毛细管电泳检测肺癌及癌旁正常组织蛋白质混合物差异

Capillary Electrophoresis Detection of Lung Cancer and Adjacent Normal Tissue Differences in Protein Mixture

以建立的毛细管电泳(CE)-激光诱导荧光(LIF)检测蛋白质的方法对提取肺癌及癌旁正常组织蛋白质混合物(变性/活性)差异进行检测.采用异硫氰酸荧光素(FITC)为衍生剂,电泳缓冲液为1×TBE(TBE为Tris-硼酸-EDTA,变性电泳pH 10.0,活性电泳为pH 8.3且含有2 mg/L考马斯亮蓝),分离电压15 kV,柱温15℃,电动进样(10 kV×10 s),激发波长/发射波长=488/520 nm检测时,肺癌及癌旁正常组织蛋白质混合物样品得到较好分离且有明显差异.与目前常用蛋白分析方法:变性SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)以及活性蓝绿温和胶电泳(BN-PAGE)进行比较.BN-PAGE结果显示肺癌组织相比正常组织有较明显蛋白种类差异;SDS-PAGE结果表明一些蛋白质表达量差异也是肺癌及癌旁正常组织的显著差别,且主要集中在20～116 kDa.CE-LIF检测结果与PAGE结果大致相同,且CE-LIF检测蛋白质的灵敏度高于PAGE,能更准确反映肺癌及癌旁正常组织的蛋白质差异.结论是CE-LIF可用于蛋白质差异检测,时间短,效果较好,对活性蛋白质进行分析体现了其优点:可提供较强的动力——电压,及强动力下良好的温度稳定性.

Protein mixture （denatured and native） differences of extracted from lung cancer and adjacent normal tissue by established method of capillary electrophoresis （CE）-laser-induced fluorescence （LIF） detection of protein was detected using the fluorescein isothiocyanate （FITC） as a derivative agent. When detected by the 1 × TBE electrophoresis buffer solution （pH 10.0 of denaturing gel electrophoresis and pH 8.3 of native gel electrophoresis containing 2 mg/L Coomassie brilliant blue）, separation voltage of 15 kV, column temperature of 15 ℃, electric injection （10 kV X 10 s）, and excitation wavelength/emission wavelength of 488/520 nm, lung cancer and adjacent normal tissue protein mixture samples obtained a better separation and there was significant difference. Comparing with the current commonly used analysis methods, the SDS-denatured polyacrylamide gel electrophoresis （SDS-PAGE） as well as the Blue Native polyacrylamidegel electrophoresis （BN-PAGE）, the BN-PAGE results showed that compared to normal tissue, the lung cancer tissue had more significant protein type differences. But the SDS-PAGE results showed that some differences of protein expression had significant difference between the lung cancer and adjacent normal tissue, and mainly concentrated in 20~116 kDa. CE-LIF detection results were roughly the same with PAGE, and CE-LIF is more sensitive than PAGE and it can more accurately reflect protein differences of lung cancer and adjacent normal tissue. We concluded that the CE-LIF could be used to detect protein differences. And the time was shorter, effect was better, and analysis of active protein reflected its advantage of providing a strong driving force-voltage and a well temperature stability by strong motivation.