Nagai-Okatani Chiaki, Zou Xia, Matsuda Atsushi, Itakura Yoko, Toyoda Masashi, Zhang Yan, Kuno Atsushi
Glycoscience and Glycotechnology Research Group, Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan.
Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai, China.
Methods Mol Biol. 2022;2460:161-180. doi: 10.1007/978-1-0716-2148-6_10.
Lectin microarray (LMA) is a high-sensitive glycan analysis technology used to obtain global glycomic profiles of both N- and O-glycans attached not only to purified glycoproteins but also to crude glycoprotein samples. Through additional use of laser microdissection (LMD) for tissue collection, we developed an LMA-based glycomic profiling technique for a specific type of cells in a tiny area of formalin-fixed paraffin-embedded (FFPE) tissue sections. This LMD-LMA method makes it possible to obtain reproducible tissue glycomic profiles that can be compared with each other, using a unified protocol for all procedures, including FFPE tissue preparation, tissue staining, protein extraction and labeling, and LMA analysis. Here, we describe the standardized LMD-LMA procedure for a "tissue glycome mapping" approach, which facilitates an in-depth understanding of region- and tissue-specific protein glycosylation. We also describe potential applications of the spatial tissue glycomic profiles, including histochemical analysis for evaluating distribution of lectin ligands and a fluorescence LMD-LMA method for cell type-selective glycomic profiling using a cell type-specific probe, composed of a lectin and an antibody. The protocols presented here will accelerate the effective utilization of FFPE tissue specimens by providing tissue glycome maps for the discovery of the biological roles and disease-related alterations of protein glycosylation.
凝集素微阵列（LMA）是一种高灵敏度的聚糖分析技术，用于获取不仅与纯化糖蛋白而且与粗糖蛋白样品相连的N-聚糖和O-聚糖的整体糖组图谱。通过额外使用激光显微切割（LMD）进行组织采集，我们开发了一种基于LMA的糖组分析技术，用于福尔马林固定石蜡包埋（FFPE）组织切片微小区域内特定类型的细胞。这种LMD-LMA方法使得能够获得可相互比较的可重复组织糖组图谱，该图谱使用统一的方案进行所有程序，包括FFPE组织制备、组织染色、蛋白质提取和标记以及LMA分析。在这里，我们描述了一种用于“组织糖组图谱绘制”方法的标准化LMD-LMA程序，该程序有助于深入了解区域和组织特异性蛋白质糖基化。我们还描述了空间组织糖组图谱的潜在应用，包括用于评估凝集素配体分布的组织化学分析以及使用由凝集素和抗体组成的细胞类型特异性探针进行细胞类型选择性糖组分析的荧光LMD-LMA方法。本文介绍的方案将通过提供组织糖组图谱以发现蛋白质糖基化的生物学作用和疾病相关改变，加速FFPE组织标本的有效利用。
