Study on sample preparation for the metabolomics of <i>Metarhizium</i> based on GC-MS

YANG Hua; XU Jinzhu; ZHAO Danyang; QIU Hualong; TIAN Longyan; QIN Changsheng

doi:10.7671/j.issn.1001-411X.202010022

Journal of South China Agricultural University > 2021 > 42(5): 69-79. > DOI: 10.7671/j.issn.1001-411X.202010022

YANG Hua, XU Jinzhu, ZHAO Danyang, et al. Study on sample preparation for the metabolomics of Metarhizium based on GC-MS[J]. Journal of South China Agricultural University, 2021, 42(5): 69-79. DOI: 10.7671/j.issn.1001-411X.202010022

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Study on sample preparation for the metabolomics of Metarhizium based on GC-MS

1.
College of Agriculture, South China Agricultural University, Guangzhou 510642, China
2.
Guangdong Academy of Forestry/Guangdong Provincial Key Laboratory of Silviculture, Protection and Utilization, Guangzhou 510520, China

More Information

Received Date: October 22, 2020
Available Online: May 17, 2023

Graphical Abstract

Abstract

Abstract

Objective
The metabolite pretreatment technology was optimized based on GC-MS to establish a rapid, accurate sample preparation protocol for metabolomics analysis in Metarhizium.

Method
Several sample preparation steps, including cell quenching, metabolite extraction, derivatization and detection were optimized, and the stability of this method was also determined.

Result
The quenching effect of 40% cold ethanol was better than that of other quenching solutions, and the recoveries of nucleic acid and protein of Metarhizium were 9.63% and 11.61% respectively. Total 109 metabolites were obtained by cold methanol, more than those by other methods. The longer derivation time was, the more metabolites could be obtained, and 1.5 h was the best. Too high initial temperature of gas chromatography was not conducive to acquisition of metabolites, and 50 ℃ was the best. The optimal sample preparation conditions were as follows: After quenching with 40% cold ethanol, the supernatant was extracted with 2 mL cold methanol. After centrifugation, the supernatant was dried with N₂, and then added with 80 μL 20 mg/mL methoxylamine hydrochloride pyridine solution. After severe oscillation for 30 s, the supernatant was reacted at 37 ℃ for 90 min, and cooled to room temperature. Then 80 μL BSTFA derivatization agent with 1%(φ) TMCS was added, the derivatization reaction continued for 1.5 h at 70 ℃, and solution cooled to room temperature.

Conclusion
The method is simple, convenient and reproducible, it is conducive to carry out more in depth studies on metabolic mechanisms, and provides references for related studies on metabolic groups of pathogenic microorganisms in agriculture and forestry.
- metabolomics,
- Metarhizium,
- quenching,
- extraction,
- pretreatment,
- GC-MS

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References (44)

