Abstract An important goal of metabolomics is to characterize the changes in metabolic networks in cells or various tissues of an organism in response to external perturbations or pathologies. The profiling of metabolites and their steady state concentrations does not directly provide information regarding the architecture and fluxes through metabolic networks. This requires tracer approaches. NMR is especially powerful as it can be used not only to identify and quantify metabolites in an unfractionated mixture such as biofluids or crude cell/tissue extracts, but also determine the positional isotopomer distributions of metabolites derived from a precursor enriched in stable isotopes such as 13C and 15N via metabolic transformations. In this article we demonstrate the application of a variety of 2-D NMR editing experiments to define the positional isotopomers of compounds present in polar and non-polar extracts of human lung cancer cells grown in either [Uâ??13C]-glucose or [Uâ??13C,15N]-glutamine as source tracers. The information provided by such experiments enabled unambiguous reconstruction of metabolic pathways, which is the foundation for further metabolic flux modeling.
Content Type Journal Article
Pages 1-14
DOI 10.1007/s10858-011-9484-6
Authors
Teresa W-M. Fan, Department of Chemistry, University of Louisville, Louisville, KY USA
Andrew N. Lane, Center for Regulatory Environmental Analytical Metabolomics, University of Louisville, Louisville, KY USA
Cell-free expression and stable isotope labelling strategies for membrane proteins
Cell-free expression and stable isotope labelling strategies for membrane proteins
Abstract Membrane proteins are highly underrepresented in the structural data-base and remain one of the most challenging targets for functional and structural elucidation. Their roles in transport and cellular communication, furthermore, often make over-expression toxic to their host, and their hydrophobicity and structural complexity make isolation and reconstitution a complicated task, especially in cases where proteins are targeted to inclusion bodies. The development of cell-free expression systems...
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01-09-2011 12:46 PM
Dynamics and interactions of glycoconjugates probed by stable-isotope-assisted NMR spectroscopy.
Dynamics and interactions of glycoconjugates probed by stable-isotope-assisted NMR spectroscopy.
Dynamics and interactions of glycoconjugates probed by stable-isotope-assisted NMR spectroscopy.
Methods Enzymol. 2010;478:305-22
Authors: Yamaguchi Y, Kato K
Unique advantages offered by nuclear magnetic resonance (NMR) spectroscopy provide high-resolution information not only on structures but also on dynamics and interactions of glycoconjugates in solution. These benefits are further enhanced by applying stable-isotope-labeling techniques, which we...
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12-31-2010 07:03 PM
[NMR paper] Micromixer-based time-resolved NMR: applications to ubiquitin protein conformation.
Micromixer-based time-resolved NMR: applications to ubiquitin protein conformation.
Related Articles Micromixer-based time-resolved NMR: applications to ubiquitin protein conformation.
Anal Chem. 2003 Feb 15;75(4):956-60
Authors: Kakuta M, Jayawickrama DA, Wolters AM, Manz A, Sweedler JV
Time-resolved NMR spectroscopy is used to studychanges in protein conformation based on the elapsed time after a change in the solvent composition of a protein solution. The use of a micromixer and a continuous-flow method is described where the contents of...
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11-24-2010 09:01 PM
An economical method for producing stable-isotope labeled proteins by the E. coli cel
An economical method for producing stable-isotope labeled proteins by the E. coli cell-free system
Abstract Improvement of the cell-free protein synthesis system (CF) over the past decade have made it one of the most powerful protein production methods. The CF approach is especially useful for stable-isotope (SI) labeling of proteins for NMR analysis. However, it is less popular than expected, partly because the SI-labeled amino acids used for SI labeling by the CF are too expensive. In the present study, we developed a simple and inexpensive method for producing an SI-labeled protein...
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Proteins
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11-07-2010 02:47 PM
Stable-isotope-assisted NMR approaches to glycoproteins using immunoglobulin G as a m
Stable-isotope-assisted NMR approaches to glycoproteins using immunoglobulin G as a model system.
Related Articles Stable-isotope-assisted NMR approaches to glycoproteins using immunoglobulin G as a model system.
Prog Nucl Magn Reson Spectrosc. 2010 May;56(4):346-59
Authors: Kato K, Yamaguchi Y, Arata Y
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10-19-2010 04:51 PM
[NMR paper] Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for
Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for NMR analysis.
Related Articles Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for NMR analysis.
J Biomol NMR. 1995 Sep;6(2):129-34
Authors: Kigawa T, Muto Y, Yokoyama S
For the application of multidimensional NMR spectroscopy to larger proteins, it would be useful to perform selective labeling of one of the 20 amino acids. For some amino acids, however, amino acid metabolism drastically reduces the efficiency and selectivity...
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08-22-2010 03:50 AM
Stable-isotope-assisted NMR approaches to glycoproteins using immunoglobulin G as a m
Stable-isotope-assisted NMR approaches to glycoproteins using immunoglobulin G as a model system
Publication year: 2010
Source: Progress in Nuclear Magnetic Resonance Spectroscopy, In Press, Accepted Manuscript, Available online 19 March 2010</br>
Koichi, Kato , Yoshiki, Yamaguchi , Yoji, Arata</br>
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NMR-based stable isotope resolved metabolomics in systems biochemistry
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