用于蛋白质组分析的两种马铃薯块茎蛋白质提取方法的比较
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摘要
【目的】进行两种提取方法下马铃薯块茎蛋白质组结果差异分析,并确定最佳马铃薯块茎蛋白质提取方法.【方法】采用酚提取法和三氯乙酸/丙酮沉淀与乙酸铵/甲醇沉淀相结合的两步沉淀法提取马铃薯块茎蛋白质,进行2-DE分离,对差异表达蛋白质进行MALDI-TOF-MS质谱鉴定.【结果】两步沉淀法提取的马铃薯块茎蛋白质较酚提取法获得蛋白质纯度和浓度及蛋白质点数目均明显增加,双向电泳凝胶图谱分辨率较高.质谱鉴定结果表明,两步沉淀法中鉴定的差异表达蛋白质丰度均显著高于酚提取法,并鉴定到一些新出现的蛋白质包括假定的线粒体NAD依赖的苹果酸脱氢酶、酸性磷酸酶类似物和蛋白酶抑制剂II前体类型B.【结论】提取马铃薯块茎蛋白质时两步沉淀法优于酚提取法.
【Objective】 To analyze the difference potato tuber proteomic under two extraction methods,as well as the optimal extraction method of potato tuber protein.【Method】 Potato tuber protein was extracted with phenol extraction method and two-step precipitation method,and then analyzed using two-dimension gel electrophoresis(2-DE),respectively.The differentially expressed proteins were identified by MALDI-TOF/TOF mass spectrometry.【Result】 The purity and concentration of protein extracted with two-step precipitation method was significantly high than that of phenol extraction method,and the number of protein spots and the resolution of 2-DE gel maps were also increased.Mass spectrometry identification results indicated that compared with phenol extraction method,the abundance of protein extracted with two-steps precipitation extraction method was significantly enhanced.And some new proteins were only identified in two-steps precipitation extraction method,including putative mitochondrial NAD-dependent malate dehydrogenase,acid phosphatase-like and proteinase inhibitor II precursor type B.【Conclusion】 Potato tuber protein extracted with two-step precipitation method is better than phenol extraction method.
【Objective】 To analyze the difference potato tuber proteomic under two extraction methods,as well as the optimal extraction method of potato tuber protein.【Method】 Potato tuber protein was extracted with phenol extraction method and two-step precipitation method,and then analyzed using two-dimension gel electrophoresis(2-DE),respectively.The differentially expressed proteins were identified by MALDI-TOF/TOF mass spectrometry.【Result】 The purity and concentration of protein extracted with two-step precipitation method was significantly high than that of phenol extraction method,and the number of protein spots and the resolution of 2-DE gel maps were also increased.Mass spectrometry identification results indicated that compared with phenol extraction method,the abundance of protein extracted with two-steps precipitation extraction method was significantly enhanced.And some new proteins were only identified in two-steps precipitation extraction method,including putative mitochondrial NAD-dependent malate dehydrogenase,acid phosphatase-like and proteinase inhibitor II precursor type B.【Conclusion】 Potato tuber protein extracted with two-step precipitation method is better than phenol extraction method.
引文
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[2] Rode C,Winkelmann T,Braun H P,et al.Difference gel electrophoresis (DIGE):methods and protocols[M].Totowa:Humana Press,2012.
[3] Wang X,Shi M,Lu X,et al.A method for protein extraction from different subcellular fractions of laticifer latex in Hevea brasiliensis compatible with 2-DE and MS[J].Proteome Science,2010,8(1):35-45.
[4] Sebastiana M,Figueiredo A,Monteiro F,et al.A possible approach for gel-based proteomic studies in recalcitrant woody plants[J].Springerplus,2013,2(1):210.
[5] Faurobert M,Pelpoir E,Cha b J.Plant proteomics:methods and protocols[M].Totowa:Humana Press,2007.
[6] He C,Wang Y.Protein extraction from leaves of Aloe vera L.,a succulent and recalcitrant plant,for proteomic analysis[J].Plant Molecular Biology Reporter,2008,26(4):292-300.
