Abstract
Proximity labeling has emerged as a powerful approach for identifying protein-protein interactions within living systems, particularly those involving weak or transient associations. Here, we present a comprehensive proximity labeling study of five conserved Caenorhabditis elegans proteins-NEKL-2, NEKL-3, MLT-2, MLT-3, and MLT-4-that form two NEKL-MLT kinase-scaffold subcomplexes involved in membrane trafficking and actin regulation. Using endogenously expressed TurboID fusions and a data-independent acquisition (DIA) mass spectrometry (MS) pipeline, we profiled NEKL-MLT interactomes across 23 experiments, including several methodological variations, applying stringent controls and filtering strategies. By analyzing and comparing experimental outcomes, we develop a set of intuitive quantitative metrics to assess experimental outcomes and quality. We demonstrate that DIA-based workflows produce sensitive physiologically relevant findings, even in the presence of experimental noise and variability across biological replicates. Our approach is validated through the identification of known NEKL-MLT binding partners and conserved genetic suppressors of nekl-mlt mutant phenotypes. Gene ontology enrichment further supports the involvement of newly identified NEKL-MLT interactors in processes including membrane trafficking, cytoskeletal regulation, and cell adhesion. Additionally, several novel proximate interactors were functionally validated using genetic assays. Our findings underscore the utility of DIA-MS in proximity labeling applications and highlight the value of incorporating internal controls, quantitative metrics, and biological validation to enhance confidence in candidate interactors. Overall, this study provides a scalable, organismal-level strategy for probing endogenous protein networks and offers practical guidelines for proximity labeling in multicellular systems.