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Extending the range of SLIM-labeling applications: from human cell lines in culture to Caenorhabditis elegans whole-organism labeling

ABSTRACT: 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 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 labelled 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]-labelled bacteria (Eschericha 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.

INSTRUMENT(S): Orbitrap Fusion, Q Exactive

ORGANISM(S): Homo Sapiens (human) Escherichia Coli Caenorhabditis Elegans

SUBMITTER: Laurent Lignières

LAB HEAD: Jean-Michel Camadro

PROVIDER: PXD028880 | Pride | 2023-06-23

REPOSITORIES: Pride

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