NDLI: Sample preparation of Mycobacterium tuberculosis extracts for nuclear magnetic resonance metabolomic studies.

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Year: 2012 Issue: 67

Sample preparation of Mycobacterium tuberculosis extracts for nuclear magnetic resonance metabolomic studies.

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Sample preparation of Mycobacterium tuberculosis extracts for nuclear magnetic resonance metabolomic studies.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Zinniel, Denise K. Fenton, Robert J. Halouska, Steven Powers, Robert Barletta, Raul G.
Description	Country affiliation: United States Author Affiliation: Zinniel DK ( School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, USA.)
Abstract	Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment, harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 °C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) H NMR and two-dimensional (2D) H-(13)C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.
File Format	HTM / HTML
e-ISSN	1940087X
DOI	10.3791/3673
Journal	Journal of Visualized Experiments
Issue Number	67
Language	English
Publisher	MyJove Corp.
Publisher Date	2012-09-03
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Discipline Physical Sciences Discipline Life Sciences Discipline Medicine Metabolomics Mycobacterium Tuberculosis Chemistry Metabolism Nuclear Magnetic Resonance, Biomolecular Research Support, N.i.h., Extramural Research Support, Non-u.s. Gov't Video-audio Media
Content Type	Text
Resource Type	Article

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