GlycoBIST: A System for Automatic Glycan Profiling of a Target Protein Using Milli‐Bead Array in a Tip

Abstract

Lectin is a biomolecule that recognizes a specific part of glycans and, thus, has been used widely as a probe for glycoprotein analysis. Owing to the wide repertoire in nature combined with the recent two decades of advances in microarray technology, the multiplexed use of lectins has been widely used for glycan profiling of endogenous proteins. Because protein glycosylation is recognized as being biologically important and is expected to be a reliable disease marker, lectin microarray analysis with highly sensitive detection has been used to discover disease‐relevant glycosylation alterations. However, the conventional system is limited to research purposes; thus, its implementation in clinical settings is warranted. Here, we provide an automatic glycan profiling method using GlycoBIST. A unique array format is used for 10‐plexed lectin–glycoprotein interaction analysis on 1‐mm‐sized beads, which are arranged vertically in a capillary‐shaped plastic tip. Using a one‐boxed autopipetting machine, the whole process (including interaction, washing, and detection) is performed automatically and serially, resulting in reproducible measurements. In this article, a typical method for glycan profiling of a purified glycoprotein and the fabrication of GlycoBIST tips is explained. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Fabrication of a GlycoBIST tip Basic Protocol 2: Automatic profiling of a target glycoprotein using GlycoBIST

Auto-Lectin Dotcoding by Two Octopuses: Rapid Analysis of Fluorescence-Labeled Glycoproteins by an 8-channel Fully-Automatic Bead Array Scanner with a Rolling-Circle Detector

“…The GlycoBIST tip was synthesized as previously described, with slight modifications . Procedures of tip fabrication and analyte labeling are reported in the Supporting Information with an explanatory diagram and table (Figures S1, S2, and Table S1).…”

“…The measurement process consisted of three steps: the interaction between the Cy3-labeled analyte and lectin beads, washing, and fluorescence scanning of the GlycoBIST tips. All processes were performed using LuBEA GT VIII Type F, an improved machine from the previous chemiluminescence scanner LuBEA GT-VIII for fluorescence scanning.…”

“…A washing step was also added to each reaction. All processes were performed using LuBEA GT-VIII as previously described …”

“…In the first attempt to automate this process, we have previously reported the milli-sized lectin bead array system GlycoBIST, 13 enabling minimum glycan profiling with 10lectins handled by simple robotics, with potential automatic data processing for signal quantification. 14 Notably, it is suitable for a large-scale experiment, as up to 1000 lectin beads for one lectin are prepared simultaneously and maintained in the same product batch before GlycoBIST tip production. However, this system is based on chemiluminescence detection assisted by overlaid antibodies, which is a time-consuming measurement process.…”

“…The GlycoB-IST tip was synthesized as previously described, with slight modifications. 14 Procedures of tip fabrication and analyte labeling are reported in the Supporting Information with an explanatory diagram and table (Figures S1, S2, and Table S1). The measurement process consisted of three steps: the interaction between the Cy3-labeled analyte and lectin beads, washing, and fluorescence scanning of the GlycoBIST tips.…”