Abstract
1H-Nuclear magnetic resonance (1H-NMR) spectroscopy is a powerful technique to analyze the composition of complex mixtures based on the particular proton fingerprint of each molecule. Here we describe a protocol for exometabolome analysis of mammalian cells using this technique, including sample preparation, spectra acquisition, and integration. The potential of this technique is exemplified by application to cultures of a Chinese hamster ovary (CHO) cell line. The average error associated to this method is under 3% and the limit of quantification for all metabolites analyzed is below 180 μM.
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References
Sauer PW, Burky JE, Wesson MC et al (2000) A high-yielding, generic fed-batch cell culture process for production of recombinant antibodies. Biotechnol Bioeng 67(5):585–597
Wurm FM (2004) Production of recombinant protein therapeutic in cultivated mammalian cells. Nat Biotechnol 22(11):1393–1398
Gatti MDL, Wlaschin KF, Nissom PM et al (2007) Comparative transcriptional analysis of mouse hybridoma and recombinant Chinese hamster ovary cells undergoing butyrate treatment. J Biosci Bioeng 103(1):82–91
Kim N, Lee G (2001) Overexpression of bcl-2 inhibits sodium butyrate-induced apoptosis in Chinese hamster ovary cells resulting in enhanced humanized antibody production. Biotechnol Bioeng 71(3):184–193
Yee JC, Gerdtzen ZP, Hu W-S (2009) Comparative transcriptome analysis to unveil genes affecting recombinant protein productivity in mammalian cells. Biotechnol Bioeng 102(1): 246–263
Yoon SK, Kim SH, Lee GM (2003) Effect of low culture temperature on specific productivity and transcription level of anti-4-1BB antibody in recombinant Chinese hamster ovary cells. Biotechnol Prog 19(4):1383–1386
Yoon SK, Hwang SO, Lee GM (2004) Enhancing effect of low culture temperature on specific antibody productivity of recombinant Chinese hamster ovary cells: clonal variation. Biotechnol Prog 20(6):1683–1688
Ozturk SS, Palsson BO (1991) Effect of medium osmolarity on hybridoma growth, metabolism and antibody production. Biotechnol Bioeng 37:989–993
Sellick CA, Croxford AS, Maqsood AR et al (2011) Metabolite profiling of recombinant CHO cells: designing tailored feeding regimes that enhance recombinant antibody production. Biotechnol Bioeng 108(12):3025–3031
Khoo SHG, Al-Rubeai M (2009) Metabolic characterization of a hyper-productive state in an antibody producing NSO myeloma cell line. Meth Eng 11:199–211
Dunn WB, Broadhurst DI, Atherton HJ et al (2011) Systems level studies of mammalian metabolomes: the roles of mass spectrometry and nuclear magnetic resonance spectroscopy. Chem Soc Rev 40:387–426
Bradley SA, Ouyang A, Purdie J et al (2010) Fermentanomics: monitoring mammalian cell cultures with NMR spectroscopy. J Am Chem Soc 132:9531–9533
Wilson DM, Burlingame AL (1974) Deuterium and carbon-13 tracer studies of ethanol metabolism in the rat by 2H, 1H-decoupled 13C nuclear magnetic resonance. Biochem Biophys Res Commun 56(3):828–835
Dauner M, Bailey JE, Sauer U (2001) Metabolic flux analysis with a comprehensive isotopomer model in Bacillus subtilis. Biotechnol Bioeng 76(2):144–156
Gout E, Blingny R, Genix P et al (1992) Effect of glyphosate on plant cell metabolism. 31P and 13C NMR studies. Biochimie 74(9–10):875–882
Alves PM, Fonseca LL, Peixoto CC et al (2000) NMR studies on energy metabolism of immobilized primary neurons and astrocytes during hypoxia, ischemia and hypoglycemia. NMR Biomed 13:438–448
Weljie AM, Zang P, Bondareva A et al (2011)1H-NMR metabolomics identification of markers of hypoxia-induced metabolic shifts in a breast cancer model system. J Biomol NMR 49(3–4):185–193
Viant MR, Lyeth BG, Miller MG et al (2005) An NMR metabolomic investigation of early metabolic disturbances following traumatic brain injury in a mammalian model. NMR Biomed 18:507–516
Lindon JC, Nicholson JK, Holmes E et al (2000) Metabonomics: metabolic processes studied by NMR spectroscopy of biofluids. Conc Magn Res 12(5):289–320
Griffin JL, Shockcor JP (2004) Metabolic profiles of cancer cells. Nat Rev 4:451–461
Weljie AM, Newton J, Mercier P et al (2006) Targeted profiling: quantitative analysis of 1H NMR metabolomics data. Anal Chem 78:4430–4442
Wishart DS (2008) Quantitative metabolomics using NMR. Anal Chem 27:228–237
Jansen JFA, Backes WH, Nikolay K et al (2006) 1H NMR spectroscopy of the brain: absolute quantification of metabolites. Radiology 240(2):318–332
Duarte IF, Marques J, Ladeirinha AF et al (2009) Analytical approaches toward successful human cell metabolome studies by NMR spectroscopy. Anal Chem 82:5023–5032
Aranibar N, Borys M, Mackin NA et al (2011) NMR-based metabolomics of mammalian cell and tissue cultures. J Biomol NMR 49:195–206
Mercier P, Lewis MJ, Chang D et al (2010) Towards automatic metabolic profiling of high-resolution one-dimensional proton NMR spectra. J Biomol NMR 49(3–4):307–323
Acknowledgements
This work was supported by Fundação para a Ciência e a Tecnologia (FCT): projects PTDC/EBB-EBI/102750/2008 and PTDC/EBB-BIO/119501/2010; fellowships SFRH/BD/81553/2011 and SFRH/BPD/80514/2011. FCT is also acknowledged for supporting the National NMR Network (REDE/1517/RMN/2005).
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Duarte, T.M., Carinhas, N., Silva, A.C., Alves, P.M., Teixeira, A.P. (2014). 1H-NMR Protocol for Exometabolome Analysis of Cultured Mammalian Cells. In: Pörtner, R. (eds) Animal Cell Biotechnology. Methods in Molecular Biology, vol 1104. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-733-4_16
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DOI: https://doi.org/10.1007/978-1-62703-733-4_16
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Publisher Name: Humana Press, Totowa, NJ
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Online ISBN: 978-1-62703-733-4
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