糖蛋白N-糖链释放及荧光标记衍生物的电喷雾质谱研究
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摘要
糖基化是蛋白质翻译后修饰的重要过程之一,糖基化蛋白糖链在蛋白质折叠、定位及蛋白间的相互作用中具有重要的意义,而要深入研究糖链在此过程中的功能和调控机制,就首先需要对糖蛋白糖链进行解离。对于O-糖链目前较好的解离方法是化学方法,而N-糖链多用特异性糖苷酶等进行解离。本研究在已有的N-糖蛋白糖链释放方法基础上建立了一种非特异性的链酶蛋白酶E (Pronase E)酶解释放N-糖链,优化酶解条件得到仅带一个氨基酸的糖氨酸,9-氯甲酸芴甲酯(Fmoc-Cl)及苯异硫氰酸酯(FITC)对糖氨酸进行衍生,新酶对糖氨酸衍生物酶解回收还原性糖链的方法。得到了以下结论：
1. Pronase E酶解释放N-糖链：非特异性蛋白酶Pronase E代替N-糖苷酶F酶解糖蛋白牛胰核糖核酸酶B和鸡白蛋白,优化酶解条件选择当糖蛋白与蛋白酶质量比为1：1时,得到只带一个天冬氨酸(Asn)的闭环N-糖链,称其为糖氨酸(Glycan-Asn),这样实现了对糖蛋白N-糖链的释放,同时为糖链引入了天然的-NH2活性基团,还保持了糖链原有的还原端闭环结构。
2.酶解糖氨酸衍生物及LC-MS分析：以9-氯甲酸芴甲酯(Fmoc-Cl)为衍生试剂对解离后的糖氨酸进行衍生,采用高效液相色谱-电喷雾质谱联用技术(HPLC-ESI/MS)对Fmoc-Cl糖氨酸衍生物进行分析,并对HPLC完全分离后的各糖链分管收集,能够得到纯度较高的单一寡糖链,进一步酶解糖氨酸衍生物。
3.苯异硫氰酸酯(FITC)标记糖链及糖氨酸：还原性寡糖由于没有天然的伯氨基,因此以对氨基苯作为手臂化合物,通过还原氨化法引入伯氨基与FITC反应,合成寡糖的荧光探针。以乳糖为标准寡糖链,优化FITC标记寡糖对氨基苯衍生物的条件,发现在样品溶液中加入过量的FITC吡啶丙酮溶液、三乙胺调pH至11.0、在40℃反应6h后用电喷雾质谱进行分析,寡糖衍生化完全；同时,进一步成功地将该荧光素衍生寡糖的方法应用于其他类型寡糖的衍生(包括壳寡糖及Pronase E酶解鸡白蛋白后的N-糖氨酸)。
综上所述,本研究建立了一种非特异性蛋白酶Pronase E酶解释放N-糖链、荧光试剂Fmoc-Cl及FITC标记糖链、LC-MS实时分析同时新酶酶解回收还原性糖链的新方法。该方法对N-糖链释放完全、荧光衍生化效率高；操作简单、通用性强,对分离和制备还原性N-糖链以及研究糖链与蛋白质的相互作用具有重要的意义。
Glycosylation, an important process of the post translation of protein, plays a key role in folding、positioning and the interaction of proteins. In order to study the function and control mechanism of glycans, glycans released is necessary. O-linked glycans could be released from glycoprotein using chemical method while emzyme digestion is an available method used for N-linked glycans. In this study a non-specific enzymatic digestion of N-glycoprotein was developed, Under the optimized digestion conditions N-glycans bearing a single asparagine residue, keeping the close ring structure at reducing end,was released. ESI-MS was used to analyze the glycan-acid. New enzyme was used to digest glycan-Asn in order to recovery the reducing glycans. A brief summary is described as following:
1. A non-specific enzymatic digestion of glycopeptides to release N-glycans from glycoprotein:Pronase E instead of the traditional PNGase F was used to release glycopeptides from Ribo B and Chicken Albumin, Under the optimized digestion conditions that the quantity ratio of the Pronase E to glycoprotein was 1:1, the closed-ring oligosaccharides named glycans-Asn with a single amino acid (Asn) were obtained. This method not only remains the native structure of N-glycans, but also provides possibility of fluorescent derivation with primary amine as function group.
2. A modified 9-Fluorenylmethyl chloroformate (Fmoc-Cl) pre-column derivatization procedure has also been successfully applied to these glycans-Asn:the Fmoc-Cl labeled glycans-Asn products were characterized by high performance liquid chromatography/electrospray ionization mass spectrometry (HPLC-ESI/MS). to prepare pured glycans we collected glycans respectively after HPLC analysis, then a new enzyme was introduced to digest the collected glycans to get reducing glycans.
3. Glycans and glycan-acid derivatized by Fluorescein Isothiocyanate:For some reducative glycans without natural amine group, we used an indirect method which p-Phenylenediamine was chosen as a linker and introduced an free amino group to oligosaccharide through reductive amination reaction, which was ready for further derivatized with FITC. The conditions for FITC labeling were optimized as derivative reaction at 40℃for 6 h (pH=11), FITC dissolved in acetone. Using this procedure, we have prepared fluorescein derivatives of Chitoosaccharides and Glycan-Asn (Chicken Albumin digested by Pronase E)
In conclusion, we established a new method about which glycoprotein was digested by Pronase E to release glycans, glycans was derivated by Fmoc-Cl/FITC to analyze with on-line HPLC and glycan-acid derivatives were digested by new enzyme to recovery free glycans. This enzyme digestion has exhibited a high efficiency in terms of glycoprotein digestion and fluorescent derivation, and HPLC separation is in favor of the reducing glycans recovering. Futherly, it was very applicable and easy to operate, which lead to the convenience of separation and preparation of pure N-glycans and explore the interaction between glycan and proteins.
