O-糖链的非还原性解离及同时PMP衍生化的方法研究
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摘要
本文建立了以β-消除反应为基础的O-糖链的非还原性解离及同时PMP衍生化标记的新方法,得出以下结果：
1.把PMP衍生化与其它几种常见衍生化方法(AEC、ABEE、2-AP、2-AB)进行了比较,结果表明PMP衍生化方法具有明显优点：第一,PMP衍生化方法简单,衍生化产物稳定；第二,比较AEC、ABEE、2-AP、2-AB、PMP 5种衍生化产物的ESI-MS检测结果,PMP的衍生化产物质谱信号相对丰度强,检测灵敏度高；第三,PMP衍生化方法在碱性条件下进行,与β-消除反应具有相容性
2.以粘蛋白(mucin)为材料,利用PMP衍生反应体系发展非还原性O-糖链解离方法,选择氨水作为碱性介质；反应温度设置为50℃；反应时间设置为20 h；氨水：0.5 M的PMP甲醇溶液(体积比)=1：1；糖蛋白：PMP(摩尔比)=1：4。通过此新反应体系,我们得到还原性O-糖链,且其还原端在解离的同时被PMP标记,解离与衍生化反应完全,衍生产物稳定。
3.用非还原性解离新方法,我们对粘蛋白和胎牛蛋白的O-糖链进行非还原性解离,并用ESI-MS进行分析。糖链经PMP衍生化后,疏水性提高,产物无立体异构体,在245 nm处有很强的紫外吸收,可对其进行ESI-LC-MS分析,并进行方法学考察,色谱条件：流动相为22：78～30：70的乙腈-水(0.01 mol·L-1的醋酸铵缓冲液,pH=5.5),梯度洗脱60 min,流速为200μL·min-1,检测器为紫外检测器。实现了对粘蛋白中O-糖链的分离与质谱鉴定。
4.将非还原性解离O-糖链新方法应用于复杂生物样品中O-糖链的分析,对胎牛血清和人血清中的O-糖链进行解离及分析。并对解离得到的胎牛血清中的11种O-糖链和人血清中的25种O-糖链进行组成分析。表明该新反应体系适用于微量样品中O-糖链的解离和结构解析,对糖生物学方法学研究有重要意义。
We establised a non-reductive O-liked glycan released method based onβ-elimination that can release reducting O-linked glycans and simultaneously derivatization with PMP. The results were summarized as fellows:
1. By comparison of PMP derivatization with several other commonly used methods including AEC, ABEE,2-AP,2-AB derivatization, We found that PMP-derivatization method has several advantages:Firstly, the opration of PMP derivatization is simple and convinient, the derivative products are very stable. Secondly, We found that the relative abundance of PMP-derivatization products was much higher through analysis by ESI-MS. Thirdly, PMP derivatization reacted under alkaline conditions, compatibility withβ-elimination reaction.
2.We use PMP derivatization reaction system to develop new non-reductive O-glycan release method, mucin was used as a standard start material. We chose ammonia as the alkaline medium because it can provided a milder environment; release reaction temperature was set at 50℃in 20 h; ammonia:0.5 M PMP methanol solution (volume ratio)= 1:1; glycoprotein:PMP (mole ratio)= 1:4. During the procedure, we got O-linked oligosaccharides with the reducing end intact which were simultiniously labeled by PMP to protect the reducing end from peeling reaction.
3. The O-glycans of mucin released by new reaction system were separated by NP-HPLC and the PMP tag increased the sensitivity of MS detection. The optimized chromatographic conditions were:mobile phase was 22:78-30:70 of acetonitrile-water (0.01 mol.L-1 ammonium acetate buffer, pH= 5.5);gradient elution 60 min; a flow rate of 200μL·min-1; were detected by UV detector. The O-glycans released from mucin has basicly separated under this conditions and indicated O-glycans were quantitatively released.
