Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia, USA.
Nat Methods. 2011 Jan;8(1):85-90. doi: 10.1038/nmeth.1540. Epub 2010 Dec 5.
Major challenges of glycomics are to characterize a glycome and identify functional glycans as ligands for glycan-binding proteins (GBPs). To address these issues we developed a general strategy termed shotgun glycomics. We focus on glycosphingolipids (GSLs), a class of glycoconjugates that is challenging to study, recognized by toxins, antibodies and GBPs. We derivatized GSLs extracted from cells with a heterobifunctional fluorescent tag suitable for covalent immobilization. We separated fluorescent GSLs by multidimensional chromatography, quantified them and coupled them to glass slides to create GSL shotgun microarrays. Then we interrogated the microarrays with cholera toxin, antibodies and sera from individuals with Lyme disease to identify biologically relevant GSLs that we subsequently characterized by mass spectrometry. Shotgun glycomics incorporating GSLs and potentially glycoprotein-derived glycans is an approach for accessing the complex glycomes of animal cells and is a strategy for focusing structural analyses on functionally important glycans.
糖组学的主要挑战是对糖组进行特征分析，并确定功能性聚糖作为糖结合蛋白 (GBP) 的配体。为了解决这些问题，我们开发了一种称为“鸟枪法糖组学”的通用策略。我们专注于糖脂（GSLs），这是一类糖缀合物，其难以研究，被毒素、抗体和 GBP 识别。我们用一种适合共价固定的异双功能荧光标记物对从细胞中提取的 GSL 进行衍生化。我们通过多维色谱法分离荧光 GSL，对其进行定量，并将其偶联到载玻片上，以创建 GSL 鸟枪法微阵列。然后，我们用霍乱毒素、抗体和莱姆病患者的血清对微阵列进行检测，以鉴定具有生物学意义的 GSL，随后我们通过质谱法对其进行表征。包含 GSL 和潜在糖蛋白衍生聚糖的鸟枪法糖组学是一种用于研究动物细胞复杂糖组的方法，也是一种将结构分析集中在功能重要聚糖上的策略。
