应用iTRAQ结合2D LC-MS/MS技术分析哈蟆油与伪品蛙油的差异蛋白质组
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摘要
采用iT RAQ结合2D LC-MS/MS技术对哈蟆油、青蛙油、蟾蜍油、牛蛙油的蛋白质组进行研究。采用Q Exactive质谱鉴定并经MASCOT软件搜库,对所鉴定的蛋白质进行理化性质分析、层次聚类分析、Gene ontology(GO)功能注释和生物信息学分析。结果共获得2286个肽段,鉴定到1220个蛋白质,其中1212个蛋白质具有定量信息。鉴定得到的蛋白质分子量主要分布在4. 1～36. 9 kD a,占82. 79%,等电点主要分布在4. 41～9. 89,占94. 84%。对比青蛙油、蟾蜍油和牛蛙油与哈蟆油蛋白质数据库,筛选得到8个差异蛋白(比值> 1. 2或比值<0. 83,且P <0. 05),其中显著上调的蛋白质3个,显著下调的蛋白质5个。本结果在蛋白质组成上显示了哈蟆油与伪品蛙油的差异性,为进一步分析哈蟆油中蛋白质成分特点、功能奠定基础。
Isobaric tags for relative and absolute quantification( iTRAQ) and proteomic methods were used to identify different expressed proteins between the Oviductus Ranae( OR) and counterfeit oviducts( oviducts of Rana Nigromaculata,Bufo bufogargarizans and Rana catesbeiana). Q exactive mass spectrometry was used to identify the proteins,and search the database by MASCOT software. The identified proteins were analyzed the physical and chemical properties,clustering analysis,GO annotation and bioinformatics analysis. The results showed a total of 1220 proteins were identified at from 2286 unique peptide sequences,1212 of which had quantitative information. The molecular weight of identified proteins were mainly distributed in 4. 1-36. 9 kD a,accounting for 82. 79%,and the isoelectric points were mainly distributed in 4. 41-9. 89,accounting for 94. 84%. Comparing with counterfeit oviducts,8 proteins were differentially excreted by 1. 2 fold or 0. 83 fold,P < 0. 05 in OR,including 3 up-regulated and 5 down-regulated proteins. In conclusion,this data showed the specific expressed proteins in OR. The study established foundation to reveal the composition characteristics and function of proteins.
Isobaric tags for relative and absolute quantification( iTRAQ) and proteomic methods were used to identify different expressed proteins between the Oviductus Ranae( OR) and counterfeit oviducts( oviducts of Rana Nigromaculata,Bufo bufogargarizans and Rana catesbeiana). Q exactive mass spectrometry was used to identify the proteins,and search the database by MASCOT software. The identified proteins were analyzed the physical and chemical properties,clustering analysis,GO annotation and bioinformatics analysis. The results showed a total of 1220 proteins were identified at from 2286 unique peptide sequences,1212 of which had quantitative information. The molecular weight of identified proteins were mainly distributed in 4. 1-36. 9 kD a,accounting for 82. 79%,and the isoelectric points were mainly distributed in 4. 41-9. 89,accounting for 94. 84%. Comparing with counterfeit oviducts,8 proteins were differentially excreted by 1. 2 fold or 0. 83 fold,P < 0. 05 in OR,including 3 up-regulated and 5 down-regulated proteins. In conclusion,this data showed the specific expressed proteins in OR. The study established foundation to reveal the composition characteristics and function of proteins.
引文
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[10]Goulet F,Hélie P,Vachon P.Eugenol anesthesia in African clawed frogs(Xenopus laevis)of different body weights[J].J Am Assoc Lab Anim Sci,2010,49:460-463.
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[12]Zhou Y S,Lamrani M,Chan-Park M B,et al.i TRAQ-coupled two-dimensional liquid chromatography/tandem mass spectrometric analysis of protein profile in Escherichia coli incubated with human neutrophil peptide 1-potential in antimicrobial strategy[J].Rapid Commun Mass Spectrom,2010,24(18):2787-2790.
[13]Golovan S P,Hakimov H A,Verschoor C P,et al.Analysis of Sus scrofa liver proteome and identification of proteins differentially expressed between genders,and conventional and genetically enhanced lines[J].Comp Biochem Physiol Part D:Genomics Proteomics,2008,3(3):234-242.
[14]刘靖宇.草菇不同生长发育时期的比较蛋白质组学分析[D].福州:福建农林大学,2012.
[15]Zhang S F,Zhang Y,Xie Z X,et al.i TRAQ-based quantitative proteomic analysis of a toxigenic dinoflagellate Alexandrium catenella and its non-toxic mutant[J].Proteomics,2015,15(23-24):4041-4050.
