基于气质和液质联用技术的烟草鲜烟叶代谢组学分析流程
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摘要
为进一步推广普及烟草代谢组学研究技术,规范烟草代谢组学数据的采集流程,提高烟草代谢组学数据的可比性和可用性,建立了适用于新鲜烟叶代谢组学研究的样品采集和质谱检测方法。将采集后的鲜烟叶迅速用锡箔纸包裹并在液氮中冷冻;将充分冷冻的烟叶样品在干冰包埋下转移至实验室,真空冷冻干燥;将干燥后的烟叶样品打碎成粉末,提取代谢物;采用9种主要基于GC-MS和LC-MS技术的检测方法收集代谢组数据,分别是6种靶向检测(植物色素、游离氨基酸、有机酸、生物碱及多酚和萜类化合物)和3种非靶向检测(脂类、基于GC-MS的全成分分析和基于LC-MS全成分分析)方法。采用所建方法对不同产区、不同品种及不同生长发育时期的第十叶位鲜烟叶进行分析。结果表明:①共定性定量500余种代谢物,涉及烟草主要初生、次生代谢物,其中绝对定量101种;②主成分分析表明不同生长发育期鲜烟叶的代谢组差异较大,如大多数胺类(包括游离氨基酸)代谢物随生长发育期变化其质量分数逐渐降低;同一期鲜烟叶代谢组的生态差异大于品种差异。该方法具有稳定性和可重复性高、数据量大且可靠、覆盖代谢物多等特点,可用于烟草代谢组学研究。
To promote metabolomics technology in tobacco research, to standardize tobacco metabolomics data acquisition process, and to improve data's comparability and usability, a tobacco metabolomics workflow was established to cover fresh leave collection and mass spectrometry analysis. Fresh tobacco leaves were quickly wrapped using tin foil after picking, and then frozen in liquid nitrogen. Fully frozen tobacco leaves were embedded with dry ice and transferred to laboratories, and then grounded into powder after being lyophilized in vacuum for metabolite extraction. Tobacco metabolomics data were collected with nine different methods based mainly on GC-MS and LC-MS, including six targeted methods(on phytochromes, free amino acids, organic acids, alkaloids, polyphenols and terpenoids) and three untargeted methods(lipids, all components analysis based on GC-MS or LC-MS). Fresh tenth tobacco leaves(counted from bottom to top) sampled from different cultivars and planting areas at different growth stages were analyzed using the established methods. The results showed that: 1) Up to five hundred metabolites including mostly primary and secondary metabolites were qualified and quantified, of which 101 metabolites were absolutely quantified. 2) The metabolome of fresh tobacco leaves, indicated by principal component analysis(PCA) method, differed greatly at different growth stages, e.g. the contents of most amines(including free amino acids) in tobacco leaves gradually decreased during growing. At the same growth stage, ecological differences were greater than variety differences in the metabolome of fresh tobacco leaves. This method is stable, reliable and suitable for tobacco metabolomics research with the characteristics of high repeatability, rich data and broad coverage of metabolites in tobacco leaves.
To promote metabolomics technology in tobacco research, to standardize tobacco metabolomics data acquisition process, and to improve data's comparability and usability, a tobacco metabolomics workflow was established to cover fresh leave collection and mass spectrometry analysis. Fresh tobacco leaves were quickly wrapped using tin foil after picking, and then frozen in liquid nitrogen. Fully frozen tobacco leaves were embedded with dry ice and transferred to laboratories, and then grounded into powder after being lyophilized in vacuum for metabolite extraction. Tobacco metabolomics data were collected with nine different methods based mainly on GC-MS and LC-MS, including six targeted methods(on phytochromes, free amino acids, organic acids, alkaloids, polyphenols and terpenoids) and three untargeted methods(lipids, all components analysis based on GC-MS or LC-MS). Fresh tenth tobacco leaves(counted from bottom to top) sampled from different cultivars and planting areas at different growth stages were analyzed using the established methods. The results showed that: 1) Up to five hundred metabolites including mostly primary and secondary metabolites were qualified and quantified, of which 101 metabolites were absolutely quantified. 2) The metabolome of fresh tobacco leaves, indicated by principal component analysis(PCA) method, differed greatly at different growth stages, e.g. the contents of most amines(including free amino acids) in tobacco leaves gradually decreased during growing. At the same growth stage, ecological differences were greater than variety differences in the metabolome of fresh tobacco leaves. This method is stable, reliable and suitable for tobacco metabolomics research with the characteristics of high repeatability, rich data and broad coverage of metabolites in tobacco leaves.
