基于气相色谱质谱联用的酵母菌代谢组学分析方法的建立
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摘要
近年来微生物代谢组学逐渐成为一种新兴的分析手段.微生物虽然有着结构简单、营养需求单一的特点,但也存在自身种类繁多、细胞内代谢物质理化性质各不相同的弊端.因此,在前期样品处理方面仍存在很大的不足.基于GC-MS的分析方法,建立了一种用于酵母菌S288C的代谢组学分析方法,通过考察甲醇(-20℃)、V(乙腈):V(水)=1:1(-20℃)、V(乙腈):V(甲醇):V(水)=2:2:1(-20℃)、V(水):V(异丙醇):V(甲醇)=2:2:5(-20℃)、75%乙醇(煮沸):5种溶剂对胞内物提取的影响,确定最佳的提取溶剂为V(水):V(异丙醇):V(甲醇)=2:2:5(-20℃).该方法可检测348种代谢物,通过数据库检索可定性出142种物质,包括46种氨基酸类代谢物、9种糖类代谢物、30种有机酸代谢物、17种醇类代谢物以及23种酯类代谢物.
In recent years,microbial metabolomics has gradually become a new analysis method.Although microorganisms have the characteristics of simple structure and single nutritional requirement,they also have many kinds of their own.The physical and chemical properties of intracellular metabolites are different.Therefore,there are still a lot of deficiencies in the pre-sample treatment.Based on GC-MS,a metabolomics analysis method for yeast S288 C has been established in this paper.The effects of five solvents,including methanol(-20 ℃),V(acetonitrile)■V(water)=1■1(-20 ℃),V(acetonitrile)■V(methanol/water)=2■2■1(-20 ℃),V(water)■V(isopropanol/methanol)=2■2■5(-20 ℃) and 75% ethanol(boiling) on the extraction of intracellular substances are investigated.The optimum solvent is V(water)■V(isopropanol)■V(methanol)=2■2■5(-20 ℃).The method can detect 348 metabolites,and 142 metabolites are identified by NIST library searching,including 46 amino acids,9 carbohydrates,30 organic acids,17 alcohols and 23 esters.
In recent years,microbial metabolomics has gradually become a new analysis method.Although microorganisms have the characteristics of simple structure and single nutritional requirement,they also have many kinds of their own.The physical and chemical properties of intracellular metabolites are different.Therefore,there are still a lot of deficiencies in the pre-sample treatment.Based on GC-MS,a metabolomics analysis method for yeast S288 C has been established in this paper.The effects of five solvents,including methanol(-20 ℃),V(acetonitrile)■V(water)=1■1(-20 ℃),V(acetonitrile)■V(methanol/water)=2■2■1(-20 ℃),V(water)■V(isopropanol/methanol)=2■2■5(-20 ℃) and 75% ethanol(boiling) on the extraction of intracellular substances are investigated.The optimum solvent is V(water)■V(isopropanol)■V(methanol)=2■2■5(-20 ℃).The method can detect 348 metabolites,and 142 metabolites are identified by NIST library searching,including 46 amino acids,9 carbohydrates,30 organic acids,17 alcohols and 23 esters.
引文
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[2] Lee M Y,Park H M,Son G H,et al.Liquid chromatography-mass spectrometry-based chemotaxonomic classification of Aspergillus spp.and evaluation of the biological activity of its unique metabolite,neosartorin[J].Journal of Microbiology & Biotechnology,2013,23(7):932.
[3] 王希越,田媛媛,李秋颖,等.微生物代谢组学样品前处理方法研究进展[J].科技展望,2016,26(36):71.
[4] Castrillo J I,Hayes A,Mohammed S,et al.An optimized protocol for metabolome analysis in yeast using direct infusion electrospray mass spectrometry[J].Phytochemistry(Amsterdam),2003,62(6):929-937.
[5] Koning W D,Dam K V.A method for the determination of changes of glycolytic metabolites in yeast on a subsecond time scale using extraction at neutral pH[J].Analytical Biochemistry,1992,204(1):118-123.
[6] Dam J C V,Eman M R,Frank J,et al.Analysis of glycolytic intermediates in Saccharomyces cerevisiae using anion exchange chromatography and electrospray ionization with tandem mass spectrometric detection[J].Analytica Chimica Acta,2002,460(2):209-218.
[7] Ewald J C,Stéphanie heux,Zamboni N.High-Throughput Quantitative Metabolomics:Workflow for Cultivation,Quenching,and Analysis of Yeast in a Multiwell Format[J].Analytical Chemistry,2009,81(9):3623-3629.
[8] Buchholz A,Takors R,Wandrey C.Quantification of Intracellular Metabolites in Escherichia coli K12 Using Liquid Chromatographic-Electrospray Ionization Tandem Mass Spectrometric Techniques[J].Analytical Biochemistry,2001,295(2):129-137.
[9] Moritz B,Striegel K,Graaf A A D,et al.Kinetic properties of the glucose-6-phosphate and 6 phosphogluconate dehydrogenases from Corynebacterium glutamicum and their application for predicting pentose phosphate pathway flux in vivo[J].Febs Journal,2010,267(12):3442-3452.
[10] Alrabiah H,Xu Y,Rattray N J W,et al.Multiple metabolomics of uropathogenic E.coli reveal different information content in terms of metabolic potential compared to virulence factors[J].The Analyst,2014,139(17):4193.
[11] Bolten C J,Kiefer P,Letisse F,et al.Sampling for metabolome analysis of microorganisms.[J].Analytical Chemistry,2007,79(10):3843-9.
[12] Faijes M,Mars A E,Smid E J.Comparison of quenching and extraction methodologies for metabolome analysis of Lactobacillus plantarum[J].Microbial Cell Factories,2007,6(1):27-27.
[13] Jensen N,Jokumsen K,Villadsen J.Determination of the phosphorylated sugars of the Embden-Meyerhoff-Parnas Pathway in Lactococcus lactis using a fast sampling technique and solid phase extraction[J].Biotechnology & Bioengineering,2015,63(3):356-362.
[14] Wittmann C,Kr?mer JO,Kiefer P,et al.Impact of the cold shock phenomenon on quantification of intracellular metabolites in bacteria[J].Analytical Biochemistry,2004,327(1):135-139.
[15] 王希越.基于色谱质谱的代谢组学技术对大肠杆菌代谢表型的研究[D].大连:中国科学院大连化学物理研究所,2015.