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

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。

Objective To 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. Methods Mitochondrial 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 Software. Results The 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. Conclusion Acetone 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 11 kD. So Au chips can get better result by integrated use of the two precipitation methods.