Sensitive analysis of O-linked sugar chains by fluorescence labeling

通过荧光标记对 O-连接糖链进行灵敏分析

• 批准号: 01470147
• 负责人: HASE Sumihiro
• 金额: $ 4.35万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1989
• 资助国家: 日本
• 起止时间: 1989 至 1990
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-01470147/
• 关键词: Sugar chain; HPLC; Fluorescence labeling; Glycoprotein; O-Sugar chain; Hydrazinolysis; Pyridylamination; Oー配糖体; ヒドラジン分解; 2次元マップ

Sugar chains linked to serine or threonine residues of glycoproteins through and O-glycosidic bond are found in mucin, immunoglobulin, hormonos, etc. To liberate O-linked sugar chains, a method based on alkaline-borohydride treatment is mostly used. We have developed a fluorescence labeling method for highly sensitive detection of sugar chains and applied the method to analyze N-linked sugar chains. In this study we applied the method to Oーlinked sugar chains.Sugar chains liberated by alkaline-borohydride have no reducing end aldehyde group, therefore fluorescence labeling of the sugar chains is not possible. So a new method is required to tag the reducing end of sugar chains with 2-aminopyridine. Fot this purpose we used hydrazinolysis. To analyze the liberation of sugar chains quantitatively, we prepared glycopeptide having two O-linked sugar chains and fluorescence labeled sugar chains. These structures were identified by gas chromatography-mass spectrometry. Sugar chains were liberated by hydrazinolysis of the glycopeptide and sugar chains thus obtained were analyzed as their pyridylamino derivatives by HPLC.Under the established conditions, sugar chains were liberated from glycopeptides in a yield of 80%. The method was applied to an glycoprotein and pyridylamino derivatives of sugar chains were detected by HPLC.

糖链与糖蛋白的丝氨酸或苏氨酸残基通过O-糖苷键连接在一起，存在于粘蛋白、免疫球蛋白、激素等中。为了释放O-连接的糖链，通常采用基于碱性-硼氢化物处理的方法。我们建立了一种高灵敏的检测糖链的荧光标记方法，并将该方法应用于N-连接糖链的分析。在本研究中，我们将该方法应用于O-ー连接的糖链，碱性-硼氢化物释放的糖链没有还原的末端醛基团，因此不可能对糖链进行荧光标记。因此，需要一种新的方法来标记糖链的还原末端2-氨基吡啶。为此，我们采用了肼分解法。为了定量分析糖链的释放，我们制备了具有两个O-连接糖链的糖肽和荧光标记的糖链。用气相色谱-质谱法对其结构进行了鉴定。糖肽通过肼裂解得到糖链，用高效液相色谱仪分析得到的糖链是它们的吡啶氨基衍生物。在所建立的条件下，糖肽以80%的产率从糖链中释放出来。将该方法应用于一种糖蛋白，并用高效液相色谱法检测了糖链上的吡啶氨基衍生物。

项目成果

A.Kondo: "Improved Method for Fluorescence Labeling of Sugar Chains with Sialic Acid Residues" Agric.Biol.Chem.54. 2169-2170 (1990)

A.Kondo：“唾液酸残基糖链荧光标记的改进方法”Agric.Biol.Chem.54。

H.Oku: "Separation of OligomannoseーType Sugar Chains Having One to Five Mannose Residues by HighーPerformance Liquid Chromatography as Their Pyridylamino Derivatives" Anal.Biochem.185. 331-334 (1990)

H.Oku：“通过高效液相色谱分离具有一到五个甘露糖残基的寡甘露糖型糖链作为吡啶氨基衍生物”Anal.Biochem.185（1990）。

J.Suzuki: "Analysis by Highーperformance Anionーexchange Chromatography of Component Sugars as Their Fluorescent Pyridylamino Derivatives" Agric.Biol.Chem.55. 283-284 (1991)

J.Suzuki：“通过高效阴离子交换色谱分析组分糖作为荧光吡啶氨基衍生物”Agric.Biol.Chem.55 (1991)。

A. Kondo: "Improved Method for Fluorescence Labeling of Sugar Chains with Sialic Acid Residues" Agric. Biol. Chem.54-8. 2169-2170 (1990)

A. Kondo：“唾液酸残基糖链荧光标记的改进方法”Agric。

長谷純宏: "(糖関連化学物質・糖鎖微量分析)研究室で役に立つ新しい試薬" 医学書院, 2 (1989)

Sumihiro Hase：“实验室中有用的新试剂（糖相关化学物质和糖链的痕量分析）” Igakushoin，2 (1989)