Single-tissue proteomics in Caenorhabditis elegans reveals proteins resident in intestinal lysosome-related organelles

Abstract The nematode intestine is the primary site for nutrient uptake and storage as well as the synthesis of biomolecules; lysosome-related organelles known as gut granules are important for many of these functions. Aspects of intestine biology are not well understood, including the export of the nutrients it imports and the molecules it synthesizes, as well as the complete functions and protein content of the gut granules. Here, we report a mass spectrometry-based proteomic analysis of the intestine of the Caenorhabditis elegans and of its gut granules. Overall, we identified approximately 5,000 proteins each in the intestine and the gonad and showed that most of these proteins can be detected in samples extracted from a single worm, suggesting the feasibility of individual-level genetic analysis using proteomes. Comparing proteomes and published transcriptomes of the intestine and the gonad, we identified proteins that appear to be synthesized in the intestine and then transferred to the gonad. To identify gut granule proteins, we compared the proteome of individual intestines deficient in gut granules to the wild-type. The identified gut granule proteome includes proteins known to be exclusively localized to the granules and additional putative novel gut granule proteins. We selected two of these putative gut granule proteins for validation via immunohistochemistry, and our successful confirmation of both suggests that our strategy was effective in identifying the gut granule proteome. Our results demonstrate the practicability of single tissue mass-spectrometry- based proteomic analysis in small organisms and in its utility for making discoveries. Significance statement We show that tissue-specific proteomic analysis is achievable and can be done efficiently at an individual level in a small nematode, with resolution sufficient for genetic analysis on a single animal basis. With data collected from single animals, we produced high-quality sets of proteins that described the proteomes of the gonad and the intestine. Comparison of these proteomes with the organs’ transcriptomes improved our understanding of interorgan protein transport. We applied single-tissue proteomic to describe the proteome of the gut granules in the nematode intestine, a specialized lysosome-related organelle with important functions but which is not well characterized, identifying proteins not previously known to be associated with LROs and verifying two by subcellular localization.