal transduction, immune response and inflammatory reaction. Aberrant glycosylation has been recognized to be associated with several disease states such as cancer, inflammatory diseases, and congenital disorders, etc. Since the research of glycosylation has potential for diagnostic and therapeutic purposes, interest in investigating glycoproteins has greatly increased in recent years and numerous efforts have been focused on improving the efficiency of glycoprotein analysis. However, it is difficult to analyze due to the presence of other high abundant proteins and wide dynamic range in complex biological samples. Therefore, efficient isolation and enrichment of glycoproteins are indispensable in order to obtain an in-depth understanding of glycoproteins. In this work, a new method was proposed to prepare three kinds of concanavalin a chelating magnetic nanoparticles for the selective and specific enrichment of glycoprotein.(1) Firstly, superparamagnetic nanoparticles(MNPs) were prepared through hydrothermal method; Secondly, γ-aminopropyltriethoxysilane(APTES) was derivatized on the surface of MNPs to obtain-NH2 functionalized MNPs(NH2-MNPs); And finally, Con A was immobilized on NH2-MNPs through chelating interaction using Cu2+ as bridge group. The as-prepared Con A chelating magnetic nanoparticles(Con A-Cu-NH2-MNPs) were characterized by transmission electron microscope(TEM), fourier transform infrared spectroscopy(FT-IR) and thermogravimetric analysis(TG). The Con A-Cu-NH2-MNPs showed strong mangnetic responsiveness to an applied magnetic field, and the content of Con A on the beads was about 20 wt%.(2) 1,6-Hexanediamine(HMD) functionalized MNPs(HMD-MNPs) were prepared by one-pot chemical co-precipitation method, and then immobilize Con A on HMD-MNPs through chelating interaction using Cu2+ as bridge group. The prepared Con A chelating magnetic nanoparticles(Con A-Cu-HMD-MNPs) were characterized by TEM, FT-IR and TG. The prepared Con A-Cu-HMD-MNPs showed strong mangnetic responsiveness to an applied magnetic field, and the content of Con A on the beads was about 23 wt%.(3) Ethylenediamine tetraacetic acid(EDTA)-functionalized MNPs(EDTA-MNPs) were prepared by one-pot chemical co-precipitation method, and then immobilize Con A on EDTA-MNPs through chelating interaction using Cu2+ as bridge group. The prepared Con A chelating magnetic nanoparticles(Con A-Cu-EDTA-MNPs) were characterized by TEM, FT-IR and TG. The prepared Con A-Cu-EDTA-MNPs showed strong mangnetic responsiveness to an applied magnetic field, and the content of Con A on the beads was about 28 wt%.The capacity, selectivity and specificity of the three kinds Con A chelating MNPs were investigated using standard protein mixtures and real egg white samples. The results testified the as-prepared Con A chelating MNPs could selective enrich glycoprotein from 150, 500 and 600 times non-glycoprotein samples, as well as the real egg white samples. Above results showed the specificity and selectivity of as-prepared Con A-MNPs were equal or better than those of previous reports. By proposed method in this article, Con A was immobilized on the absorbents based on chelation mechanism, the procedure was soft and non-destructive, and the bioactivity of Con A can be kept, which endowed the as-prepared MNPs excellent performance to enrich glycoproteins from complex samples. And more, it also found that the more chelating ligands have, the bigger amount of immobilized Con A was, the higher adsorption capacity and selectivity reached. It provides a novel procedure to prepare magnetic nano-adsorbents for glycoprotein enrichment.