The glycocalyx comprises glycosylated proteins and lipids and fcorms the outermost layer of cells. It is involved in fundamental inter- and intracellular processes, including non-self-cell and self-cell recognition, cell signaling, cellular structure maintenance, and immune protection. Characterization of the glycocalyx is thus essential to understanding cell physiology and elucidating its role in promoting health and disease. This protocol describes how to comprehensively characterize the glycocalyx N-glycans and O-glycans of glycoproteins, as well as intact glycolipids in parallel, using the same enriched membrane fraction. Profiling of the glycans and the glycolipids is performed using nanoflow liquid chromatography–mass spectrometry (nanoLC-MS). Sample preparation, quantitative LC–tandem MS (LC-MS/MS) analysis, and data processing methods are provided. In addition, we discuss glycoproteomic analysis that yields the site-specific glycosylation of membrane proteins. To reduce the amount of sample needed, N-glycan, O-glycan, and glycolipid analyses are performed on the same enriched fraction, whereas glycoproteomic analysis is performed on a separate enriched fraction. The sample preparation process takes 2–3 d, whereas the time spent on instrumental and data analyses could vary from 1 to 5 d for different sample sizes. This workflow is applicable to both cell and tissue samples. Systematic changes in the glycocalyx associated with specific glycoforms and glycoconjugates can be monitored with quantitation using this protocol. The ability to quantitate individual glycoforms and glycoconjugates will find utility in a broad range of fundamental and applied clinical studies, including glycan-based biomarker discovery and therapeutics.

糖萼包含糖基化的蛋白质和脂质，并聚集在细胞的最外层。它参与基本的细胞间和细胞内过程，包括非自我细胞和自我细胞识别，细胞信号传导，细胞结构维持和免疫保护。因此，糖萼的表征对于理解细胞生理学以及阐明其在促进健康和疾病中的作用至关重要。该协议描述了如何使用相同的富集膜级分全面表征糖蛋白的糖萼N-聚糖和O-聚糖以及平行的完整糖脂。使用纳流液相色谱-质谱（nanoLC-MS）对聚糖和糖脂进行分析。样品制备，定量LC级联质谱（LC-MS / MS）分析，提供了数据处理方法。另外，我们讨论了糖蛋白组学分析，该分析产生了膜蛋白的位点特异性糖基化。为了减少所需的样品量，对相同的富集馏分进行N-聚糖，O-聚糖和糖脂分析，而对单独的富集馏分进行糖蛋白组学分析。样品制备过程需要2到3 d，而对于不同的样品量，仪器和数据分析所花费的时间可能从1到5 d不等。此工作流程适用于细胞和组织样品。使用此方案可以定量监测与特定糖型和糖缀合物相关的糖萼的系统变化。