1. Pronase E酶解释放N-糖链：非特异性蛋白酶Pronase E代替N-糖苷酶F酶解糖蛋白牛胰核糖核酸酶B和鸡白蛋白,优化酶解条件选择当糖蛋白与蛋白酶质量比为1：1时,得到只带一个天冬氨酸(Asn)的闭环N-糖链,称其为糖氨酸(Glycan-Asn),这样实现了对糖蛋白N-糖链的释放,同时为糖链引入了天然的-NH2活性基团,还保持了糖链原有的还原端闭环结构。
2.酶解糖氨酸衍生物及LC-MS分析：以9-氯甲酸芴甲酯(Fmoc-Cl)为衍生试剂对解离后的糖氨酸进行衍生,采用高效液相色谱-电喷雾质谱联用技术(HPLC-ESI/MS)对Fmoc-Cl糖氨酸衍生物进行分析,并对HPLC完全分离后的各糖链分管收集,能够得到纯度较高的单一寡糖链,进一步酶解糖氨酸衍生物。
3.苯异硫氰酸酯(FITC)标记糖链及糖氨酸：还原性寡糖由于没有天然的伯氨基,因此以对氨基苯作为手臂化合物,通过还原氨化法引入伯氨基与FITC反应,合成寡糖的荧光探针。以乳糖为标准寡糖链,优化FITC标记寡糖对氨基苯衍生物的条件,发现在样品溶液中加入过量的FITC吡啶丙酮溶液、三乙胺调pH至11.0、在40℃反应6h后用电喷雾质谱进行分析,寡糖衍生化完全；同时,进一步成功地将该荧光素衍生寡糖的方法应用于其他类型寡糖的衍生(包括壳寡糖及Pronase E酶解鸡白蛋白后的N-糖氨酸)。
综上所述,本研究建立了一种非特异性蛋白酶Pronase E酶解释放N-糖链、荧光试剂Fmoc-Cl及FITC标记糖链、LC-MS实时分析同时新酶酶解回收还原性糖链的新方法。该方法对N-糖链释放完全、荧光衍生化效率高；操作简单、通用性强,对分离和制备还原性N-糖链以及研究糖链与蛋白质的相互作用具有重要的意义。
Glycosylation, an important process of the post translation of protein, plays a key role in folding、positioning and the interaction of proteins. In order to study the function and control mechanism of glycans, glycans released is necessary. O-linked glycans could be released from glycoprotein using chemical method while emzyme digestion is an available method used for N-linked glycans. In this study a non-specific enzymatic digestion of N-glycoprotein was developed, Under the optimized digestion conditions N-glycans bearing a single asparagine residue, keeping the close ring structure at reducing end,was released. ESI-MS was used to analyze the glycan-acid. New enzyme was used to digest glycan-Asn in order to recovery the reducing glycans. A brief summary is described as following:
1. A non-specific enzymatic digestion of glycopeptides to release N-glycans from glycoprotein:Pronase E instead of the traditional PNGase F was used to release glycopeptides from Ribo B and Chicken Albumin, Under the optimized digestion conditions that the quantity ratio of the Pronase E to glycoprotein was 1:1, the closed-ring oligosaccharides named glycans-Asn with a single amino acid (Asn) were obtained. This method not only remains the native structure of N-glycans, but also provides possibility of fluorescent derivation with primary amine as function group.
2. A modified 9-Fluorenylmethyl chloroformate (Fmoc-Cl) pre-column derivatization procedure has also been successfully applied to these glycans-Asn:the Fmoc-Cl labeled glycans-Asn products were characterized by high performance liquid chromatography/electrospray ionization mass spectrometry (HPLC-ESI/MS). to prepare pured glycans we collected glycans respectively after HPLC analysis, then a new enzyme was introduced to digest the collected glycans to get reducing glycans.
3. Glycans and glycan-acid derivatized by Fluorescein Isothiocyanate:For some reducative glycans without natural amine group, we used an indirect method which p-Phenylenediamine was chosen as a linker and introduced an free amino group to oligosaccharide through reductive amination reaction, which was ready for further derivatized with FITC. The conditions for FITC labeling were optimized as derivative reaction at 40℃for 6 h (pH=11), FITC dissolved in acetone. Using this procedure, we have prepared fluorescein derivatives of Chitoosaccharides and Glycan-Asn (Chicken Albumin digested by Pronase E)
In conclusion, we established a new method about which glycoprotein was digested by Pronase E to release glycans, glycans was derivated by Fmoc-Cl/FITC to analyze with on-line HPLC and glycan-acid derivatives were digested by new enzyme to recovery free glycans. This enzyme digestion has exhibited a high efficiency in terms of glycoprotein digestion and fluorescent derivation, and HPLC separation is in favor of the reducing glycans recovering. Futherly, it was very applicable and easy to operate, which lead to the convenience of separation and preparation of pure N-glycans and explore the interaction between glycan and proteins.
引文
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