【作者】 刘影；

【导师】 黄琳娟；

【作者基本信息】 西北大学 ， 细胞生物学， 2010， 硕士

【摘要】 糖生物学是21世纪生命科学研究的重要组成部分。要深入研究糖链在生命活动中的功能和调控机制,首要任务是获得并分析生物糖组。电喷雾电离质谱(ESI-MS)是目前分析微量糖组的强大工具,但对样品的前处理要求高。本文针对目前存在的问题建立了微量复杂生物样品糖蛋白N-糖链电喷雾电离质谱分析前处理的方法。本研究先以牛胰核糖核酸酶B和鸡卵清白蛋白二种常见的糖蛋白为研究材料,对几种N-糖链解离、富集纯化方法进行了考察,得到以下结论：1.N-糖链解离方法：选用十二烷基磺酸钠(SDS)和碘乙酰胺(DTT)作蛋白质的变性剂,100℃反应10 min；加入磷酸钠缓冲液和10%乙基苯基聚乙二醇(NP40),然后加入N-糖苷酶F (PNGase F),37℃反应24 h。这种方法解离效果好,效率最高,操作简单。2.N-糖链富集纯化方法：我们将富集纯化方法联合使用,对解离样品进行质谱分析前处理,结果表明,除了微晶纤维素和阳离子交换树脂混合装柱法外,其余4种方法(微晶纤维素柱结合阳离子交换树脂,C18柱结合活性炭柱,C18柱结合阳离子交换树脂和活性炭柱结合微晶纤维素柱)均可以达到满意的富集纯化效果。我们将初步建立的方法用胎牛血清,健康小鼠血清,健康人血清,小鼠HAbI8 IgG以及HAbI8 IgG(腹水法)这5种生物样品进一步验证,并针对微量样品进行了改进：3.PVDF膜辅助N-糖链解离方法：我们利用PVDF膜能够结合蛋白质的特性,在释放糖链前对复杂生物样品进行粗纯化,再按照上述糖链解离步骤,直接在膜上完成糖蛋白变性和糖链解离过程。实验证明,这种方法能同时提高微量样品糖链纯化效果。4.改进的N-糖链富集纯化方法：上述4种富集纯化方法中,只有活性炭柱联合微晶纤维素柱纯化生物来源N-糖链解离样品的效果最好。在用活性炭柱进行纯化时,为了避免糖链洗脱不完全,我们用含0.1%三氟乙酸的50%的乙腈溶液为洗脱剂；微晶纤维素柱的平衡时间与填料多少成正比,平衡效果好坏直接影响纯化效果,而微量样品只需要少量的微晶纤维素,因此我们采用1000μL移液器标配枪头作一次性层析柱,可以大大减少干扰物,提高纯化效率。综上所述,本研究在电喷雾质谱检测技术的基础上,建立了一个更适合于微量生物来源样品糖蛋白N-糖链解离,富集纯化的新方法——采用PVDF膜辅助N-糖链解离方法以及活性炭柱联合微晶纤维素柱的富集纯化方法。这种方法快速,稳定,通用性更强,能够通量化应用于ESI-MS检测前μg级别生物样品糖蛋白N-糖链的制备,有利于糖组的后续研究。更多还原

【Abstract】 Glycobiology is an important part of life science research in 21st century. To have in-depth study of the functions and regulatory mechanism of glycans in life activities, the primary task is to obtain and analyze glycome. Electrospray Ionization Mass Spectrometry (ESI-MS) is a powerful tool to analyze micro-scale glycomes, but the pretreatment of sample is necessary. In this paper, we established a pretreatment procedure for N-linked glycans released from glycoproteins in micro-scale bio-samples before ESI-MS analysis in view of the exsting problems.This study firstly used two common glycoproteins, bovine ribonuclease B and chicken albumin, as research materials, and had an initial investigation on several releasing, purifying and enriching N-linked glycans methods and obtained the following conclusions:1. N-linked glycans releasing:we chose sodium dodecyl sulfate (SDS) and iodine acetamide (DTT) as protein denaturing agents. Glycoproteins were dissolved in the buffer and denatured at 100℃for 10 min. Then added sodium pyrophosphate buffer,10% ethyl-phenyl-polyethyleneglycol (NP40), PNGase F,37℃24 h. This approach is effective, efficient and easy to operate.2. N-linked glycans purification and enrichment:the enzymatic samples were pretreated separately by five enrichment and purification methods used in conjunction before mass spectrometry analysis. The results showed, in addition to the method of using microcrystalline cellulose and cation exchange resin in tandem, the other four kinds (combination of microcrystalline cellulose and cation exchange resin column, combination of C18 column and graphite carbon column, combination of C18 column and cation exchange resin and combination of graphite carbon column and microcrystalline cellulose column) were able to achieve a satisfactory effect of purification and enrichment.Then, we used five bio-samples, fetus bovine serum, control mouse serum, control human serum, mouse HAbI8 IgG and HAbI8 IgG (ascites method) for further verification. Moreover, we improved this method focusing on micro-scale samples:3. PVDF membrane assisted N-linked glycans releasing:the PVDF membrane has the characteristic of absorbing proteins. We used it to roughly purify the complex bio-samples prior to PNGase F digestion, and then in accordance with the above steps completed glycoprotein denaturation and enzymatic digestion directly on the membrane. The results showed that this method can also improve the purification effect of glycans from micro-scale samples.4. Improved procedure of purifying and enriching.N-linked glycans:In the above mentioned four kinds of enrichment and purification methods, only the combination of graphite carbon column and microcrystalline cellulose column had the best purifying and enriching effect. We used 0.1% trifluoroacetic acid containing 50% acetonitrile solution as eluting agent of graphite carbon column to avoid eluting free glycans incompelely; the pretreatment time of microcrystalline cellulose powder was related with quantities, and the effect of pretreatment would have a direct impact on glycans analysis. The micro-scale samples only required a small amount of powder, so we made use of the 1000μL standard pipettes as the disposable chromatography columns and greatly reduced impurities, improved the efficiency of purification.This study, based on ESI-MS technology, established a new procedure which was more suitable for purifying and enriching N-linked glycans from micro-scale bio-samples glycoproteins--PVDF membrane assisted N-linked glycans releasing, purifying and enriching free glycans by graphite carbon column and microcrystalline cellulose column. This method was rapid, stable, versatile and appliable to high-throughput N-linked glycans production before ESI-MS analysis fromμg grade bio-samples glycoproteins and propitious to subsequent glycomic research.更多还原