Synthesis and Optimization of Lectin Functionalized Nanoprobes for the Selective Recovery of Glycoproteins from Human Body Fluids

Abstract

Biomedical sciences, and in particular biomarker research, demand efficient glycoprotein enrichment platforms. Herein magnetic nanoprobes (MNP), after being coated with three broad-spectrum lectins-concanavalin A (ConA), wheat germ agglutinin (WGA), and Maackia amurensis lectin (MA)-were utilized to selectively capture glycoproteins from human body fluids. Additionally, a new methodology, based on protection of the lectins with their target sugars prior to coupling with MNPs, was proposed to overcome the nonspecific nature of conjugation. This approach contributed to preserve lectin conformation, increasing by 40% and 90% the affinity of ConA and MA for glycoproteins in relation to synthesis with nonprotected lectins. Optimal operating conditions (temperature, time) and maximum binding capacities were further determined for each lectin by use of fetuin as a reference. The enhanced performance of lectin-based nanoplatforms was demonstrated by comparing MNP@ConA with conventional Sepharose@ConA. These experiments have shown that ConA immobilized on MNP exhibited 5 times higher affinity for fetuin and ovalbumin when compared with Sepharose@ConA with the same amount of immobilized lectin. MNP@Lectins were then applied to human serum, saliva, and urine and the recovered proteins were digested with trypsin and analyzed by nano-HPLC MALDI-TOF/TOF. This allowed the identification of 180 proteins, 90% of which were found to be glycosylated by use of bioinformatics tools, therefore revealing low levels of unspecific binding. Thus, MNP@lectins have proved to be a valuable tool for glycoproteomic studies, particularly when dealing with minute amounts of material.

Carrageenan-grafted magnetite nanoparticles as recyclable sorbents for dye removal

“…Magnetite nanoparticles (MNPs) were prepared following the co-precipitation method, as previously reported (Ferreira et al 2011;Salgueiro et al 2013). Typically, 4.43 g (16.4 mmol) FeCl 3 .6H 2 O and 1.63 g (8.2 mmol) of FeCl 2 Á4H 2 O were dissolved in 190 mL of distilled water at room temperature, under nitrogen atmosphere, and mechanical stirring.…”

Post-translational Modifications and Mass Spectrometry Detection

“…Protein glycosylation is one of the most ubiquitous and complex PTMs, playing a key role in protein stability, folding and solubility, and modulating cellular processes such as cell adhesion and differentiation (142,143). Alterations in glycosylation have been associated with different pathological conditions.…”

“…Moreover, lectins can be immobilized in distinct solid supports (e.g. silica (157), agarose (158) or sepharose (159)), monolithic columns (160), and in microdimensional systems (146,153,156,161,162), including magnetic particles (142,153). These magnetic beads coupled to lectins have been recently used for capturing glycoproteins in complex biological samples, taking advantage of their high surface area and high mobility in solution (142,161).…”

Recent Developments in Liquid Chromatography and Capillary Electrophoresis for the Analysis of Glycoprotein Glycans

“…70,71 Lectin-immobilized nanoparticles have been used to entrap glycoproteins in human body fluids. 72 The capture of glycoprotein glycans using hydrazide functionalized phases was first introduced by Zhang's group. 73,74 cis-Diols on the glycan moiety are oxidized to aldehydes using sodium periodate (NaIO4), and then coupled to a hydrazide resin as hydrazones, the non-glycosylated peptides flow through and the bound N-linked glycans are released using PNGase F. This method can be used to find glycosylation sites.…”