[16]张哲文.中国林蛙皮肤转录组和蛋白质组学研究[D].大连:辽宁师范大学,2010.
[17]倪洋.中国林蛙皮肤分泌物蛋白质组学研究[D].大连:辽宁师范大学,2012.
[18]Dong Weibing,Sun Yue,Shang Dejing.Interactions between chensinin-1,a natural antimicrobial peptide derived from Rana chensinensis,and lipopolysaccharide[J].Biopolymers,2015,103(12):719-726.
[19]Shang D,Li X,Sun Y,et al.Design of Potent,Non-Toxic Antimicrobial Agents Based upon the Structure of the Frog Skin Peptide,Temporin-1CEb from Chinese Brown Frog,Rana chensinensis[J].Chemical Biology&Drug Design,2012,79(5):653-662.
[2]于洋,姜大成,张炜煜.哈蟆油对免疫力低下小鼠体内脂肪酸含量的影响[J].长春中医药大学学报,2008,24(2):150-151.
[3]瞿新明,李芸彤,张惠,等.哈蟆油非水溶性成分及其酶解物的抗氧化功效研究[J].食品科技,2012,37(10):211-213.
[4]李津明,宋茜,孙仁爽,等.林蛙油酶解前后抗疲劳作用对比研究[J].黑龙江医药,2008,21(2):30-34.
[5]王涵,赵雨,张梅,等.哈蟆油非水溶性成分抗疲劳保健功能研究[J].安徽农业大学,2011,39(20):12233-12234.
[6]张梅,赵雨,李云彤,等.哈蟆油非水溶性成分酶解前后降血脂功效比较研究[J].时珍国医国药,2012,23(2):384-385.
[7]张梅,赵雨,杨士慧,等.哈蟆油酶解前后耐缺氧活性比较研究[J].江苏中医药,2011,43(6):87-88.
[8]Wu Wells W,Wang Guanghui,Baek Seung Joon,et al.Comparative Study of Three Proteomic Quantitative Methods,DIGE,c ICAT,and i TRAQ,Using 2D Gel-or LC-MALDI TOF/TOF[J].Journal of Proteome Research,2006,5(3):651-658.
[9]GB/T 19507-2008,地理标志产品吉林长白山中国林蛙油[S].?
[10]Goulet F,Hélie P,Vachon P.Eugenol anesthesia in African clawed frogs(Xenopus laevis)of different body weights[J].J Am Assoc Lab Anim Sci,2010,49:460-463.
[11]刘靖宇,江玉姬,谢宝贵,等.应用i TRAQ结合2D LC-MS/MS技术分析草菇同核和异核菌丝蛋白质组[J].食用菌学报,2012,19(1):1-12.
[12]Zhou Y S,Lamrani M,Chan-Park M B,et al.i TRAQ-coupled two-dimensional liquid chromatography/tandem mass spectrometric analysis of protein profile in Escherichia coli incubated with human neutrophil peptide 1-potential in antimicrobial strategy[J].Rapid Commun Mass Spectrom,2010,24(18):2787-2790.
[13]Golovan S P,Hakimov H A,Verschoor C P,et al.Analysis of Sus scrofa liver proteome and identification of proteins differentially expressed between genders,and conventional and genetically enhanced lines[J].Comp Biochem Physiol Part D:Genomics Proteomics,2008,3(3):234-242.
[14]刘靖宇.草菇不同生长发育时期的比较蛋白质组学分析[D].福州:福建农林大学,2012.
[15]Zhang S F,Zhang Y,Xie Z X,et al.i TRAQ-based quantitative proteomic analysis of a toxigenic dinoflagellate Alexandrium catenella and its non-toxic mutant[J].Proteomics,2015,15(23-24):4041-4050.
[16]张哲文.中国林蛙皮肤转录组和蛋白质组学研究[D].大连:辽宁师范大学,2010.
[17]倪洋.中国林蛙皮肤分泌物蛋白质组学研究[D].大连:辽宁师范大学,2012.
[18]Dong Weibing,Sun Yue,Shang Dejing.Interactions between chensinin-1,a natural antimicrobial peptide derived from Rana chensinensis,and lipopolysaccharide[J].Biopolymers,2015,103(12):719-726.
[19]Shang D,Li X,Sun Y,et al.Design of Potent,Non-Toxic Antimicrobial Agents Based upon the Structure of the Frog Skin Peptide,Temporin-1CEb from Chinese Brown Frog,Rana chensinensis[J].Chemical Biology&Drug Design,2012,79(5):653-662.