多维液相色谱质谱组合体系在志贺菌蛋白质组学研究中的应用

发布时间：2018-04-15 18:44

  本文选题：福氏志贺菌 + 基质辅助激光解吸电离　； 参考：《中国病原生物学杂志》2016年08期

【摘要】：目的建立多维液相色谱质谱组合体系,用以获得福氏志贺菌更完整的蛋白质组信息。方法对福氏2a志贺菌301株(Sf2a301)全菌蛋白进行预分离,顺序抽提胞浆蛋白和膜蛋白。应用二维液相色谱基质辅助激光解吸附飞行时间串联质谱(2DLC-MALDI-TOF/TOF)和二维液相色谱电喷雾串联质谱(2DLC-ESI-MS/MS)构成的多维液相色谱质谱组合体系进行蛋白分离和鉴定,分别应用MASCOT和SEQUEST软件搜索NCBI上Sf2a301的蛋白质数据库,并对两种串联质谱的互补性及组合后优势进行分析。结果 MALDI-TOF/TOF和ESI-MS/MS分别鉴定到Sf2a301株的960个蛋白和729个蛋白,总共鉴定1 231个蛋白;两种技术方法鉴定蛋白质的平均氨基酸序列覆盖率分别为14.3%和13.9%,组合鉴定后覆盖率升高为15.7%。MALDI和ESI两种离子化方式具有互补性,表现为MALDI更倾向于离子化分子质量偏小、碱性、胰酶消化后羧基末端为精氨酸的肽段;ESI更倾向于离子化分子质量偏大、疏水、胰酶消化后羧基末端为赖氨酸的肽段。结论应用2DLC-MALDI-TOF/TOF和2DLC-ESI-MS/MS鉴定Sf2a301全蛋白质组,既互相确认又相互补充。多维液相色谱质谱组合体系无论在蛋白质鉴定数量还是可信程度上都优于单一串联质谱,可作为复杂样品的蛋白质组学研究技术平台。
[Abstract]:Objective to establish a multi-dimensional liquid chromatography-mass spectrometry system to obtain more complete proteome information of Shigella flexneri.Methods the whole bacterial protein of Shigella flexneri 301 strain Sf2a301) was preisolated and cytosolic protein and membrane protein were extracted sequentially.Two dimensional liquid chromatographic matrix assisted laser desorption time of flight mass spectrometry (2DLC-MALDI-TOF / TOF / TOF) and two-dimensional liquid chromatography / electrospray tandem mass spectrometry (2DLC-ESI-MS / MS) were used to isolate and identify proteins.The protein database of Sf2a301 on NCBI was searched by MASCOT and SEQUEST software, and the complementarities and advantages of the two tandem mass spectrometry were analyzed.Results 960 proteins and 729 proteins of Sf2a301 strain were identified by MALDI-TOF/TOF and ESI-MS/MS respectively, and 1 231 proteins were identified.The average amino acid sequence coverage of the two methods for protein identification was 14.3% and 13.9%, respectively. After combination identification, the average amino acid sequence coverage increased to 15.7%.MALDI and ESI, which were complementary to each other. The results showed that MALDI was more inclined to ionization molecular weight and alkalinity.The peptide segment with arginine end after trypsin digestion is more inclined to ionization molecular mass and hydrophobicity, and the carboxyl end after trypsin digestion is lysine peptide segment.Conclusion 2DLC-MALDI-TOF/TOF and 2DLC-ESI-MS/MS were used to identify the whole Sf2a301 proteome, which were mutually confirmed and complementary.Multidimensional liquid chromatography-mass spectrometry combination system is superior to single tandem mass spectrometry in terms of protein identification quantity and reliability. It can be used as a proteomics research platform for complex samples.
【作者单位】： 华北理工大学生命科学学院;
【基金】：国家自然科学基金项目(No.81302323) 河北省高等学校科学技术研究项目(No.QN20131059) 河北省自然科学基金项目(No.2013209194) 华北理工大学培育基金项目(No.GP201518);华北理工大学博士科研启动项目
【分类号】：R446.5
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本文编号：1755324