蛋白质构象以及分子间非共价相互作用的冷喷雾质谱研究

【作者】 郭娜；

【导师】 再帕尔·阿不力孜； 庾石山； 石建功；

【作者基本信息】 中国协和医科大学 ， 药物分析， 2008， 博士

【摘要】 蛋白质的结构与其功能有着紧密的联系。蛋白质通过分子内非共价键作用折叠形成蛋白质高级结构,这其中可能包含多种不同的构象体;同时蛋白质还常与其它分子形成非共价复合物,这些都是蛋白质行使不同生物功能的基础。因此,表征蛋白质构象及其复合物结构是从分子水平上阐述生命现象、药物机理的重要基础,并可为药物筛选提供依据。质谱技术,尤其是电喷雾质谱技术（ESI-MS）在该领域的应用日益受到重视,然而ESI的离子化条件使其在表征非共价相互作用方面存在一定局限性。为了解决这一难题,本研究利用冷喷雾质谱技术（CSI-MS）的低温喷雾有利于保持溶液中非共价相互作用的特点,通过考察CSI-MS谱的电荷价态分布,建立了能够表征蛋白质天然活性构象及其与小分子药物非共价复合物的新型CSI-MS分析方法。另外,探索并建立了有机盐类药物快速、简便的CSI-MS新检测方法,实现了有机盐类药物形成整体簇合物结构的质谱表征。本论文研究的具体内容主要包括四部分:1.表征蛋白质天然构象的CSI-MS分析方法研究通过考察喷雾电压、锥孔电压、喷雾温度等关键仪器参数及溶剂表面张力等实验条件对于表征蛋白质构象的影响,合理选择仪器参数及溶液条件,建立了在酸性水体系条件下开展蛋白质天然构象研究的CSI-MS分析方法。结果表明,仪器参数对蛋白质CSI-MS谱产生一定的影响,其中,锥孔电压和喷雾温度的影响比较明显。此外,不同溶剂表面张力的研究结果证明及支持了溶剂表面张力对蛋白质电荷价态分布不产生影响的观点。同时表明纯水溶剂以外的其他不同溶剂表面张力体系中,雷利方程对蛋白质最大电荷价态的预测具有不合理性。2.酸诱导蛋白质构象变化及复合物的CSI-MS研究系统考察了不同pH值的溶液体系中三个标准蛋白质（细胞色素c、泛素、肌红蛋白）以及亲环蛋白A（免疫抑制剂靶蛋白）等的CSI-MS谱,并对CSI-MS谱和ESI-MS谱进行了细致的比较分析,开展了酸诱导蛋白质折叠构象转变的定量研究。此外,利用CSI-MS方法实现了亲环蛋白A与其小分子配基环孢素所形成的非共价复合物的表征。通过本研究发现CSI-MS谱的电荷价态分布窄,且平均或最高价态低;CSI-MS技术的低温喷雾等特点能够更好地维持及反映蛋白质的天然构象,可以比较真实地表征蛋白质在溶液中的构象变化,同时可观察到完整的蛋白复合物。另外,细胞色素c在pH 2.0-3.0范围内通常存在双模式峰分布,利用CSI-MS方法定量分析其折叠平衡转变,所得结果与其它光谱数据相吻合。表明CSI-MS能准确定量分析蛋白质的折叠平衡转变情况。3.表征AB蛋白与Dactylorhin B非共价复合物结构的CSI-MS方法研究β-淀粉样蛋白（Ap）是阿尔茨海默症（AD）患者脑中老年斑的重要组分,本研究考察了Aβ₁（-40）的“老化”过程,利用CSI-MS分析方法获得了Aβ_1-40蛋白构象及聚合物随时间变化的相关信息,发现了Aβ_1-40与抗AD活性天然产物Dactylorhin B（DHB）形成的非共价复合物。同时重点考察了DHB对Aβ_1-40构象及其聚合物的影响,证明了DHB能够在一定程度上抑制Aβ_1-40蛋白的聚合,为解释其药效作用机制提供了一定科学依据。4.表征有机盐类药物整体结构的CSI-MS方法研究选择了几种类型的有机盐类药物,包括硫酸盐类、磺酸盐类及盐酸盐类等,对其进行了CSI-MS中关键测定参数和溶液条件的系统考察。在正离子检测模式的CSI-MS谱中,发现这些盐类药物主要以1:1或者2:1比例结合形成特征的簇合物离子。本研究证明CSI-MS方法能有效地表征盐类药物的整体结构,从而建立了表征盐类药物整体簇合物结构的快速、简便的新型CSI-MS分析方法,解决了有机盐类药物结构中正负电荷部分难以同时表征的难题。更多还原

