嗜酸氧化亚铁硫杆菌细胞壁相关蛋白表达图谱的构建
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摘要
嗜酸氧化亚铁硫杆菌(Acidithiobacillus ferrooxidans),是目前使用最广泛,研究最深入的浸矿微生物。细菌吸附至矿物表面无论对于细菌在自然环境中的生存,还是对于生物浸出过程都具有十分重要的意义。本文通过构建嗜酸氧化亚铁硫杆菌细胞壁相关蛋白表达图谱,从蛋白质组学角度探讨浸矿微生物表面结构,为微生物与矿物表面的吸附机理研究提供一些依据。
本论文研究中,提取了嗜酸氧化亚铁硫杆菌细胞壁相关蛋白,采用液相色谱—串联质谱技术先对酶解后的蛋白进行分离,分离后的肽段直接进入质谱仪离子源进行一级和二级质谱的分析。由于可以得到的关于A.ferrooxidans的肽质量指纹图谱(PMF)数据库还不健全,我们利用生物信息学技术构建了用于质谱蛋白质组学分析的A.ferrooxidans的PMF数据库。将得到245个肽质量指纹图谱用Mascot软件进行数据库搜寻得到蛋白相关信息,然后对每一个蛋白的输出信号及亚细胞定位进行预测,目前共得到97个目标蛋白。这些已鉴定的蛋白中42.3%有Sec-type信号肽,7.2%具有双精氨酸转运信号肽(Tat),18.6%的蛋白具有脂蛋白信号肽(Lipo),21.6%的蛋白为非经典分泌蛋白(non-classically secreted proteins),根据亚细胞定位预测还有19.6%的蛋白也被认为是目标蛋白。按其功能分类,43.3%的细胞壁相关蛋白质为细胞包被,16.5%的蛋白为假设蛋白,6.2%的蛋白为保守的假设蛋白,还有6.2%蛋白为功能未知蛋白,其他功能的蛋白含量都很少;这些蛋白质的分子量主要分布在4000～118000Da,pH值主要分布在4～12之间,其中31.96%的蛋白质的pH值小于7,68.04%的蛋白质pH值大于7,10.31%的蛋白pH大于10。通过这种方法构建的A.ferrooxidans细胞壁相关蛋白表达图谱,为以后各种环境中A.ferrooxidans细胞壁相关蛋白的研究提供了有价值的基础性研究。
本研究还采用差异蛋白质组学的方法,对不同培养条件下(单质硫和Fe~(2+)为营养源)A.ferrooxidans细胞壁相关蛋白差异表达谱进行了研究。采用双向电泳的方法分离蛋白,结合基质辅助激光解吸/电离飞行时间质谱(MALDI-TOF/MS)对分离蛋白进行鉴定。双向电泳结果经PDQuest软件分析后表明有104个差异蛋白出现,目前已经鉴定出18个差异蛋白,其中14个为表达上调,4个为表达下调。
At present,Acidithiobacillus ferrooxidans is the most widely applied and investigated bacteria in biometallurgy.The interaction at the interface between microbe and mineral plays an important role in this process.In this thesis,the cell wall related proteins of A.ferrooxidans have been studied.The surface structures researched at the aspect of Proteomics would be helpful to clarify the mechanisms of A.ferrooxidans attaching to minerals.
In this study,the cell wall related proteins of A.ferrooxidans were extracted and separated by SDS-PAGE.Liquid chromatography coupled with tandem mass spectrometry was applied to identify the proteins. Firstly,the proteins which were subjected to in-gel digestion with trypsin, were separated by liquid chromatography,and then were online analyzed by the coupled tandem mass spectrometry.As the peptide mass fingerprint(PMF)database has not been consummated,we construct our local putative A.ferrooxidans protein PMF database to be used in the mass spectrometry proteomisc analysis.The acquired 245 MS and MS/MS spectras were sent for searching in the proteins database with Mascot search engine for protein identification.The export signal and subcellular location of each obtained protein was also predicted.Portion of the aimed proteins were obtained.Among the identified proteins, 42.3%of them possess export of the Sec type,7.2%of them show a twin arginine translocation(Tat)signal,18.6%have lipoprotein signal peptides. 21.6%are non-classically secreted proteins,and 19.6%could be exported with their subcellular location prediction.According to their functional categories,43.3%of them belong to Cell Envelope,16.5%to Hypothetical Proeteins,6.2%to Conserved Hypothetical Proteins,and 6.2%belonged to Unknown Functions.Also,there are a few other functional proteins.Furthermore,the molecular weights of these proteins are distributed in the range of 4000 to 118000Da.Their isoelectric points are between 4 and 12,among which 31.96%of which show the pH values below 7,68.04%higher than 7,and 10.31%over pH values of 10.This work provides fundamental research of the cell wall related protein of A. ferrooxidans by revealing the total expressed proteins.
