Multiplex glycan bead array for high throughput and high content analyses of glycan binding proteins

Abstract

Glycan-binding proteins (GBPs) play critical roles in diverse cellular functions such as cell adhesion, signal transduction and immune response. Studies of the interaction between GBPs and glycans have been hampered by the availability of high throughput and high-content technologies. Here we report multiplex glycan bead array (MGBA) that allows simultaneous analyses of 384 samples and up to 500 glycans in a single assay. The specificity, sensitivity and reproducibility of MGBA are evaluated using 39 plant lectins, 13 recombinant anti-glycan antibodies, and mammalian GBPs. We demonstrate the utility of this platform by the analyses of natural anti-glycan IgM and IgG antibodies in 961 human serum samples and the discovery of anti-glycan antibody biomarkers for ovarian cancer. Our data indicate that the MGBA platform is particularly suited for large population-based studies that require the analyses of large numbers of samples and glycans.

DNA Encoded Glycan Libraries as a next-generation tool for the study of glycan-protein interactions

“…A similar diversity of results was observed in a related MGBA assay as well. [9] Results may be improved if higher concentrations of lectins were used. For instance, SNA lectin did not show significant binding when used at a concentration of 10 µg/mL, but at 30 µg/mL, the signal was much improved without compromising the specificity of the binding (Figure 8).…”

“…and clinical research. [4,9,11,[47][48][49][50][51] Recognizing the importance of glycans, research has focused on synthesis and isolation of glycans from different sources, including animals and plants. [12,27,[52][53][54] Synthetic efforts have led to the availability of a large number of glycan structures for study, and the number is increasing quickly.…”

“…Currently, ELISA and glycan microarray are the prominent methods for glycan-GBP studies [4][5][6][7][8][9][10][11]. In ELISA studies, single types of glycans can be immobilized, allowing individual GBPs to be screened against glycans of known identity.…”

“…[2,6,12,13] More recently, a method called multiplex glycan bead array was reported to use colored luminex beads for high-throughput and high-content detection of GBP interactions. [9] However, this strategy also required special instrumentation, and detection was limited by low commercial availability of differently colored beads. The above methods have facilitated the study of limited glycan-GBP interactions, but leave room for improvements in the areas of high-content and high-throughput analyses.…”

Protein-Specific Analysis of Invertebrate Glycoproteins

“…Positive and negative controls and pull-downs to ‘pre-clear’ endogenous biotinylated proteins, as well as Western blots with and without lectins/antibodies (i.e., just secondary reagents) or after glycosidase digestions should be considered for data interpretation. The ‘mini-description’ of the epitopes in Table 1 is based on determination of binding of the antibodies, lectins or pentraxins to standard ligands; these determinations are by no means exhaustive as invertebrate standards are rarely tested (13,16). Nevertheless, anti-horseradish peroxidase is valuable for screening of core β1,2-xylose and core α1,3-fucose (17), but the anti-xylose and anti-fucose components of the antisera are difficult to properly separate.…”