References

[1]	NICHOLSON J K, LINDON J C. System biology-metabonomics[J]. Nature, 2008, 455(7216): 1054-1056. doi: 10.1038/4551054a
[2]	PATTI G J, YANES O, SIUZDAK G. Metabolomics: The apogee of the omics trilogy[J]. Nature Reviews Molecular Cell Biology, 2012, 13(4): 263-269. doi: 10.1038/nrm3314
[3]	KARPE A V, BEALE D J, MORRISON P D, et al. Untargeted metabolic profiling of Vitis vinifera during fungal degradation[J]. FEMS Microbiology Letters, 2015, 362(10): fnv060.
[4]	VIPUL S B, PRASUN B, GIRISH H R, et al. Amelioration of biomass and lipid in marine alga by an endophytic fungus Piriformospora indica[J]. Biotechnology for Biofuels, 2019, 12: 176. https://doi.org/10.1186/s13068-019-1516-6.
[5]	OSMAN S M, FARIBA T, SICONG Z, et al. Correlations between LC-MS/MS-detected glycomics and NMR-detected metabolomics in Caenorhabditis elegans development[J]. Frontiers in Molecular Biosciences, 2019(6): 49.
[6]	SHEN Y, FATEMEH T, TANG L, et al. Quantitative metabolic network profiling of Escherichia coli: An overview of analytical methods for measurement of intracellular metabolites[J]. Trac-Trends in Analytical Chemistry, 2016, 75: 141-150. doi: 10.1016/j.trac.2015.07.006
[7]	SPURA J, CHRISTIAN REIMER L, WIELOCH P, et al. A method for enzyme quenching in microbial metabolome analysis successfully applied to gram-positive and gram-negative bacteria and yeast[J]. Analytical Biochemistry, 2009, 394(2): 192-201. doi: 10.1016/j.ab.2009.07.016
[8]	WINDER C L, DUNN W B, SCHULER S, et al. Global metabolic profiling of Escherichia coli cultures: An evaluation of methods for quenching and extraction of intracellular metabolites[J]. Analytical Chemistry, 2008, 80(8): 2939-2948. doi: 10.1021/ac7023409
[9]	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: 27. doi: 10.1186/1475-2859-6-27
[10]	CANELAS A B, TEN PIERICK A, RAS C, et al. Quantitative evaluation of intracellular metabolite extraction techniques for yeast metabolomics[J]. Analytical Chemistry, 2009, 81(17): 7379-7389. doi: 10.1021/ac900999t
[11]	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, 2003, 62(6): 929-937. doi: 10.1016/S0031-9422(02)00713-6
[12]	MAHARJAN P R, FERENCI T. Global metabolite analysis: The influence of extraction methodology on metabolome profiles of Escherichia coli[J]. Analytical Biochemistry, 2003, 313(1): 145-154. doi: 10.1016/S0003-2697(02)00536-5
[13]	MARCINOWSKA R, TRYGG J, WOLF-WATZ H, et al. Optimization of a sample preparation method for the metabolomic analysis of clinically relevant bacteria[J]. Journal of Microbiological Methods, 2011, 87(1): 24-31. doi: 10.1016/j.mimet.2011.07.001
[14]	CAJKA T, FIEHN O. Toward merging untargeted and targeted methods in mass spectrometry-based metabolomics and lipidomics[J]. Analytical Chemistry, 2015, 88(1): 524-545.
[15]	ARTHURS S, DARA S K. Microbial biopesticides for invertebrate pests and their markets in the United States[J]. Journal of Invertebrate Pathology, 2019, 165: 13-21. doi: 10.1016/j.jip.2018.01.008
[16]	任春光, 谭玉梅, 任秀秀, 等. 冠突曲霉veA基因缺失型与野生型的差异代谢物研究[J]. 菌物学报, 2018, 37(2): 193-204.
[17]	罗飞飞, 李淑林, 陈龙云, 等. 代谢组学方法鉴定球孢白僵菌孢子萌发和杀虫毒力相关的标记物[J]. 微生物学报, 2014, 54(1): 33-41.
[18]	PARK S J, HYUN S, SUH H W, et al. Biochemical characterization of cultivated Cordyceps bassiana mycelia and fruiting bodies by 1H nuclear magnetic resonance spectroscopy[J]. Metabolomics, 2013, 9(1): 236-246. doi: 10.1007/s11306-012-0442-4
[19]	de BEKKER C, SMITH P B, PATTERSON A D, et al. Metabolomics reveals the heterogeneous secretome of two entomopathogenic fungi to ex vivo cultured insect tissues[J]. PLos One, 2013, 8(8): e70609. doi: 10.1371/journal.pone.0070609
[20]	SAJED T, MARCU A, RAMIREZ M, et al. ECMDB 2.0: A richer resource for understanding the biochemistry of E. coli[J]. Nucleic Acids Research, 2016, 44(D1): D495-D501. doi: 10.1093/nar/gkv1060
