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韵雪雪, 杨永长, 姜伟, 肖代雯, 刘华, 闫慧, 黄文芳. 样品沉淀方法对线粒体蛋白指纹图谱分析影响[J]. 中国公共卫生, 2011, 27(9): 1119-1121. DOI: 10.11847/zgggws2011-27-09-21
引用本文: 韵雪雪, 杨永长, 姜伟, 肖代雯, 刘华, 闫慧, 黄文芳. 样品沉淀方法对线粒体蛋白指纹图谱分析影响[J]. 中国公共卫生, 2011, 27(9): 1119-1121. DOI: 10.11847/zgggws2011-27-09-21
YUN Xue-xue, YANG Yong-chang, JIANG Wei, . Different precipitation methods affect mitochondrial protein fingerprinting[J]. Chinese Journal of Public Health, 2011, 27(9): 1119-1121. DOI: 10.11847/zgggws2011-27-09-21
Citation: YUN Xue-xue, YANG Yong-chang, JIANG Wei, . Different precipitation methods affect mitochondrial protein fingerprinting[J]. Chinese Journal of Public Health, 2011, 27(9): 1119-1121. DOI: 10.11847/zgggws2011-27-09-21

样品沉淀方法对线粒体蛋白指纹图谱分析影响

Different precipitation methods affect mitochondrial protein fingerprinting

  • 摘要: 目的比较不同方法沉淀乳腺癌线粒体蛋白对Au芯片分析的影响,为Au芯片研究亚细胞蛋白奠定基础。方法提取体外培养乳腺癌细胞MCF-7的线粒体蛋白,分别采用丙酮沉淀,三氯醋酸-丙酮(TCA-丙酮)沉淀,饱和(NH4)2SO4沉淀处理样品,点样Au蛋白芯片,表面增强激光解析蛋白飞行时间质谱(SELDI-TOF-MS)分析蛋白指纹图谱,Biomaker Wizard Software分析软件收集数据。结果每种方法均重复试验20次,在质荷比(m/z)2000~100 000范围内检测蛋白点数目,统计结果分别为TCA-丙酮沉淀39个,丙酮沉淀71个,饱和(NH4)2SO4沉淀14个;选择8.6、10.0、11.3、14.0和15.6kD 5个含量较高的蛋白作为标志,采用q检验进行两两分析,差异有统计学意义(P<0.05)。结论对分子量(Mr)<11 kD的蛋白,丙酮沉淀处理可以获得较好的蛋白指纹图谱,对Mr>11 kD的蛋白TCA-丙酮沉淀处理结果好,综合使用2种方法分别处理样品上样Au芯片可以获得好的线粒体蛋白分析结果。

     

    Abstract: ObjectiveTo compare different precipitation methods for breast cancer mitochondrial protein analysis with Au chips and to lay a foundation for subcellular protein analysis with Au chips.MethodsMitochondrial protein was abstracted from breast cancer cell line MCF-7 and precipitated by acetone,trichloroacetic acid-acetone(TCA-Acetone),and ammonium sulfate,respectively.The samples were added to Au chips and analyzed with surface enhanced laser desorption/ ionization time-of-flight mass spectra(SELDI-TOF-MS).The results were collected by Biomaker Wizard Softw are.ResultsThe number of protein peaks was 39,71,and 14 at 2 kD-100 kD disposed by TCA-acetone,acetone,ammonium sulfate, respectively.Five protein peaks at 8.6 kD,10.0 kD,11.3 kD,14.0 kD,and 15.6 kD with high abundance were selected and compared by q test.ConclusionAcetone precipitation is the best way for proteins of molecular weight(Mr)less than 11 kD.While TCA-acetone precipitation is better for proteins of Mr over 11kD.So Au chips can get better result by integrated use of the two precipitation methods.

     

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