Metabolic stable isotope labeling

Kolkman, A., Dieksen, E. H., Slijpee, M., Heck, A. J. (2005). Double standards in quantitative proteomics direct comparative assessment of difference in gel electrophoresis and metabolic stable isotope labeling. Mol. Cell Proteomics 4, 255-266. 

Metabolic labeling is well-suited when subcellular fractionation is necessary or complexes are to be compared, and it is likely to be an important tool for comparative proteomics in the future. However, it should be pointed out that cell lines, derived from multicellular organism, will have significant differences from the intact organism. An excellent review by Beynon and Pratt discusses the experimental issues with metabolic stable isotope labeling and describes a large number of studies published. 

Hendrickson, E. L. Xia, Q. Wang, T. Leigh, J. A. Hackett, M. Comparison of spectral counting and metabolic stable isotope labeling for use with quantitative microbial proteomics. Analyst 2006,131, 1335-1341. 

The approach recruited to chemical proteomics in Reference [17] is called SILAC (stable isotope labeling with amino acids in cell culture) and is important in comparative proteomics (Figure 1). SILAC works well with cultured mammalian cells, but prokaryotes defeat it by metabolizing the label (usually supplied in lysine and arginine) into other amino acids. For applications beyond cultured eukaryotic cells, the reductive methylation route to differential labeling [18] is among the alternatives [15]-... 

A polyketide/fatty acid-type metabolic route using three propionate units has been shown by stable isotope-labeled probes and MS to the biosynthetic route towards the production of the insect pheromone (Sl-d-methyl-d-heptanone in the ant Harpegnathos saltator. 

Acylcarnitine analysis using stable-isotope-labeled internal standards provides quantitative data for acylcarnitine species [14]. However, to provide meaningful results to referring healthcare providers, it is critical to complement analytical proficiency with in-depth interpretation of results, as is true for many other examples of complex metabolic profiles [39]. 

Figure 4.10. Proteomic analysis by SILAC. Proteomic analysis by SILAC or stable isotope labeling of amino acids in cell culture utilize de novo metabolic incorporation of stable-isotope-labeled amino acids during protein synthesis. Cells can be cultured with various combinations of stable-isotope-labeled amino acids such as lysine or arginine. Tyrosine has been used in phosphoprotein studies of tyrosine residues. About five or six cell divisions are needed for complete labeling of proteins in cell cultures prior to experimentation. Labeled cells from control and treatment(s) lysates are combined and digested. Quantitation and identification are performed by LC-MS/MS.

The biosynthetic sequence of alkaloid production in M. cordata was also determined by means of HC using both whole plants and root pieces. It is advantageous in the HC study of metabolic sequences in plants to feed small amounts of compounds (2 mg in 5 ml of solution in this experiment) and stable isotopically labeled compounds instead of radioisotopes. 

Systemic bioavailability is the product of fraction of dose absorbed (/a), fraction of dose escaping gut metabolism (/g), and fraction of dose escaping first-pass metabolism (F ). Permeability class is based upon /a, which may be estimated either in vivo or in vitro by direct measurement of mass transfer across human intestinal epithelium. In vivo methods include (i) mass balance studies using unlabeled, stable-isotope labeled, or a radiolabeled drug substance (ii) oral bioavailability using a reference intravenous dose or (iii) intestinal perfusion studies either in humans or an acceptable animal model. Suitable in vitro methods involve the use of either excised human/animal intestinal tissues or cultured epithelial monolayers. All of these methods are deemed appropriate for drugs whose absorption is controlled by passive mechanisms. 

Recent progress in the fields of NMR spectroscopy and mass spectrometry has allowed stable isotope labelling to become an important technique in metabolic research for determining the biological behaviour of small molecules. Stable isotopomers are more easily prepared and handled than their radioisotopic... 

This review is intended to survey those uses of stable isotopes which are of particular importance in medicinal chemistry. These include their use in 1) structure elucidation, 2) studies of drug metabolism, 3) pharmacokinetic analysis and 4) drugs used in therapy. The use of stable isotopes for investigating intermediary metabolism, and their rapidly expanding use in clinical chemistry will not be reviewed since these topics have been covered elsewhere. Synthesis and biosynthesis with stable isotopes have also been previously surveyed >10 and many articles appear in each issue of the Journal of Labelled Compounds and a few have appeared in Biomedical Mass Spectrometry in the past year. In addition, one recent article has appeared on the use of plants to continously produce stable isotope labeled compounds of pharmacological interest. ... 

There can be little doubt that the application of stable isotope labelling methodologies has made a major impact on the development of biosynthetic and metabolic studies in a wide range of systems. As biosynthetic work becomes increasingly focused on studies at the enzymatic level, there will be continued scope for the further development of these applications particularly making use of sensitive mass spectral detection methods rather than the NMR methods which have dominated whole cell studies. We look forward to the results of these studies with anticipation. 

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