[21]	ZAKHARTSEV M, VIELHAUER O, HORN T, et al. Fast sampling for quantitative microbial metabolomics: New aspects on cold methanol quenching: Metabolite co-precipitation[J]. Metabolomics, 2015, 11(2): 286-301. doi: 10.1007/s11306-014-0700-8
[22]	刘阳, 邓静, 吴华昌, 等. 盐胁迫对枯草芽孢杆菌发酵代谢产物的影响[J]. 食品与发酵工业, 2015, 41(7): 29-33.
[23]	王洪彬, 杨泓喆, 杨霁菡, 等. 枯草芽孢杆菌代谢组样品前处理方法的比较研究[J]. 分析化学, 2015, 43(8): 1169-1174.
[24]	LUO F, WANG Q, YIN C, et al. Differential metabolic responses of Beauveria bassiana cultured in pupae extracts, root exudates and its interactions with insect and plant[J]. Journal of Invertebrate Pathology, 2015, 130: 154-164. doi: 10.1016/j.jip.2015.01.003
[25]	TSUCHIDO T, NISHINO T, KATO Y, et al. Involvement of membrane lipids in cold shock-induced autolysis of Bacillus subtilis cells[J]. Bioscience, Biotechnology, and Biochemistry, 1995, 59(9): 1636-1640. doi: 10.1271/bbb.59.1636
[26]	VILLAS-BÔAS S G, BRUHEIM P. Cold glycerol-saline: The promising quenching solution for accurate intracellular metabolite analysis of microbial cells[J]. Analytical Biochemistry, 2007, 370(1): 87-97. doi: 10.1016/j.ab.2007.06.028
[27]	明明. 基于GC-MS代谢组学方法的建立及对酿酒酵母乙醇耐受性机制研究[D]. 吉林: 吉林化工学院, 2019.
[28]	胡志宏, 常旭念, 代探, 等. 基于GC-MS的灰葡萄孢菌代谢组分析[J]. 分析测试学报, 2017, 36(5): 633-639. doi: 10.3969/j.issn.1004-4957.2017.05.009
[29]	郭刚, 唐丹, 田萍萍, 等. 基于GC-MS的阿维链霉菌代谢物组学研究方法的建立[J]. 生物技术通报, 2015(5): 61-67.
[30]	孙茂成, 李艾黎, 霍贵成, 等. 乳酸菌代谢组学研究进展[J]. 微生物学通报, 2012, 39(10): 1499-1505.
[31]	张剑霜, 喻浩, 钟欣, 等. 基于GC-MS代谢组学技术比较冬虫夏草与蝉花的质量[J]. 中国实验方剂学杂志, 2018, 24(18): 23-29.
[32]	李娟, 任路静, 孙冠男, 等. 气相色谱−质谱联用技术及其在代谢组学中的应用[J]. 生物工程学报, 2013, 29(4): 434-446.
[33]	WERF M J V D, OVERKAMP K M, MUILWIJK B, et al. Microbial metabolomics: Toward a platform with full metabolome coverage[J]. Analytical Biochemistry, 2007, 370(1): 17-25. doi: 10.1016/j.ab.2007.07.022
[34]	VILLAS-BÔAS S G, MAS S, ÅKESSON M, et al. Mass spectrometry in metabolome analysis[J]. Mass Spectrometry Reviews, 2005, 24(5): 613-646. doi: 10.1002/mas.20032
[35]	BUZIOL S, BASHIR I, BAUMEISTER A, et al. New bioreactor-coupled rapid stopped-flow sampling technique for measurements of metabolite dynamics on a subsecond time scale[J]. Biotechnology and Bioengineering, 2002, 80(6): 632-636. doi: 10.1002/bit.10427
[36]	WITTMANN C, KRÖMER J O, 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. doi: 10.1016/j.ab.2004.01.002
[37]	VILLAS-BÔAS S G, HØJER-PEDERSEN J, ÅKESSON M, et al. Global metabolite analysis of yeast: Evaluation of sample preparation methods[J]. Yeast, 2005, 22(14): 1155-1169. doi: 10.1002/yea.1308
[38]	CANELAS A B, RAS C, TEN PIERICK A, et al. Leakage-free rapid quenching technique for yeast metabolomics[J]. Metabolomics, 2008, 4(3): 226-239. doi: 10.1007/s11306-008-0116-4
[39]	ÁLVAREZ-SÁNCHEZ B, PRIEGO-CAPOTE F, CASTRO M D L D. Metabolomics analysis: II: Preparation of biological samples prior to detection[J]. Trac-Trends in Analytical Chemistry, 2010, 29(2): 120-127. doi: 10.1016/j.trac.2009.12.004
[40]	KIM H K, VERPOORTE R. Sample preparation for plant metabolomics[J]. Phytochemical Analysis, 2010, 21(1): 4-13. doi: 10.1002/pca.1188
[41]	MEYER H, LIEBEKE M, LALK M. A protocol for the investigation of the intracellular Staphylococcus aureus metabolome[J]. Analytical Biochemistry, 2010, 401(2): 250-259. doi: 10.1016/j.ab.2010.03.003
[42]	熊喜悦, 盛小奇, 王华, 等. 代谢组学气相色谱-质谱分析方法中样品衍生化技术的新进展[J]. 化学通报, 2015, 78(7): 602-607.
[43]	NASUTION U, van GULIK W M, KLEIJN R J, et al. Measurement of intracellular metabolites of primary metabolism and adenine nucleotides in chemostat cultivated Penicillium chrysogenum[J]. Biotechnology and Bioengineering, 2006, 94(1): 159. doi: 10.1002/bit.20842
[44]	KANANI H, CHRYSANTHOPOULOS P K, KLAPA M I. Standardizing GC-MS metabolomics[J]. Journal of Chromatography B, 2008, 871(2): 191-201. doi: 10.1016/j.jchromb.2008.04.049

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