Stable isotopic labeling is the most reliable and accurate method for quantitative proteomics and metabolomics. Proteomics studies on uniformly 15N-labeled rats have been previously reported using 15N-labeled algae as protein source for Stable Isotope Metabolic Labeling in Mammals (SILAM).
If the goal is to detect and relatively quantify a large number of metabolites in an untargeted manner, lower organisms can be fed or mammalian cells grown in culture with a diet or media, respectively where 12C is replaced with the stable 13C isotope. The generated tissue or cellular material can then be used as isotope-labeled internal standard reference. This approach has been successfully established for microorganisms and was subsequently extended to plants and small animals.
Preliminary studies by us have shown that the partially 13C-labeled mouse reference material can be used for the relative quantification of amino acids in human plasma samples. We envision that this approach can be extended to the relative quantification of other small molecules of interest in metabolomics research.
Metabolomics Study using the Partially 13C-labeled SILAM Mouse Diet
We have shown that amino acids in human plasma samples can be relatively quantified using reference material from mice fed for 14 days with the partially 13C-labeled diet.
Figure 1 shows the 13C enrichment of free amino acids in mouse plasma, heart tissue and plasma protein hydrolysates mapped on a simplified metabolic pathway. The overall average 13C enrichment was ~ 65%. Essential amino acids showed particularly high 13C isotope enrichment. This is due to the fact that essential amino acids cannot be produced by the organism itself and proteins from the partially 13C-enriched feed serve as the main protein source.
For the relative quantification of amino acids in human plasma samples, the partially 13C-labeled mouse reference turned out to be a very valuable source. Since mice share many metabolic pathways with humans it is conceivable that our approach can be further extended to other small molecules of interest in human metabolomics research.
In addition, mouse 13C-labeled blood and tissue material can serve as valuable reference for metabolomics studies of mouse models, to verify biomarker candidates and aid in their identification.
