Sojitra Mirat, Schmidt Edward N, Lima Guilherme M, Carpenter Eric J, McCord Kelli A, Atrazhev Alexey, Macauley Matthew S, Derda Ratmir
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.
Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada.
Nat Protoc. 2025 Apr;20(4):989-1019. doi: 10.1038/s41596-024-01070-3. Epub 2024 Oct 16.
Glycans constitute a significant fraction of biomolecular diversity on cellular surfaces across all kingdoms of life. As the structure of glycans is not directly encoded by the organism's DNA, it is impossible to use high-throughput DNA technologies to study the role of cellular glycosylation or to understand how glycocalyx is recognized by glycan-binding proteins (GBPs). To address this gap, we recently described a liquid glycan array (LiGA) platform that allows profiling of glycan-GBP interactions on the surface of live cells in vitro and in vivo using next-generation sequencing. LiGA is a library of DNA-barcoded bacteriophages, where each clonal bacteriophage displays 5-1,500 copies of a glycan and the distinct DNA barcode inside each bacteriophage clone encodes the structure and density of the displayed glycans. Deep sequencing of the glycophages associated with live cells yields a glycan-binding profile of GBPs expressed on the surface of cells. This protocol provides detailed instructions for how to use LiGA to probe cell surface receptors and includes information on the preparation of glycophages, analysis by MALDI-TOF mass spectrometry, the assembly of a LiGA library and its deep sequencing. Using this protocol, we measure glycan-binding profiles of the immunomodulatory sialic acid-binding immunoglobulin-like lectins‑1, -2, -6, -7 and -9 expressed on the surface of different cell types. Compared with existing methods that require complex specialist equipment, this method allows users with basic molecular biology expertise to measure the precise glycan-binding profile of GBPs on the surface of any cell type expressing exogenous GBP within 2-3 d.
聚糖构成了所有生命王国细胞表面生物分子多样性的重要部分。由于聚糖的结构并非由生物体的DNA直接编码，因此无法使用高通量DNA技术来研究细胞糖基化的作用，也无法了解聚糖结合蛋白（GBP）如何识别糖萼。为了填补这一空白，我们最近描述了一种液体聚糖阵列（LiGA）平台，该平台能够利用下一代测序技术在体外和体内对活细胞表面的聚糖-GBP相互作用进行分析。LiGA是一个DNA条形码噬菌体文库，每个克隆噬菌体展示5至1500个聚糖拷贝，每个噬菌体克隆内独特的DNA条形码编码所展示聚糖的结构和密度。对与活细胞相关的糖噬菌体进行深度测序，可得出细胞表面表达的GBP的聚糖结合图谱。本方案提供了关于如何使用LiGA探测细胞表面受体的详细说明，包括糖噬菌体的制备、基质辅助激光解吸电离飞行时间质谱分析、LiGA文库的组装及其深度测序的信息。使用本方案，我们测量了在不同细胞类型表面表达的免疫调节性唾液酸结合免疫球蛋白样凝集素-1、-2、-6、-7和-9的聚糖结合图谱。与需要复杂专业设备的现有方法相比，该方法使具有基础分子生物学专业知识的用户能够在2至3天内测量任何表达外源性GBP的细胞类型表面GBP的精确聚糖结合图谱。
