By a simple procedure we have shown complete incorporation of isotope labeled lysine into C. elegans by feeding the nematode on prelabeled lysine auxotroph E. coli for one generation. Furthermore, we have rendered this E. coli to allow efficient gene knockdown by RNAi in C. elegans. Traditionally, SILAC is based on labeling with lysine and arginine, which provides one label per peptide after digestion with trypsin. However, in C. elegans such quantification is troubled by arginine to proline conversion, so we employed only labeled lysine and used lysyl endopeptidase (Lys-C) for subsequent digestion. Even though the resulting peptides are longer than tryptic peptides and less suited for mass spectrometry, we were able to quantify a vast number of proteins due to intensive peptide fractionation. Larance et al. (2011) included arginine labeling of C. elegans and prevented conversion by RNAi-mediated knockdown of ornithine transaminase, orn-1. However, knockdown of orn-1 may have unwanted effects in, e.g., metabolic or developmental studies, and RNAi against an additional gene may reduce knockdown efficiency (Kamath et al., 2001).
Our present setup allows unbiased comparison of up to three populations of C. elegans to study proteomic changes upon treatment, mutations, or gene knockdown (Figure 1). Furthermore, in combination with enrichment of posttranslational modifications signaling cascades can be studied and we are confident that this approach will facilitate characterization of protein functions in C. elegans.
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