Integrating endogenous TurboID and data-independent acquisition mass spectrometry for in vivo proximity labeling.

ABSTRACT: Proximity labeling has emerged as a powerful approach for identifying protein-protein interaction networks within living systems, particularly those involving weak or transient associations. Here, we present a comprehensive revised proximity labeling workflow, integrating TurboID labeling of endogenously expressed fusion proteins and data-independent acquisition (DIA) mass spectrometry (MS). We benchmark this pipeline with a 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. Profiling of NEKL-MLT interactomes across 23 experiments validated our approach through the identification of known NEKL-MLT binding partners and conserved nekl-mlt ge

SUBMITTER: Fay DS 

PROVIDER: S-EPMC12811337 | biostudies-literature | 2026 Jan

REPOSITORIES: biostudies-literature