Evaluation of sample extraction methods for proteomic analysis of coniferous seeds

【作者】 甄艳； 施季森；

【Author】 Yan Zhen Jisen Shi Key Laboratory of Forest Genetics and Biotechnology of the Ministry of Education of the people’s Republic of China,Nanjing Forestry University,159 Longpan Road,Nanjing 210037,People’s Republic of China

【机构】 Key Laboratory of Forest Genetics and Biotechnology of the Ministry of Education of the people’s Republic of China,Nanjing Forestry University；

【摘要】 <正>In this study,three methods of protein extraction from the seeds of the Chinese fir were compared by examining the quality(including the number of protein spots observed) in the two-dimensional gel electrophoresis(2-DE),obtained by isoelectric focusing(IEF) and sodium dodecyl sulfate polyacrylamide gel eletrophoresis(SDS-PAGE) of the total released protein.Three protein extraction methods were:TCA-acetone precipitation,SDS extraction/acetone precipitation and phenol extraction methanol/ammonium acetate precipitation.The results showed that TCA-acetone precipitation was the most effective method for protein extraction;it gave the highest yield of total protein(8.9 mg protein per g seed weight) and the greatest number of proteins spots(1034 spots) on the 2-DE gel.Further,several proteins were identified by liquid chromatography mass spectrometry(LC MS/MS),which are legumin-like storage protein,similar to AMP binding/acetate-CoA ligase,similar to 40S ribosomal protein S20 and unknown protein.These data demonstrates that TCA-acetone precipitation followed by 2-DE and LC MS/MS is a suitable method for proteomic analysis of coniferous species, such as Chinese fir and provides a valuable starting point for similar proteomic ananlysis of other coniferous tree species.更多还原

【Abstract】 In this study,three methods of protein extraction from the seeds of the Chinese fir were compared by examining the quality(including the number of protein spots observed) in the two-dimensional gel electrophoresis(2-DE),obtained by isoelectric focusing(IEF) and sodium dodecyl sulfate polyacrylamide gel eletrophoresis(SDS-PAGE) of the total released protein.Three protein extraction methods were:TCA-acetone precipitation,SDS extraction/acetone precipitation and phenol extraction methanol/ammonium acetate precipitation.The results showed that TCA-acetone precipitation was the most effective method for protein extraction;it gave the highest yield of total protein(8.9 mg protein per g seed weight) and the greatest number of proteins spots(1034 spots) on the 2-DE gel.Further,several proteins were identified by liquid chromatography mass spectrometry(LC MS/MS),which are legumin-like storage protein,similar to AMP binding/acetate-CoA ligase,similar to 40S ribosomal protein S20 and unknown protein.These data demonstrates that TCA-acetone precipitation followed by 2-DE and LC MS/MS is a suitable method for proteomic analysis of coniferous species, such as Chinese fir and provides a valuable starting point for similar proteomic ananlysis of other coniferous tree species.更多还原