Metabolome

An alternative approach to incorporate spectroscopic probes can be accomplished by the use of biarsenical tetracysteine system (Adams et al, 2002) or enzymatic alkylation (Keppler et al, 2003). A mutant 0 -alkylguanine-DNAalkyltansferase can efficiently transfer a benzyl group to itself when presented with 0 -benzylguanine derivatives. This enables the labeling of spectroscopic probes on to the transferase, which is fused to the target protein under investigation. 

Amino acid analogs with a reactive functionality afford cross-linking between the selected (mutated) residue and the adjacent residues. Such study may be used to infer topologies inter- and intra-molecularly within the mutant protein or between the interacting biomacromolecules. 

An interaction between biotin and avidin is one of the strongest biomolecular interacting systems. Therefore an introduction of biotin derivatives to proteins facilitates their efficient purification/characterization. 

In its relationship to functional proteomics, the global metabolite profiling offers a potentially powerful strategy to the functional assignment of enzyme networks because  

Many enzymes function as parts of large protein complexes and networks in vivo, which may be difficult to reconstract in vitro. 

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Kell DB. Metabolomics and systems biology making sense of the soup. Curr Opin Microbiol 2004 7 296-307. 

These seemingly simple questions have profound implications because they ask us to examine possibly obsolete policies and behaviors that have become deeply ingrained and unexamined habit patterns. For instance, let us consider the fields of genomics, proteomics, metabolomics, and systems biology as applied to pharmaceutical R D ... 

Metabolomics studies the entire metabolism of an organism. It is possible to consider characterising the complex pattern of cellular proteins and metabolites that are excreted in urine. Pattern recognition techniques of nuclear magnetic resonance spectra have been applied to determine the dose-response using certain classical liver and kidney toxicants (Robertson et al, 2000). This could well provide a signature of the functional state of the kidney, and perturbations in the pattern as a result of exposure to a chemical could be observed. But first it would be necessary to understand how compounds with known effects on the kidney affect these processes. 

Adjunctive to flux control analysis, other components of metabolism that contribute to product accumulation are needed including (1) substrate/precursor pool sizes (metabolomics), (2) co-factor capacities (metabolomics), (3) gene expression profiles (transcriptomics and quanfifafive real-time PCR), (4) protein profiles (pro-... 

Several observations lend credence to the rationale of monitoring cellular activities at the level of the proteins (proteomes) and metabolites (metabolomes). Some are listed below ... 

Several biochemical events occur posttranscriptionally that define the response of cells to stimuli. For instance, alternative splicing, posttrans-lational modifications, regulation of enzyme activities, distribution of metabolites between cellular compartments, necessitate analysis at the level of the proteome and the metabolome. 

Thus there is sufficient ground to cover at the functional level even after successful genetic characterizations that justifies the development of technologies for functional genomics, especially for high-throughput monitoring of proteomes and metabolomes. 

Maharjan, R. P. Ferenci,T. Global metabolite analysis the influence of extraction methodology on metabolome profiles of Escherichia coli. Anal. Biochem. 2003,313, 154-154. 

Castrillo, J. L. Hayes, A. Mohammed, S. Gaskell, S. J. Oliver, S. G. An optimized protocol for metabolome analysis in yeast using direct infusion electrospray mass spectrometry. Phytochemistry 2003, 62, 929-937. 

Porter, S.E.G., Stoll, D.R., Rutan, S.C., Carr, P.W., Cohen, J.D. (2006). Analysis of four-way two-dimensional liquid chromatography-diode array data application to metabolomics. Anal. Chem. 78, 5559-5569. 

Other applications that utilize different types of reversed-phase columns in both dimensions have been advocated by Carr (Stoll et al., 2006) for metabolomics work in small-molecule separations. These stationary phases include a pentafluorophenyl-propyl stationary phase in the first dimension and a carbon-coated zirconia material stationary phase in the second dimension. A common mistake in 2D method development is to mismatch the solvent system the two solvent systems must be miscible as discussed below. 

Recent proteomic or metabolomic analyses often require the separation/identification of several hundreds to several thousands of species in a mixture using LC-MS. It is practically impossible to achieve complete separation for a sample with complexity of... 

Tolstikov, V.V., Lommen, A., Nakanishi, K., Tanaka, N., Fiehn, O. (2003). Monolithic silica-based capillary reversed-phase liquid chromatography/electrospray mass spectrometry for plant metabolomics. Anal. Chem. 75, 6737-6740. 

Antoniewicz, M.R., Stephanopoulos, G., and Kelleher, J.K., Evaluation of regression models in metabolic physiology Predicting fluxes from isotopic data without knowledge of the pathway, Metabolomics 2,41, 2006. 

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