Li Guoyun, Li Lingyun, Tian Fang, Zhang Linxia, Xue Changhu, Linhardt Robert J
†College of Food Science and Technology, Ocean University of China, Qingdao, Shandong 266003, China.
‡Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180, United States.
ACS Chem Biol. 2015 May 15;10(5):1303-10. doi: 10.1021/acschembio.5b00011. Epub 2015 Feb 23.
Glycosaminoglycans (GAGs), a family of polysaccharides widely distributed in eukaryotic cells, are responsible for a wide array of biological functions. Quantitative disaccharide compositional analysis is one of the primary ways to characterize the GAG structure. This structural analysis is typically time-consuming (1-2 weeks) and labor intensive, requiring GAG recovery and multistep purification, prior to the enzymatic/chemical digestion of GAGs, and finally their analysis. Moreover, 10(5)-10(7) cells are usually required for compositional analysis. We report a sensitive, rapid, and quantitative analysis of GAGs present in a small number of cells. Commonly studied cell lines were selected based on phenotypic properties related to the biological functions of GAGs. These cells were lysed using a commercial surfactant reagent, sonicated, and digested with polysaccharide lyases. The resulting disaccharides were recovered by centrifugal filtration, labeled with 2-aminoacridone, and analyzed by liquid chromatography (LC)-mass spectrometry (MS). Using a highly sensitive MS method, multiple reaction monitoring (MRM), the limit of detection for each disaccharide was reduced to 0.5-1.0 pg, as compared with 1.0-5.0 ng obtained using standard LC-MS analysis. Sample preparation time was reduced to 1-2 days, and the cell number required was reduced to 5000 cells for complete GAG characterization to as few as 500 cells for the characterization of the major GAG disaccharide components. Our survey of the glycosaminoglycanomes of the 20 selected cell lines reveals major differences in their GAG amounts and compositions. Structure-function relationships are explored using these data, suggesting the utility of this method in cellular glycobiology.
糖胺聚糖（GAGs）是一类广泛分布于真核细胞中的多糖，具有多种生物学功能。定量二糖组成分析是表征GAG结构的主要方法之一。这种结构分析通常耗时较长（1 - 2周）且劳动强度大，需要在对GAG进行酶解/化学消化并最终进行分析之前，先回收GAG并进行多步纯化。此外，组成分析通常需要10⁵ - 10⁷个细胞。我们报告了一种对少量细胞中存在的GAG进行灵敏、快速且定量的分析方法。基于与GAG生物学功能相关的表型特性选择了常用的细胞系。这些细胞用市售表面活性剂试剂裂解、超声处理，并用多糖裂解酶消化。所得二糖通过离心过滤回收，用2 - 氨基吖啶标记，然后通过液相色谱（LC）-质谱（MS）分析。使用高灵敏度的质谱方法——多反应监测（MRM），与标准LC - MS分析得到的1.0 - 5.0 ng相比，每种二糖的检测限降低至0.5 - 1.0 pg。样品制备时间缩短至1 - 2天，完整表征GAG所需的细胞数量减少至5000个细胞，而表征主要GAG二糖成分所需的细胞数量则少至500个细胞。我们对20个选定细胞系的糖胺聚糖组进行的调查揭示了它们在GAG含量和组成上的主要差异。利用这些数据探索了结构 - 功能关系，表明该方法在细胞糖生物学中的实用性。