【Abstract】 The structure of protein are closely related to the function of protein.Functions of proteins are performed through protein higher order structures and the protein-drugs complex formed by inter-molecular interactions.Characterization of native conformations and non-covalent complexes of proteins are of great significance to understand a variety of biological processes,drug mechanisms and drug discoveries on the molecular level.The development of mass spectrometry,especially eletrospray ionization mass spectometry（ESI-MS） technique has been widely applied in this field. However,ESI-MS technique has limitation in characterizing precisely non-covalent interaction of proteins because of its harsh ionization conditions.The objective of this paper was to solve this difficult problem.In this paper,a novel cold-spray ionization mass spectrometry（CSI-MS） method was investigated allowing the convenient and precise characterization of native conformations and non-covalent complexes of proteins. In addition,a rapid and simple CSI-MS method was developed to characterize the cluster formation of organic salt drugs.1.Characterizing native protein conformation by CSI-MS methodThe optimum conditions were determined by changing the instrumental settings including needle voltage,orificel voltage,spray temperature and experimental conditions such as solvent surface tension.A CSI-MS method was established for characterizing the native protein conformation in water-acetic acid solution.The results showed that protein CSI-MS spectra were affected by the instrumental settings.Among these parameters,the orificel voltage and spray temperature had critical effects on protein spectra.In addition, protein CSD in CSI-MS spectra did not seem to be limited by the solvent surface tension as predicted by Rayleigh equations.2.Charactering acid-induced conformational changes and non-covalent interactions by CSI-MS methodIn this paper,the equilibrium acid-induced confonnational transitions of proteins, including cytochrome c,ubiquitin,myoglobin and cyclophilin A（CypA） were investigated using CSI-MS over a wide range of pH values in aqueous solutions. Comparisons were made between the CSI-MS and traditional ESI-MS spectra. Significant narrower charged-state distribution and a shift to lower charge state were observed in CSI-MS spectra compared to ESI-MS.In addition,non-covalent complexes were observed in case of the protein-ligand complex between CypA and cyclosporin A （CsA）.Furthermore,protein conformations characterized by CSI-MS were comparable with those obtained by other established biophysical methods.The results indicated CSI-MS method was a powerful tool to characterize the exactly acid-induced conformational transitions and could protect native conformations and protein complex structures from damages.3.Non-covalent interaction between amyloid-β-peptide_（1-40） and Dactylorhin B by using CSI-MS methodAmyloid-β-peptide_（1-40）（Aβ） is the major proteinaceous component of senile plaques formed in Alzheimer’s disease（AD） brain.Aβ’s aggregational properties and its complex formation ability with DHB were studied as a function of time. Furthermore,the Aβ-DHB complex’s influence on Aβ’s aggregation was investigated. The results showed that DHB had the ability to inhibit Aβ’s aggregation which confirmed the pharmaceutical mechanism of DHB.4.Development of a CSI-MS method for the characterization of organic salt drugs.Organic salts drugs including sulfate,hydrochloride and sulfonate were selected and detected by CSI-MS.The optimal conditions were determined under different experiment conditions and instrumental settings.In the positive ion mode of CSI,the cluster ions of these salts were mainly formed at 1:1 or 2:1 ratio.This study proved that CSI-MS can be effectively characterized the overall structure of organic salt drugs.A rapid and simple CSI-MS method was developed to characterize cluster formation of organic salt drugs.更多还原