[7] Lau B Y,Deb-Choudhury S,Morton J D,et al.Method developments to extract proteins from oil palm chromoplast for proteomic analysis[J].Springerplus,2015,4(1):791-804.
[8] Al-Obaidi J R,Saidi N B,Usuldin S R,et al.Comparison of different protein extraction methods for gel-based proteomic analysis of ganoderma spp[J].Protein Journal,2016,35(2):100-6.
[9] Calderan-Rodrigues M J,Jamet E,Douche T,et al.Cell wall proteome of sugarcane stems:comparison of a destructive and a non-destructive extraction method showed differences in glycoside hydrolases and peroxidases[J]. BMC Plant Biology,2016,16(1):14-31.
[10] Song Y,Zhang H,Wang G,et al.DMSO,an organic cleanup solvent for TCA/acetone-precipitated proteins,improves 2-DE protein analysis of rice roots[J].Plant Molecular Biology Reporter,2012,30(5):1204-1209.
[11] Lu s I M,Alexandre B M,Oliveira M M,et al.Selection of an appropriate protein extraction method to study the phosphoproteome of maize photosynthetic tissue[J].Plos One,2016,11(10):e0164387.
[12] 李成.马铃薯匍匐茎发育特性、二倍体野生种与四倍体栽培种块茎差异蛋白质组研究[D].兰州:甘肃农业大学,2015.
[13] Karppinen K,Taulavuori E,Hohtola A.Optimization of protein extraction from Hypericum perforatum tissues and immunoblotting detection of Hyp-1 at different stages of leaf development[J].Molecular Biotechnology,2010,46(3):219-26.
[14] Wang W,Vignani R,Scali M,et al.A universal and rapid protocol for protein extraction from recalcitrant plant tissues for proteomic analysis[J].Electrophoresis,2006,27(13):2782-2786.
[15] Vertommen A,Panis B,Swennen R,et al.Evaluation of chloroform/methanol extraction to facilitate the study of membrane proteins of non-model plants[J].Planta,2010,231(5):1113-1125.
[16] Jorrin J V,Maldonado A M,Castillejo M A.Plant proteome analysis:a 2006 update[J].Proteomics,2007,7(16):2947-2962.
[17] Fíla J,Apková V,Feciková J,et al.Impact of homogenization and protein extraction conditions on the obtained tobacco pollen proteomic patterns[J].Biologia Plantarum,2011,55(3):499-506.
[18] Xiang X,Ning S,Jiang X,et al.,Protein extraction from rice (Oryza sativa L.) root for two-dimensional electrophresis[J].Frontiers of Agriculture in China,2010,4(4):416-421.
[19] Raorane M L,Narciso J O,Kohli A.Recombinant proteins from plants:methods and protocols[M].New York:Springer,2016.
[20] Sghaier-Hammami B,Redondo-L pez I,Maldonado-Alconada A M,et al.A proteomic approach analysing the arabidopsis thaliana response to virulent and avirulent pseudomonas syringae strains[J].Acta Physiologiae Plantarum,2011,34(3):905-922.
[21] Jellouli N,Salem A B,Ghorbel A,et al.Evaluation of protein extraction methods for vitis vinifera leaf and root proteome analysis by two-dimensional electrophoresis[J].Journal of Integrative Plant Biology,2010,52(10):933-40.
[22] Carpentier S C,Witters E,Laukens K,et al.Preparation of protein extracts from recalcitrant plant tissues:An evaluation of different methods for two-dimensional gel electrophoresis analysis[J].Proteomics,2005,5(10):2497-2507.
[23] Wu X,Xiong E,Wang W,et al.Universal sample preparation method integrating trichloroacetic acid/acetone precipitation with phenol extraction for crop proteomic analysis[J].Nature Protocols,2014,9(2):362-374.
[24] Cilia M,Fish T,Yang X,et al.A comparison of protein extraction methods suitable for gel-based proteomic studies of aphid proteins[J].Journal of Biomolecular Techniques,2009,20(4):201-215.
[25] Delaplace P,Wal Fvd,Dierick J F,et al.Potato tuber proteomics:Comparison of two complementary extraction methods designed for 2-DE of acidic proteins[J].Proteomics,2006,24(6):6494-6497.
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