We also studied the comparative proteomics of A.ferrooxidans grown under different energy resources(Fe~(2+)or sulphur).The cell wall related proteins of A.ferrooxidans were separated by the two-dimensional gel electrophoresis(2-DE)and then identified by the matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).The resulted Images were quantitatively analyzed with the PDQuest 2D analysis software.The image analysis indicates 104 differentially-expressed protein spots,18 of which have been identified.
本论文研究中,提取了嗜酸氧化亚铁硫杆菌细胞壁相关蛋白,采用液相色谱—串联质谱技术先对酶解后的蛋白进行分离,分离后的肽段直接进入质谱仪离子源进行一级和二级质谱的分析。由于可以得到的关于A.ferrooxidans的肽质量指纹图谱(PMF)数据库还不健全,我们利用生物信息学技术构建了用于质谱蛋白质组学分析的A.ferrooxidans的PMF数据库。将得到245个肽质量指纹图谱用Mascot软件进行数据库搜寻得到蛋白相关信息,然后对每一个蛋白的输出信号及亚细胞定位进行预测,目前共得到97个目标蛋白。这些已鉴定的蛋白中42.3%有Sec-type信号肽,7.2%具有双精氨酸转运信号肽(Tat),18.6%的蛋白具有脂蛋白信号肽(Lipo),21.6%的蛋白为非经典分泌蛋白(non-classically secreted proteins),根据亚细胞定位预测还有19.6%的蛋白也被认为是目标蛋白。按其功能分类,43.3%的细胞壁相关蛋白质为细胞包被,16.5%的蛋白为假设蛋白,6.2%的蛋白为保守的假设蛋白,还有6.2%蛋白为功能未知蛋白,其他功能的蛋白含量都很少;这些蛋白质的分子量主要分布在4000～118000Da,pH值主要分布在4～12之间,其中31.96%的蛋白质的pH值小于7,68.04%的蛋白质pH值大于7,10.31%的蛋白pH大于10。通过这种方法构建的A.ferrooxidans细胞壁相关蛋白表达图谱,为以后各种环境中A.ferrooxidans细胞壁相关蛋白的研究提供了有价值的基础性研究。
本研究还采用差异蛋白质组学的方法,对不同培养条件下(单质硫和Fe~(2+)为营养源)A.ferrooxidans细胞壁相关蛋白差异表达谱进行了研究。采用双向电泳的方法分离蛋白,结合基质辅助激光解吸/电离飞行时间质谱(MALDI-TOF/MS)对分离蛋白进行鉴定。双向电泳结果经PDQuest软件分析后表明有104个差异蛋白出现,目前已经鉴定出18个差异蛋白,其中14个为表达上调,4个为表达下调。
At present,Acidithiobacillus ferrooxidans is the most widely applied and investigated bacteria in biometallurgy.The interaction at the interface between microbe and mineral plays an important role in this process.In this thesis,the cell wall related proteins of A.ferrooxidans have been studied.The surface structures researched at the aspect of Proteomics would be helpful to clarify the mechanisms of A.ferrooxidans attaching to minerals.
In this study,the cell wall related proteins of A.ferrooxidans were extracted and separated by SDS-PAGE.Liquid chromatography coupled with tandem mass spectrometry was applied to identify the proteins. Firstly,the proteins which were subjected to in-gel digestion with trypsin, were separated by liquid chromatography,and then were online analyzed by the coupled tandem mass spectrometry.As the peptide mass fingerprint(PMF)database has not been consummated,we construct our local putative A.ferrooxidans protein PMF database to be used in the mass spectrometry proteomisc analysis.The acquired 245 MS and MS/MS spectras were sent for searching in the proteins database with Mascot search engine for protein identification.The export signal and subcellular location of each obtained protein was also predicted.Portion of the aimed proteins were obtained.Among the identified proteins, 42.3%of them possess export of the Sec type,7.2%of them show a twin arginine translocation(Tat)signal,18.6%have lipoprotein signal peptides. 21.6%are non-classically secreted proteins,and 19.6%could be exported with their subcellular location prediction.According to their functional categories,43.3%of them belong to Cell Envelope,16.5%to Hypothetical Proeteins,6.2%to Conserved Hypothetical Proteins,and 6.2%belonged to Unknown Functions.Also,there are a few other functional proteins.Furthermore,the molecular weights of these proteins are distributed in the range of 4000 to 118000Da.Their isoelectric points are between 4 and 12,among which 31.96%of which show the pH values below 7,68.04%higher than 7,and 10.31%over pH values of 10.This work provides fundamental research of the cell wall related protein of A. ferrooxidans by revealing the total expressed proteins.
We also studied the comparative proteomics of A.ferrooxidans grown under different energy resources(Fe~(2+)or sulphur).The cell wall related proteins of A.ferrooxidans were separated by the two-dimensional gel electrophoresis(2-DE)and then identified by the matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).The resulted Images were quantitatively analyzed with the PDQuest 2D analysis software.The image analysis indicates 104 differentially-expressed protein spots,18 of which have been identified.
引文
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