引文
[1]杜文,谭新良,易建华,等.用烟叶化学成分进行烟叶质量评价[J].中国烟草学报,2007,13(3):25-31.DU Wen,TAN Xinliang,YI Jianhua,et al.Evaluation of leaf tobacco quality using chemical composition data[J].Acta Tabacaria Sinica,2007,13(3):25-31.
[2]陈彬,马君红,于晓娜,等.重庆烟区烟叶化学成分与感官质量关系研究[J].西南农业学报,2014,27(4):1756-1761.CHEN Bin,MA Junhong,YU Xiaona,et al.Research on relationship between chemical components and smoking quality of tobacco leaf in Chongqing[J].Southwest China Journal of Agricultural Sciences,2014,27(4):1756-1761.
[3]薛琳,朱启法,季学军,等.皖南烤烟烟叶化学成分与感官品质的相关性[J].烟草科技,2016,49(11):26-32.XUE Lin,ZHU Qifa,JI Xuejun,et al.Correlation of chemical components with overall sensory quality of tobacco leaves in south Anhui[J].Tobacco Science&Technology,2016,49(11):26-32.
[4]江厚龙,陈涛,马红辉,等.重庆烟区烤烟感官品质主要影响因子分析[J].烟草科技,2014(6):87-92.JIANG Houlong,CHEN Tao,MA Honghui,et al.Analysis of main factors influencing sensory quality of flue-cured tobacco in Chongqing growing areas[J].Tobacco Science&Technology,2014(6):87-92.
[5]吴有祥,王玉平,杨佳玫,等.清香型烟叶的感官品质风格特征及主要化学成分[J].贵州农业科学,2015(1):40-44.WU Youxiang,WANG Yuping,YANG Jiamei,et al.Sensory quality style characteristics and main chemical components of fen-flavour style tobacco[J].Guizhou Agricultural Sciences,2015(1):40-44.
[6]崔英.黔南烤烟化学成分与感官质量的典型相关分析[J].安徽农业科学,2017,45(33):21-25.CUI Ying.Canonical correlation analysis between components and smoking quality of flue-cured tobacco in Qiannan[J].Journal of Anhui Agricultural Science,2017,45(33):21-25.
[7]葛巍巍,张宏宇,顾坚.烟叶化学成分与燃烧特性的相关性[J].消防科学与技术,2012,31(10):1032-1035.GE Weiwei,ZHANG Hongyu,GU Jian.Correlation between chemical compositions and combustion characteristics of tobacco leaves[J].Fire Science and Technology,2012,31(10):1032-1035.
[8]石盼盼,宋朝鹏,王梅,等.烟草质体色素研究进展[J].湖南农业科学,2011(19):37-40.SHI Panpan,SONG Zhaopeng,WANG Mei,et al.Progress of plastid pigment in tobacco[J].Hunan Agricultural Sciences,2011(19):37-40.
[9]杨永霞,张嘉炜,贾宏昉,等.成熟期温度对烟叶质体色素积累及香气成分的影响[J].烟草科技,2016,49(5):16-22.YANG Yongxia,ZHANG Jiawei,JIA Hongfang,et al.Effects of temperature on plastid pigment accumulation and aroma components in tobacco leaves at mature stage[J].Tobacco Science&Technology,2016,49(5):16-22.
[10]徐晓燕,孙五三,王能如.烟草多酚类化合物的合成与烟叶品质的关系[J].中国烟草科学,2003,24(1):3-5.XU Xiaoyan,SUN Wusan,WANG Nengru.Synthesis of polyphenolic compounds and their effects on tobacco quality[J].Chinese Tobacco Science,2003,24(1):3-5.
