Development of the method for high-sensitivity structural analysis of carbohydrate moieties of a glycoprotein by mass spectrometry

糖蛋白碳水化合物部分质谱高灵敏度结构分析方法的开发

• 批准号: 12558077
• 负责人: TAKAO Toshifumi
• 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 Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12558077/
• 关键词: mass spectrometry; glycoprotein; carbohydrate; chemical derivatization; structural analysis; fragment ions; glycopeptide; 逆相担体; ペプチド・マス・フィンガープリント

In order to establish the method for high-sensitivity structural analysis of carbohydrate moieties of a glycoprotein, the following two approaches have been examined: 1 ) electrospray-ionization (ESI) tandem mass spectrometry (MS/MS) has been directly applied for the analysis of glycopeptides with relatively large molecular masses (3000 Da 〜), which were obtained by an in-gel digestion of a glycoprotein. 2) the chemical derivatization with 4-aminobenzoic acid 2-diethylaminoethyl ester (ABDEAE), which was developed for the high-sensitivity detection and structural analysis of carbohydrates by MS, has been applied to carbohydrates cleaved from a glycoprotein with PNGase F. As the results obtained for human transferrin 1) the two glycopeptides, which were produced by in-gel digestion with lysylendopeptidase and separated by HPLC, gave specific fragmentation at glycosidic bonds upon low-energy collision induced dissociation in ESI-MS/MS. It could clearly reveal the sequence and partial branching structure of the carbohydrate moieties. 2) the carbohydrate moieties derived from transferrin were derivatized in gel with ABDEAE. The excess reagent was found to be efficiently removed by passing a reaction mixture through a polymer-based amino-propyl resin In addition, the salts were separated from the carbohydrate derivatives by a reversed-phase resin. Consequently, the derivatives could be subjected to on-line micro-HPLC/ESI-MS, which allowed the detection of such those prepared from the gel band corresponding to ca. 10 pmol of transferrin as a charging amount to SDS-PAGE."SeqMS", a software aid for de novo sequencing by MS/MS, has been modified so as to efficiently interpret MS/MS spectra of both glycopeptides and carbohydrate derivatives.

为了建立对糖蛋白的碳含量部分进行高敏性结构分析的方法，已经研究了以下两种方法：1）电喷雾量离子化（ESI）串联质谱（MS/MS）已直接应用于与相对较大的分子群体（3000 DADAY）的分析，以分析糖蛋白酶（3000）糖蛋白。 2）用4-氨基苯甲酸2-二甲基乙酯（ABDEAE）进行化学衍生化，该化学衍生物是用于通过MS对碳氢化物对碳氢化物进行高敏性检测和结构分析的开发的，已被用于从PNGASE F. copopotin copotion裂解的碳含量。用溶液肽酶消化并通过HPLC分离，在低能量碰撞诱导的ESI-MS/MS中解离时在糖苷键处得到了特异性片段化。它可以清楚地揭示碳水化合物部分的序列和部分分支结构。 2）用Abdeae将衍生自转移的碳水化合物部分衍生在凝胶中。发现过量的试剂通过通过聚合物的氨基 - 丙基树脂将反应混合物通过反相反相 - 相位的树脂从碳水化的衍生物中分离而有效地去除。因此，衍生物可以在线微HPLC/ESI-MS进行，从而可以检测从与Ca相对应的凝胶带中制备的这些。 10 PMOL的转铁蛋白作为SDS-PAGE的充电量。“ Seqms”是通过MS/MS进行从头测序的软件辅助，以有效地解释糖肽和碳羟基衍生物的MS/MS光谱。

项目成果

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Toshifumi Takao、Yoshinori Satomi：“‘质谱’基本生化实验方法，第 3 卷（蛋白质，I.检测和结构分析方法）”东京化学同人 153-167（2001）。

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F.-Cossio, J. et al.: "Automated Interpretation of Low-Energy Collision-Induced Dissociation Spectra by "SeqAJS" , a Software Aid for De Novo Sequencing by Tandem Mass Spectrometry"Electrophoresis. 21. 1694-1699 (2000)

F.-Cossio, J. 等人：“通过“SeqAJS”自动解释低能碰撞诱导解离光谱，这是一种通过串联质谱进行从头测序的软件辅助工具”电泳。

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Betancourt, L. 等人：“胰蛋白酶消化物中 N 末端封闭肽的选择性分离和鉴定”J。