Metabolism, protein stable isotopes

Schwender, J., and J.B. Ohlrogge. 2002. Probing in vivo metabolism by stable isotope labeling of storage lipids and proteins in developing Brassica napus embryos. Plant Physiol. 130 347-361. 

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.

Incorporation of a stable isotopic tag into proteins/peptides in metabolically active cells was first described to quantify protein abundance in yeast (43). Wild-type and mutant cell populations were grown in media containing the naturally abundant isotopes of nitrogen and enriched in 15N, respectively, followed by trypsin digestion and LC-ESI-MS/MS analysis to identify and quantify relative phosphopeptide levels in both populations (43). 

Des Robert C, Le Bacquer O, Piloquet H, et al. Acute effects of intravenous glutamine supplementahon on protein metabolism in very low birth weight infants a stable isotope study. Pediatr Res 2002 51 87-93. 

The contribution of MS to identification of compounds and quantification of their concentration is complementary to other detection techniques and, despite being very practical and versatile, it remains fundamentally replaceable. However, knowledge of molecular weight is a prerequisite for techniques that rely on the synergies with stable isotopic tracers. In fact, powerful analytical methods exist to obtain important insights on cell dynamics from the ratiometric measurement of marked and not-marked species (or atoms). We cite, for example, (1) relative abundances of virtually all metabolites or proteins in two separate cultures are quantified based on the isotope dilution theory [43 5] (2) information on the mechanisms and kinetics of nonlinear chemical processes can be extracted from response tracer experiments [46 7] and (3) the labeling patterns in metabolic intermediates are used to resolve the relative rate in convergent reactions in vivo [48,49]. 

We have Illustrated the use of double cross polarization nmr to follow the formation or metabolism of particular chemical bonds of proteins in Intact biological tissue. Neither the use of radioisotopes, nor the use of other means of detecting stable Isotopes, has comparable directness and specificity. 

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