4.We successfully released、purified and analysised the O-glycans of the complex biological samples such as fetal calf serum and human serum. This method could be applied to handle micro-scale O-glycan analysis.
1.把PMP衍生化与其它几种常见衍生化方法(AEC、ABEE、2-AP、2-AB)进行了比较,结果表明PMP衍生化方法具有明显优点：第一,PMP衍生化方法简单,衍生化产物稳定；第二,比较AEC、ABEE、2-AP、2-AB、PMP 5种衍生化产物的ESI-MS检测结果,PMP的衍生化产物质谱信号相对丰度强,检测灵敏度高；第三,PMP衍生化方法在碱性条件下进行,与β-消除反应具有相容性
2.以粘蛋白(mucin)为材料,利用PMP衍生反应体系发展非还原性O-糖链解离方法,选择氨水作为碱性介质；反应温度设置为50℃；反应时间设置为20 h；氨水：0.5 M的PMP甲醇溶液(体积比)=1：1；糖蛋白：PMP(摩尔比)=1：4。通过此新反应体系,我们得到还原性O-糖链,且其还原端在解离的同时被PMP标记,解离与衍生化反应完全,衍生产物稳定。
3.用非还原性解离新方法,我们对粘蛋白和胎牛蛋白的O-糖链进行非还原性解离,并用ESI-MS进行分析。糖链经PMP衍生化后,疏水性提高,产物无立体异构体,在245 nm处有很强的紫外吸收,可对其进行ESI-LC-MS分析,并进行方法学考察,色谱条件：流动相为22：78～30：70的乙腈-水(0.01 mol·L-1的醋酸铵缓冲液,pH=5.5),梯度洗脱60 min,流速为200μL·min-1,检测器为紫外检测器。实现了对粘蛋白中O-糖链的分离与质谱鉴定。
4.将非还原性解离O-糖链新方法应用于复杂生物样品中O-糖链的分析,对胎牛血清和人血清中的O-糖链进行解离及分析。并对解离得到的胎牛血清中的11种O-糖链和人血清中的25种O-糖链进行组成分析。表明该新反应体系适用于微量样品中O-糖链的解离和结构解析,对糖生物学方法学研究有重要意义。
We establised a non-reductive O-liked glycan released method based onβ-elimination that can release reducting O-linked glycans and simultaneously derivatization with PMP. The results were summarized as fellows:
1. By comparison of PMP derivatization with several other commonly used methods including AEC, ABEE,2-AP,2-AB derivatization, We found that PMP-derivatization method has several advantages:Firstly, the opration of PMP derivatization is simple and convinient, the derivative products are very stable. Secondly, We found that the relative abundance of PMP-derivatization products was much higher through analysis by ESI-MS. Thirdly, PMP derivatization reacted under alkaline conditions, compatibility withβ-elimination reaction.
2.We use PMP derivatization reaction system to develop new non-reductive O-glycan release method, mucin was used as a standard start material. We chose ammonia as the alkaline medium because it can provided a milder environment; release reaction temperature was set at 50℃in 20 h; ammonia:0.5 M PMP methanol solution (volume ratio)= 1:1; glycoprotein:PMP (mole ratio)= 1:4. During the procedure, we got O-linked oligosaccharides with the reducing end intact which were simultiniously labeled by PMP to protect the reducing end from peeling reaction.
3. The O-glycans of mucin released by new reaction system were separated by NP-HPLC and the PMP tag increased the sensitivity of MS detection. The optimized chromatographic conditions were:mobile phase was 22:78-30:70 of acetonitrile-water (0.01 mol.L-1 ammonium acetate buffer, pH= 5.5);gradient elution 60 min; a flow rate of 200μL·min-1; were detected by UV detector. The O-glycans released from mucin has basicly separated under this conditions and indicated O-glycans were quantitatively released.
4.We successfully released、purified and analysised the O-glycans of the complex biological samples such as fetal calf serum and human serum. This method could be applied to handle micro-scale O-glycan analysis.
引文
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