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

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.

Automated summary

The simple light isotope metabolic-labeling technique utilizes U-[12C]-labeled molecules as the sole carbon source for in vivo biosynthesis of amino acids, resulting in the incorporation of U-[12C]-amino acids into proteins. This technique has advantages for mass-spectrometry-based proteomics analysis, providing more intense monoisotopic ions and a better signal-to-noise ratio. The technique has been successfully applied to eukaryotic microorganisms and human cells, as well as an indirect labeling strategy in a nematode model organism.