[11]杨志晓,王轶,任学良,等.烟草多酚类化合物合成与积累影响因素研究进展[J].河南农业科学,2012,41(10):1-5.YANG Zhixiao,WANG Yi,REN Xueliang,et al.Research advance on influencing factors of synthesis and accumulation of polyphenolic compounds in tobacco[J].Journal of Henan Agricultural Sciences,2012,41(10):1-5.
[12]黄茹,吴玉萍,夏振远,等.氮素水平对红花大金元烟叶游离氨基酸和蛋白质含量的影响[J].中国烟草科学,2017,38(5):50-55.HUANG Ru,WU Yuping,XIA Zhenyuan,et al.Effect of nitrogen level on free amino acid and protein contents of Honghuadajinyuan tobacco leaves[J].Chinese Tobacco Science,2017,38(5):50-55.
[13]武雪萍,王小彬,查燕,等.施肥对土壤腐殖质总碳和氨基酸氮含量的影响[J].中国土壤与肥料,2010(6):43-49.WU Xueping,WANG Xiaobin,ZHA Yan,et al.The effect of fertilizer on the content of total humic acid Cand amino acids[J].Soil and Fertilizer Sciences in China,2010(6):43-49.
[14]杨永吉,罗莎.不同氨基酸含量的复合肥对烤烟生长和产量·品质的影响[J].安徽农业科学,2013,41(15):6674-6676.YANG Yongji,LUO Sha.Effects of compound fertilizer with different content of amino acid on growth,yield and quality of flue-cured tobacco[J].Journal of Anhui Agricultural Science,2013,41(15):6674-6676.
[15]肖波,刘光辉,李迪秦,等.氨基酸叶面肥喷施次数对烤烟上部叶产质量的影响[J].作物研究,2013(5):445-448.XIAO Bo,LIU Guanghui,LI Diqin,et al.Effect of application of frequency of amino-acid foliar fertilizer on yield and quality of upper tobacco leaves[J].Crop Research,2013(5):445-448.
[16]武雪萍,秦艳青,刘国顺,等.有机与无机肥不同配比对烟叶氨基酸含量的影响[J].河南农业大学学报,2003,37(2):115-118,123.WU Xueping,QIN Yanqing,LIU Guoshun,et al.Effects of combined application of different ratio of organic fertilizer and chemical fertilizer on amino acids of flue-cured tobacco leaves[J].Journal of Henan Agricultural University,2003,37(2):115-118,123.
[17]Hanifah A,Maharijaya A,Putri S P,et al.Untargeted metabolomics analysis of eggplant(Solanum melongena L.)fruit and its correlation to fruit morphologies[J].Metabolites,2018,8(3):49.
[18]Sánchez de Medina V,Calderón-Santiago M,El Riachy M,et al.High-resolution mass spectrometry to evaluate the influence of cross-breeding segregating populations on the phenolic profile of virgin olive oils[J].Journal of the Science of Food and Agriculture,2014,94(15):3100-3109.
[19]Chen W,Wang W,Peng M,et al.Comparative and parallel genome-wide association studies for metabolic and agronomic traits in cereals[J].Nature Communications,2016,7:12767.
[20]Mudau S P,Steenkamp P A,Piater L A,et al.Metabolomics-guided investigations of unintended effects of the expression of the hydroxycinnamoyl quinate hydroxycinnamoyltransferase(hqt1)gene from Cynara cardunculus var.scolymus in Nicotiana tabacum cell cultures[J].Plant Physiology and Biochemistry,2018,127:287-298.
[21]王森,李健爽,杜晓燕.代谢组学在转基因作物非预期效应评价中的应用[J].食品科学,2014,35(9):312-316.WANG Sen,LI Jianshuang,DU Xiaoyan.Applications of metabolomics in evaluation of unintended effects of genetically modified crops[J].Food Science,2014,35(9):312-316.
[22]Nam K H,Shin H J,Pack I S,et al.Metabolomic changes in grains of well-watered and drought-stressed transgenic rice[J].Journal of the Science of Food and Agriculture,2016,96(3):807-814.
[23]Ghatak A,Chaturvedi P,Weckwerth W.Metabolomics in plant stress physiology[M]//Advances in biochemical engineering/biotechnology,Cham:Springer,2018,164:187-236.
