二维电泳和iTRAQ的实验比较

A comparative study between the two dimensional gel electrophoresis and iTRAQ method

对蛋白组学中的二维电泳和iTRAQ两种技术进行比较研究,为神经科学的蛋白组学研究提供选择参考。取生后1～3d C57Bl/6小鼠背根神经节进行培养,对照组加DMSO,实验组加JNJ460。分别用二维电泳和iTRAQ两种技术检测背根神经节细胞蛋白的变化,用4700 TOF/TOF串联飞行质谱仪检测变化蛋白的肽片段质量,用GPS软件检索Mascot蛋白质数据库,鉴定出匹配的蛋白。利用二维电泳技术观察到4种表达上调的蛋白,2种是结构蛋白,2种是信号转导蛋白。利用iTRAQ技术观察到22种表达上调的蛋白,其中10种是结构蛋白,3种是信号蛋白,2种是代谢蛋白,2种是降解蛋白化,2种是细胞基质蛋白及3种其它蛋白。利用二维电泳技术观察到发生表达变化的蛋白都位于胞浆内,而利用iTRAQ技术观察到的表达变化的蛋白有胞浆蛋白外,还有线粒体蛋白、膜蛋白和核蛋白。同时,利用二维电泳技术观察到的蛋白变化都在2倍以上,而利用iTRAQ技术计算出的蛋白变化在1.3至1.6倍之间。另,iTRAQ技术在鉴定大分子和小分子蛋白方面也有优势。结果表明iTRAQ技术比二维电泳技术能发现更多数量和更多种类的蛋白,但在比较胞浆蛋白的变化及蛋白量的变化方面二维电泳技术有一定的长处,对iTRAQ的实验结果有互补作用。

The present study provided a reference for the neuroproteomics study through comparison between the two dimensional gel electrophoresis （2-DE） and isobaric tags for relative and absolute quantification （iTRAQ） method. The dorsal root ganglia （DRG） were cultured from 1 to 3 d postnatal C57B1/6 mice. The DRGs in control group were incubated with DMSO, and those in the experimental group were incubated with JNJ460. The peptide masses of changed proteins were analyzed by 4700 matrix-assisted laser desorption/ionization coupled to a dual time-of-flight （TOF/TOF） mass spectrometry, and the mass spectrometric data were used to identify the proteins through the general proteomics standard （GPS） search and Mascot database. Four proteins were found to be up-regulated by 2-DE. Two of them were structural proteins, and the others were signaling proteins. 22 proteins were detected to be up-regulated from iTRAQ. These proteins included 10 structural proteins, 3 signaling transduction proteins, 2 metabolic proteins, 2 degradation protein, 2 extracellular matrix pro- teins and 3 other proteins. All up-regulated proteins found by 2-DE were located in the cytoplasm; some up-regulated proteins found by iTRAQ were located in the cytoplasm, some located in the mitochondfion, and some in the membrane and nucleus. All of the protein expression ratios above were 2 folds from 2-DE, but iTRAQ detected 1.3 to 1.6 folds of the protein expression ratios, iTRAQ had an advan- tage in the identification of proteins with large and small molecular weights compared to 2-DE. Our results showed that iTRAQ could find more proteins and more styles of proteins compared with 2-DE method. But 2-DE had an advantage in the identification of cytoplasmic proteins and in the quantification of the protein expression. The present results suggested 2-DE had the complementary virtue to iTRAQ.