SureCLIP and DrCLIP: Genetic manipulation-free, generalizable interactome mapping for cells and zebrafish

Unbiased interactome mapping currently relies on two types of approaches: co-immunoprecipitation coupled with mass spectrometry (co-IP/MS) and proximity-based tagging. Although co-IP/MS does not require genetic manipulation - thereby avoiding artifacts from exogenous protein expression and the time needed to generate stable cell or animal lines - it is increasingly being supplanted by proximity labeling techniques due to lower sensitivity and limited robustness. These drawbacks largely arise from the non-covalent nature of protein-protein interactions (PPIs) and carryover of bait and immunoglobulin (IgG) proteins in the final eluate. Here, we present a reversible crosslinker-mediated co-IP/MS workflow for endogenous proteins that (1) obviates the need for extensive lysis, wash, and elution optimization, (2) minimizes bait and IgG co-elution, and (3) captures several hundreds of interactors. We first describe subcellular-resolution, reversible crosslinker-mediated co-immunoprecipitation (SureCLIP), which enables PPI mapping of Hic-5, a focal adhesion protein, and lamin B1, a nuclear envelope protein, in cultured cells. We then introduce DrCLIP, a Danio rerio adaptation of SureCLIP, which allows comprehensive mapping of PPIs in zebrafish larvae. Using this approach, we profiled the kindlin-2 interactome in wild-type embryos without genetic manipulation. Both SureCLIP and DrCLIP exhibit high sensitivity and enable identification of novel interactors, providing a generalizable and powerful platform for defining native interactomes in 2D culture and whole animals.