[24]张晓磊,张瑞英.代谢组学及其在农作物研究中的应用[J].生物技术通讯,2018,29(3):446-450.ZHANG Xiaolei,ZHANG Ruiying.Metabolomics and its application in the crop research[J].Letters in Biotechnology,2018,29(3):446-450.
[25]Gonzalez-Dominguez A,Duran-Guerrero E,Fernandez-Recamales A,et al.An overview on the importance of combining complementary analytical platforms in metabolomic research[J].Current Topics in Medicinal Chemistry,2017,17(30):3289-3295.
[26]Deborde C,Moing A,Roch L,et al.Plant metabolism as studied by NMR spectroscopy[J].Progress in Nuclear Magnetic Resonance Spectroscopy,2017,102/103:61-97.
[27]Koo I,Wei X,Zhang X.Analysis of metabolomic profiling data acquired on GC-MS[J].Methods in Enzymology,2014,543:315-324.
[28]Zhou B,Xiao J F,Tuli L,et al.LC-MS-based metabolomics[J].Molecular BioSystems,2012,8(2):470-481.
[29]Naz S,Moreira dos Santos D C,Garcia A,et al.Analytical protocols based on LC-MS,GC-MSand CE-MS for nontargeted metabolomics of biological tissues[J].Bioanalysis,2014,6(12):1657-1677.
[30]Want E,Masson P.Processing and analysis of GC/LC-MS-based metabolomics data[J].Methods in Molecular Biology,2011,708:277-298.
[31]YC/T 382-2010烟草及烟草制品质体色素的测定高效液相色谱法[S].YC/T 382-2010 Tobacco and tobacco productsDetermination of plastid pigments-High performance liquid chromatography method[S].
[32]沈丹红,路鑫,常玉玮,等.高效液相色谱-紫外/质谱检测法联合测定新鲜烟叶中的25种酚类物质[J].色谱,2014,32(1):40-46.SHEN Danhong,LU Xin,CHANG Yuwei,et al.Determination of phenolic compounds in fresh tobacco leaves by high performance liquid chromatographyultraviolet/mass spectrometry[J].Chinese Journal of Chromatography,2014,32(1):40-46.
[33]LI Lili,LU Xin,ZHAO Jieyu,et al.Lipidome and metabolome analysis of fresh tobacco leaves in different geographical regions using liquid chromatography-mass spectrometry[J].Analytical Bioanalytical Chemistry,2015,407:5009-5020.
[34]郑庆霞,刘萍萍,陈千思,等.气相色谱-串联质谱检测新鲜烟叶中的萜类成分[J/OL].烟草科技,2019:1-11.http://kns.cnki.net/kcms/detail/41.1137.ts.20190130.1515.002.html ZHENG Qingxia,LIU Pingping,CHEN Qiansi,et al.Determination of terpenoids in fresh tobacco leaves by GC-MS/MS[J/OL].Tobacco Science&Technology,2019:1-11.http://kns.cnki.net/kcms/detail/41.1137.ts.20190130.1515.002.html.
[35]ZHAO Yanni,ZHAO Chunxia,LU Xin,et al.Investigation of the relationship between the metabolic profile of tobacco leaves in different planting regions and climate factors using a pseudotargeted method based on gas chromatography/mass spectrometry[J].Journal of Proteome Research,2013,12(11):5072-5083.
[36]李好丽,赵春霞,张俊杰,等.柱前衍生-超高效液相色谱-单重四极杆质谱法分析不同产区烤烟的20种游离氨基酸[J].色谱,2013,31(12):1182-1188.LI Haoli,ZHAO Chunxia,ZHANG Junjie,et al.Determination of twenty free amino acids in flue-cured tobacco leaves using ultra performance liquid chromatography-single quadruple mass spectrometry and pre-column derivatization[J].Chinese Journal of Chromatography,2013,31(12):1182-1188.
