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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。
Objective To establish a multidimensional liquid chromatography-mass spectrometry (LC-MS / MS) system to obtain a more complete proteome information of Shigella flexneri. Methods The whole bacterial proteins of 301 strains of Shigella flexneri (Sf2a301) were pre-separated and the cytoplasmic proteins and membrane proteins were extracted sequentially. Multidimensional liquid chromatography-mass spectrometry (MS-MS / MS) was developed using 2DLC-MALDI-TOF and 2DLC-ESI-MS / MS with two-dimensional liquid chromatography-assisted laser desorption / ionization time of flight mass spectrometry The combination system was used to isolate and identify proteins. The protein database of Sf2a301 on NCBI was searched by using MASCOT and SEQUEST software, respectively. The complementarity and post-combination superiority of the two tandem mass spectra were analyzed. Results A total of 960 proteins and 729 proteins of Sf2a301 strain were identified by MALDI-TOF / TOF and ESI-MS / MS, respectively. A total of 1 231 proteins were identified. The average amino acid sequence coverage of the two proteins was 14.3% 13.9%, the combination of identification coverage increased to 15.7%. MALDI and ESI two kinds of ionization are complementary, the performance of MALDI is more inclined to the ionization of molecular weight is small, alkaline, tryptic digestion carboxy terminal arginine peptide; ESI is more inclined to ionization molecular mass Large, hydrophobic, tryptic digestion of carboxyl-terminal lysine peptide. Conclusion The whole proteome of Sf2a301 was identified by 2DLC-MALDI-TOF / TOF and 2DLC-ESI-MS / MS, which not only confirmed each other but also complemented each other. The multidimensional liquid chromatography-mass spectrometry combination system is superior to single tandem mass spectrometry in terms of both the amount of protein identification and the degree of credibility, and can be used as a proteomics research technology platform for complex samples.