Abstract
I conducted this study to develop an improved method for glycome detection using fluorescent magnetic beads, whose surfaces were modified using lectins, for the highly sensitive detection of saccharides or glycoproteins via fluorescence quenching using a novel fluorescence emitter and quencher pair. The emitter (Cy3 fluorophore) was incorporated into magnetic beads, and a fluorescence quencher (cyanopyranyl group) was bound to glycomes via covalent bonding. The fluorescence intensities of fluorescent magnetic beads containing lectins decreased specifically in the presence of glycomes, which was a result of fluorescence quenching from Cy3 to cyanopyranyl groups due to the formation of a stable complex between lectins and glycome. Fluorescence intensities were plotted as a function of glycoprotein concentration, and good linear relationships were observed. This method enabled the fluorescent reading-out of a series of lectin-glycome interactions on the basis of recognition selectivity and affinity of immobilized lectins without tedious washing processes. Moreover, a simple profiling process was performed using this assay for diverse glycoconjugates, which not only included simple saccharides but also glycoproteins and glycome in cell lysates. These results clearly indicate that the combination of magnetic beads with the novel emitter-quencher pair enabled the highly sensitive detection of lectin-glycome interactions.
Highlights
These results clearly indicate that the combination of magnetic beads with the novel emitter-quencher pair enabled the highly sensitive detection of lectin-glycome interactions
Modification of Glycomes Using Fluorescence Quenchers (E)-2-(2-(4-(2-(2,5-dioxopyrrolidin-1-yl)-2-oxoethoxy)styryl)-6-methyl-4H-pyran-4ylidene)malononitrile (Quen-1) was labeled to glycoproteins as follows: The activatedesterized compound Quen-1 was dissolved in 50 μL dimethylsulfoxide and the mixture was added to 500 μL of PBS buffer in the presence of 1.0 mg glycoprotein
Glycoproteins were labeled with (E)-2-(2-(4-(2-(2,5dioxopyrrolidin-1-yl)-2-oxoethoxy)styryl)-6-methyl-4H-pyran-4-ylidene)malononitrile (Quen-1) through amino groups in glycoproteins and activated ester groups in Quen-1, as shown in Figure 1b, and they were reacted with lectin-modified fluorescent magnetic beads
Summary
Glycosylation is an important posttranslational modification of proteins that profoundly affects various chemical and biological processes such as apoptosis, angiogenesis, and microbial recognition [3,4,5]. Mining of the glycome for cancer-associated biomarkers represents a new paradigm for cancer diagnosis and prognosis, and the detection of glycoconjugates as biomarkers of biological events is important for the advancement of basic scientific research and for diagnostic applications. Model systems such as self-assembled monolayers and gold glyconanoparticles have been widely used to study carbohydrate-protein binding events [16,17,18]. The detection limits of previous assays are poor, and more sensitive analytical systems are needed for the detection of carbohydrate-protein interactions due to the inherent diversity, complexity, and heterogeneity of glycan structures that make glycome analysis challenging
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