[37]云南省烟草农业科学研究院,云南中烟工业有限责任公司.中国烟草总公司特色优质烟叶开发重大专项项目“清香型特色优质烟叶开发”技术报告[R].北京:国家烟草专卖局,2017.Yunnan Academy of Tobacco Agriculture Science,China Tobacco Yunnan Industrial Co.,Ltd..Technical report of major special projects for the development of characteristic and quality tobacco leaves of China National Tobacco Corporation:Development of fresh flavor type characteristic and quality tobacco leaves[R].Beijing:State Tobacco Monopoly Administration,2017.
[2]陈彬,马君红,于晓娜,等.重庆烟区烟叶化学成分与感官质量关系研究[J].西南农业学报,2014,27(4):1756-1761.CHEN Bin,MA Junhong,YU Xiaona,et al.Research on relationship between chemical components and smoking quality of tobacco leaf in Chongqing[J].Southwest China Journal of Agricultural Sciences,2014,27(4):1756-1761.
[3]薛琳,朱启法,季学军,等.皖南烤烟烟叶化学成分与感官品质的相关性[J].烟草科技,2016,49(11):26-32.XUE Lin,ZHU Qifa,JI Xuejun,et al.Correlation of chemical components with overall sensory quality of tobacco leaves in south Anhui[J].Tobacco Science&Technology,2016,49(11):26-32.
[4]江厚龙,陈涛,马红辉,等.重庆烟区烤烟感官品质主要影响因子分析[J].烟草科技,2014(6):87-92.JIANG Houlong,CHEN Tao,MA Honghui,et al.Analysis of main factors influencing sensory quality of flue-cured tobacco in Chongqing growing areas[J].Tobacco Science&Technology,2014(6):87-92.
[5]吴有祥,王玉平,杨佳玫,等.清香型烟叶的感官品质风格特征及主要化学成分[J].贵州农业科学,2015(1):40-44.WU Youxiang,WANG Yuping,YANG Jiamei,et al.Sensory quality style characteristics and main chemical components of fen-flavour style tobacco[J].Guizhou Agricultural Sciences,2015(1):40-44.
[6]崔英.黔南烤烟化学成分与感官质量的典型相关分析[J].安徽农业科学,2017,45(33):21-25.CUI Ying.Canonical correlation analysis between components and smoking quality of flue-cured tobacco in Qiannan[J].Journal of Anhui Agricultural Science,2017,45(33):21-25.
[7]葛巍巍,张宏宇,顾坚.烟叶化学成分与燃烧特性的相关性[J].消防科学与技术,2012,31(10):1032-1035.GE Weiwei,ZHANG Hongyu,GU Jian.Correlation between chemical compositions and combustion characteristics of tobacco leaves[J].Fire Science and Technology,2012,31(10):1032-1035.
[8]石盼盼,宋朝鹏,王梅,等.烟草质体色素研究进展[J].湖南农业科学,2011(19):37-40.SHI Panpan,SONG Zhaopeng,WANG Mei,et al.Progress of plastid pigment in tobacco[J].Hunan Agricultural Sciences,2011(19):37-40.
[9]杨永霞,张嘉炜,贾宏昉,等.成熟期温度对烟叶质体色素积累及香气成分的影响[J].烟草科技,2016,49(5):16-22.YANG Yongxia,ZHANG Jiawei,JIA Hongfang,et al.Effects of temperature on plastid pigment accumulation and aroma components in tobacco leaves at mature stage[J].Tobacco Science&Technology,2016,49(5):16-22.
[10]徐晓燕,孙五三,王能如.烟草多酚类化合物的合成与烟叶品质的关系[J].中国烟草科学,2003,24(1):3-5.XU Xiaoyan,SUN Wusan,WANG Nengru.Synthesis of polyphenolic compounds and their effects on tobacco quality[J].Chinese Tobacco Science,2003,24(1):3-5.
[11]杨志晓,王轶,任学良,等.烟草多酚类化合物合成与积累影响因素研究进展[J].河南农业科学,2012,41(10):1-5.YANG Zhixiao,WANG Yi,REN Xueliang,et al.Research advance on influencing factors of synthesis and accumulation of polyphenolic compounds in tobacco[J].Journal of Henan Agricultural Sciences,2012,41(10):1-5.
