Department of Biochemistry, Boston University, Boston, Massachusetts.
Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia.
Curr Protoc. 2021 Mar;1(3):e84. doi: 10.1002/cpz1.84.
Identification of N- and O-glycosylation on specific sites of proteins, along with glycan structural information, is necessary to determine the roles glycoproteins play in normal and pathologic cellular functions. Because such glycosylation is macro- and micro-heterogeneous and alters the dissociation behavior of glycopeptides, specific sample preparation, mass spectrometry, and data analysis techniques are required. Advanced tandem mass spectrometry-based glycoproteomics coupled with powerful data mining algorithms are key elements for characterization of protein glycosylation. This article includes the detailed, streamlined sample preparation method for liquid chromatography-mass spectrometry data acquisition and subsequent bioinformatics-based data annotation using the publicly available GlycReSoft program for highly efficient identification and quantification of glycoprotein glycosylation. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Characterization of glycans and site occupancy on purified glycoprotein Support Protocol 1: In-gel digestion of glycoproteins Support Protocol 2: Detection of glycoproteins from cells/tissue through glycopeptide enrichment Basic Protocol 2: Acquisition of glycopeptides through high-resolution nano-LC-MS/MS Basic Protocol 3: Identification and quantification of glycopeptides using GlycReSoft.
鉴定蛋白质特定部位的 N-和 O-糖基化以及聚糖结构信息，对于确定糖蛋白在正常和病理细胞功能中的作用是必要的。由于这种糖基化具有宏观和微观异质性，并改变了糖肽的解离行为，因此需要特定的样品制备、质谱和数据分析技术。基于高级串联质谱的糖蛋白质组学与强大的数据挖掘算法相结合，是糖蛋白特征描述的关键要素。本文包括详细、简化的样品制备方法，用于液相色谱-质谱数据采集，以及使用公开的 GlycReSoft 程序进行基于生物信息学的数据注释，用于高效鉴定和定量糖蛋白的糖基化。© 2021 威利父子公司。基本方案 1：纯化糖蛋白上聚糖的特征描述和位点占有率支持方案 1：糖蛋白的胶内消化支持方案 2：通过糖肽富集从细胞/组织中检测糖蛋白基本方案 2：通过高分辨率纳升 LC-MS/MS 获得糖肽基本方案 3：使用 GlycReSoft 鉴定和定量糖肽。
