Royle Louise, Mattu Taj S, Hart Edmund, Langridge James I, Merry Anthony H, Murphy Neil, Harvey David J, Dwek Raymond A, Rudd Pauline M
Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom.
Anal Biochem. 2002 May 1;304(1):70-90. doi: 10.1006/abio.2002.5619.
A sensitive, rapid, quantitative strategy has been developed for O-glycan analysis. A structural database has been constructed that currently contains analytical parameters for more than 50 glycans, enabling identification of O-glycans at the subpicomole level. The database contains the structure, molecular weight, and both normal and reversed-phase HPLC elution positions for each glycan. These observed parameters reflect the mass, three-dimensional shape, and hydrophobicity of the glycans and, therefore, provide information relating to linkage and arm specificity as well as monosaccharide composition. Initially the database was constructed by analyzing glycans released by mild hydrazinolysis from bovine serum fetuin, synthetic glycopeptides, human glycophorin A, and serum IgA1. The structures of the fluorescently labeled sugars were determined from a combination of HPLC data, mass spectrometric composition and mass fragmentation data, and exoglycosidase digestions. This approach was then applied to human neutrophil gelatinase B and secretory IgA, where 18 and 25 O-glycans were identified, respectively, and the parameters of these glycans were added to the database. This approach provides a basis for the analysis of subpicomole quantities of O-glycans from normal levels of natural glycoproteins.
已开发出一种用于O-聚糖分析的灵敏、快速、定量策略。构建了一个结构数据库，目前包含50多种聚糖的分析参数，能够在亚皮摩尔水平鉴定O-聚糖。该数据库包含每种聚糖的结构、分子量以及正相和反相HPLC洗脱位置。这些观察到的参数反映了聚糖的质量、三维形状和疏水性，因此提供了有关连接和臂特异性以及单糖组成的信息。最初，该数据库是通过分析从牛血清胎球蛋白、合成糖肽、人血型糖蛋白A和血清IgA1中经温和肼解释放的聚糖构建的。荧光标记糖的结构由HPLC数据、质谱组成和质量碎片数据以及外切糖苷酶消化的组合确定。然后将该方法应用于人中性粒细胞明胶酶B和分泌型IgA，分别鉴定出18种和25种O-聚糖，并将这些聚糖的参数添加到数据库中。该方法为分析来自正常水平天然糖蛋白的亚皮摩尔量O-聚糖提供了基础。