[12]黄茹,吴玉萍,夏振远,等.氮素水平对红花大金元烟叶游离氨基酸和蛋白质含量的影响[J].中国烟草科学,2017,38(5):50-55.HUANG Ru,WU Yuping,XIA Zhenyuan,et al.Effect of nitrogen level on free amino acid and protein contents of Honghuadajinyuan tobacco leaves[J].Chinese Tobacco Science,2017,38(5):50-55.
[13]武雪萍,王小彬,查燕,等.施肥对土壤腐殖质总碳和氨基酸氮含量的影响[J].中国土壤与肥料,2010(6):43-49.WU Xueping,WANG Xiaobin,ZHA Yan,et al.The effect of fertilizer on the content of total humic acid Cand amino acids[J].Soil and Fertilizer Sciences in China,2010(6):43-49.
[14]杨永吉,罗莎.不同氨基酸含量的复合肥对烤烟生长和产量·品质的影响[J].安徽农业科学,2013,41(15):6674-6676.YANG Yongji,LUO Sha.Effects of compound fertilizer with different content of amino acid on growth,yield and quality of flue-cured tobacco[J].Journal of Anhui Agricultural Science,2013,41(15):6674-6676.
[15]肖波,刘光辉,李迪秦,等.氨基酸叶面肥喷施次数对烤烟上部叶产质量的影响[J].作物研究,2013(5):445-448.XIAO Bo,LIU Guanghui,LI Diqin,et al.Effect of application of frequency of amino-acid foliar fertilizer on yield and quality of upper tobacco leaves[J].Crop Research,2013(5):445-448.
[16]武雪萍,秦艳青,刘国顺,等.有机与无机肥不同配比对烟叶氨基酸含量的影响[J].河南农业大学学报,2003,37(2):115-118,123.WU Xueping,QIN Yanqing,LIU Guoshun,et al.Effects of combined application of different ratio of organic fertilizer and chemical fertilizer on amino acids of flue-cured tobacco leaves[J].Journal of Henan Agricultural University,2003,37(2):115-118,123.
[17]Hanifah A,Maharijaya A,Putri S P,et al.Untargeted metabolomics analysis of eggplant(Solanum melongena L.)fruit and its correlation to fruit morphologies[J].Metabolites,2018,8(3):49.
[18]Sánchez de Medina V,Calderón-Santiago M,El Riachy M,et al.High-resolution mass spectrometry to evaluate the influence of cross-breeding segregating populations on the phenolic profile of virgin olive oils[J].Journal of the Science of Food and Agriculture,2014,94(15):3100-3109.
[19]Chen W,Wang W,Peng M,et al.Comparative and parallel genome-wide association studies for metabolic and agronomic traits in cereals[J].Nature Communications,2016,7:12767.
[20]Mudau S P,Steenkamp P A,Piater L A,et al.Metabolomics-guided investigations of unintended effects of the expression of the hydroxycinnamoyl quinate hydroxycinnamoyltransferase(hqt1)gene from Cynara cardunculus var.scolymus in Nicotiana tabacum cell cultures[J].Plant Physiology and Biochemistry,2018,127:287-298.
[21]王森,李健爽,杜晓燕.代谢组学在转基因作物非预期效应评价中的应用[J].食品科学,2014,35(9):312-316.WANG Sen,LI Jianshuang,DU Xiaoyan.Applications of metabolomics in evaluation of unintended effects of genetically modified crops[J].Food Science,2014,35(9):312-316.
[22]Nam K H,Shin H J,Pack I S,et al.Metabolomic changes in grains of well-watered and drought-stressed transgenic rice[J].Journal of the Science of Food and Agriculture,2016,96(3):807-814.
[23]Ghatak A,Chaturvedi P,Weckwerth W.Metabolomics in plant stress physiology[M]//Advances in biochemical engineering/biotechnology,Cham:Springer,2018,164:187-236.
[24]张晓磊,张瑞英.代谢组学及其在农作物研究中的应用[J].生物技术通讯,2018,29(3):446-450.ZHANG Xiaolei,ZHANG Ruiying.Metabolomics and its application in the crop research[J].Letters in Biotechnology,2018,29(3):446-450.
