NeuCode Labeling in Nematodes: Proteomic and Phosphoproteomic Impact of Ascaroside Treatment in Caenorhabditis elegans

Abstract

The nematode Caenorhabditis elegans is an important model organism for biomedical research. We previously described NeuCode stable isotope labeling by amino acids in cell culture (SILAC), a method for accurate proteome quantification with potential for multiplexing beyond the limits of traditional stable isotope labeling by amino acids in cell culture. Here we apply NeuCode SILAC to profile the proteomic and phosphoproteomic response of C. elegans to two potent members of the ascaroside family of nematode pheromones. By consuming labeled E. coli as part of their diet, C. elegans nematodes quickly and easily incorporate the NeuCode heavy lysine isotopologues by the young adult stage. Using this approach, we report, at high confidence, one of the largest proteomic and phosphoproteomic data sets to date in C. elegans: 6596 proteins at a false discovery rate < 1% and 6620 phosphorylation isoforms with localization probability >75%. Our data reveal a post-translational signature of pheromone sensing that includes many conserved proteins implicated in longevity and response to stress. Molecular & Cellular

Conserved phosphorylation hotspots in eukaryotic protein domain families

“…Phosphorylation data for 6 Drosophila species ( Drosophila ananassae , Drosophila melanogaster , Drosophila pseudoobscura , Drosophila simulans , Drosophila virilis , and Drosophila yakuba ) was obtained from the iProteinDB in FlyDB database 44 . Two additional metazoan phosphoproteomes were obtained from published studies for Caenorhabditis elegans 45 and Trichoplax adhaerens 46 . Phosphosites for 18 fungal species ( S. cerevisiae , Saccharomyces paradoxus , Saccharomyces mikatae , Saccharomyces kudriavzevii , Saccharomyces bayanus , Naumovozyma castellii , Candida glabrata , Vanderwaltozyma polyspora , Zygosaccharomyces rouxii , Kluyveromyces lactis , Lachancea kluyveri , Lachancea waltii , Lachancea thermotolerans , Komagataella (Pichia) pastoris , Meyerozyma guilliermondii , Candida albicans , Scheffersomyces stipitis , Schizosaccharomyces pombe ) were obtained from Studer and colleagues 9 .…”

Phosphoproteomics in the Age of Rapid and Deep Proteome Profiling

“… 131 − 138 Recently, Rhoads et al implemented an in vivo labeling strategy with NeuCode to study the phosphoproteome in Caenorhabditis elegans . 139 This study provided one of the largest phosphoproteomic data sets to date for C. elegans (6 620 phosphorylation isoforms), revealing a post-translational signature of pheromone sensing in the organism.…”

Multiplexed proteome analysis with neutron-encoded stable isotope labeling in cells and mice

“…Since 2013, NeuCode SILAC has been applied to a number of different experimental models including yeast 4,11 , adherent cell lines 19 , nematodes 20 , and mice 21 . In this protocol we highlight the use of NeuCode for adherent cell lines and mouse models; however, application of the method to other model systems is straight forward and simply requires the dietary replacement of normal lysine with NeuCode lysine isotopologues.…”