Elucidation of Caenorhabditis elegans germline protein-protein interaction networks by in vivo photo-crosslinking

Understanding protein interaction networks in their native environment is crucial for elucidating biological roles. Incorporating unnatural amino acids (unAAs) labeled with fluorescent dyes enables direct protein labeling in live cells, facilitating the study of protein context. Meanwhile, chemical crosslinking allows the identification of proximal amino acids in native protein structures, aiding in structural biology. However, system-wide crosslinking poses challenges in peptide identification due to complex sample backgrounds. In this study, we propose a novel approach combining biolabeling and crosslinking mass spectrometry (CLMS) to study protein-protein interactions (PPIs) in live Caenorhabditis elegans. We employ photoactivatable amino acids for crosslinking, enabling precise timing and unaffected translation. This innovative pipeline promises to generate a comprehensive PPI network in a living organism, enhancing in vivo CLMS depth and capturing transient PPIs. By utilizing this strategy, we aim to establish protocols for studying PPI networks and protein complexes in C. elegans, particularly in isolated gonad nuclei, and extend this to the entire germline. The integration of biolabeling, CLMS, and pull-down assays offers a multifaceted approach to unraveling complex protein interactions within a live organism, paving the way for deeper insights into cellular mechanisms.