[25]Gonzalez-Dominguez A,Duran-Guerrero E,Fernandez-Recamales A,et al.An overview on the importance of combining complementary analytical platforms in metabolomic research[J].Current Topics in Medicinal Chemistry,2017,17(30):3289-3295.
[26]Deborde C,Moing A,Roch L,et al.Plant metabolism as studied by NMR spectroscopy[J].Progress in Nuclear Magnetic Resonance Spectroscopy,2017,102/103:61-97.
[27]Koo I,Wei X,Zhang X.Analysis of metabolomic profiling data acquired on GC-MS[J].Methods in Enzymology,2014,543:315-324.
[28]Zhou B,Xiao J F,Tuli L,et al.LC-MS-based metabolomics[J].Molecular BioSystems,2012,8(2):470-481.
[29]Naz S,Moreira dos Santos D C,Garcia A,et al.Analytical protocols based on LC-MS,GC-MSand CE-MS for nontargeted metabolomics of biological tissues[J].Bioanalysis,2014,6(12):1657-1677.
[30]Want E,Masson P.Processing and analysis of GC/LC-MS-based metabolomics data[J].Methods in Molecular Biology,2011,708:277-298.
[31]YC/T 382-2010烟草及烟草制品质体色素的测定高效液相色谱法[S].YC/T 382-2010 Tobacco and tobacco productsDetermination of plastid pigments-High performance liquid chromatography method[S].
[32]沈丹红,路鑫,常玉玮,等.高效液相色谱-紫外/质谱检测法联合测定新鲜烟叶中的25种酚类物质[J].色谱,2014,32(1):40-46.SHEN Danhong,LU Xin,CHANG Yuwei,et al.Determination of phenolic compounds in fresh tobacco leaves by high performance liquid chromatographyultraviolet/mass spectrometry[J].Chinese Journal of Chromatography,2014,32(1):40-46.
[33]LI Lili,LU Xin,ZHAO Jieyu,et al.Lipidome and metabolome analysis of fresh tobacco leaves in different geographical regions using liquid chromatography-mass spectrometry[J].Analytical Bioanalytical Chemistry,2015,407:5009-5020.
[34]郑庆霞,刘萍萍,陈千思,等.气相色谱-串联质谱检测新鲜烟叶中的萜类成分[J/OL].烟草科技,2019:1-11.http://kns.cnki.net/kcms/detail/41.1137.ts.20190130.1515.002.html ZHENG Qingxia,LIU Pingping,CHEN Qiansi,et al.Determination of terpenoids in fresh tobacco leaves by GC-MS/MS[J/OL].Tobacco Science&Technology,2019:1-11.http://kns.cnki.net/kcms/detail/41.1137.ts.20190130.1515.002.html.
[35]ZHAO Yanni,ZHAO Chunxia,LU Xin,et al.Investigation of the relationship between the metabolic profile of tobacco leaves in different planting regions and climate factors using a pseudotargeted method based on gas chromatography/mass spectrometry[J].Journal of Proteome Research,2013,12(11):5072-5083.
[36]李好丽,赵春霞,张俊杰,等.柱前衍生-超高效液相色谱-单重四极杆质谱法分析不同产区烤烟的20种游离氨基酸[J].色谱,2013,31(12):1182-1188.LI Haoli,ZHAO Chunxia,ZHANG Junjie,et al.Determination of twenty free amino acids in flue-cured tobacco leaves using ultra performance liquid chromatography-single quadruple mass spectrometry and pre-column derivatization[J].Chinese Journal of Chromatography,2013,31(12):1182-1188.
[37]云南省烟草农业科学研究院,云南中烟工业有限责任公司.中国烟草总公司特色优质烟叶开发重大专项项目“清香型特色优质烟叶开发”技术报告[R].北京:国家烟草专卖局,2017.Yunnan Academy of Tobacco Agriculture Science,China Tobacco Yunnan Industrial Co.,Ltd..Technical report of major special projects for the development of characteristic and quality tobacco leaves of China National Tobacco Corporation:Development of fresh flavor type characteristic and quality tobacco leaves[R].Beijing:State Tobacco Monopoly Administration,2017.
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