Chang Po-Chih, Reddy P Muralidhar, Ho Yen-Peng
Department of Chemistry, National Dong Hwa University, Hualien, 97401, Taiwan, Republic of China.
Anal Bioanal Chem. 2014 Sep;406(22):5339-46. doi: 10.1007/s00216-014-7965-7. Epub 2014 Jun 27.
Stable-isotope dimethyl labeling was applied to the quantification of genetically modified (GM) soya. The herbicide-resistant gene-related protein 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) was labeled using a dimethyl labeling reagent, formaldehyde-H2 or -D2. The identification and quantification of CP4 EPSPS was performed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The CP4 EPSPS protein was separated from high abundance proteins using strong anion exchange chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, the tryptic peptides from the samples and reference were labeled with formaldehyde-H2 and formaldehyde-D2, respectively. The two labeled pools were mixed and analyzed using MALDI-MS. The data showed a good correlation between the peak ratio of the H- and D-labeled peptides and the GM soya percentages at 0.5, 1, 3, and 5 %, with R (2) of 0.99. The labeling reagents are readily available. The labeling experiments and the detection procedures are simple. The approach is useful for the quantification of GM soya at a level as low as 0.5 %.
采用稳定同位素二甲基标记法对转基因大豆进行定量分析。使用二甲基标记试剂甲醛-H₂或甲醛-D₂对除草剂抗性基因相关蛋白5-烯醇丙酮酸莽草酸-3-磷酸合酶（CP4 EPSPS）进行标记。采用基质辅助激光解吸/电离质谱（MALDI-MS）对CP4 EPSPS进行鉴定和定量分析。通过强阴离子交换色谱和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳从高丰度蛋白中分离出CP4 EPSPS蛋白。然后，分别用甲醛-H₂和甲醛-D₂对样品和参比品中的胰蛋白酶肽段进行标记。将两个标记好的混合样品进行混合，并用MALDI-MS进行分析。数据表明，H标记和D标记肽段的峰面积比与转基因大豆含量在0.5%、1%、3%和5%时具有良好的相关性，R²为0.99。标记试剂易于获得。标记实验和检测程序简单。该方法可用于低至0.5%水平的转基因大豆定量分析。
