Extending the Range of SLIM-Labeling Applications: From Human Cell Lines in Culture to Caenorhabditis elegans...

The simple light isotope metabolic-labeling technique relies on the in vivo biosynthesis of amino acids from U-[¹²C]-labeled molecules provided as the sole carbon source. The incorporation of the resulting U-[¹²C]-amino acids into proteins presents several key advantages for mass-spectrometry-based proteomics analysis, as it results in more intense monoisotopic ions, with a better signal-to-noise ratio in bottom-up analysis. In our initial studies, we developed the simple light isotope metabolic (SLIM)-labeling strategy using prototrophic eukaryotic microorganisms, the yeasts Candida albicans and Saccharomyces cerevisiae, as well as strains with genetic markers that lead to amino-acid auxotrophy. To extend the range of SLIM-labeling applications, we evaluated (i) the incorporation of U-[¹²C]-glucose into proteins of human cells grown in a complex RPMI-based medium containing the labeled molecule, considering that human cell lines require a large number of essential amino-acids to support their growth, and (ii) an indirect labeling strategy in which the nematode Caenorhabditis elegans grown on plates was fed U-[¹²C]-labeled bacteria (Escherichia coli) and the worm proteome analyzed for ¹²C incorporation into proteins. In both cases, we were able to demonstrate efficient incorporation of ¹²C into the newly synthesized proteins, opening the way for original approaches in quantitative proteomics.