HIT-MAP: A scalable approach to multimodal mapping of subcellular organization.

Multimodal mapping of subcellular protein organization through imaging and interaction proteomics has been mostly confined to large consortia, owing to high per-target costs, reliance on target-specific antibodies or libraries of epitope-tagged cDNAs, and the lack of unified pipelines for coordinated data acquisition. Here, we present HIT-MAP (high-throughput integrated tagging for cell mapping), an end-to-end framework that couples endogenous epitope tagging with optimized wide-field immunofluorescence imaging and affinity purification-mass spectrometry (AP-MS). Applied to a pilot set of 16 representative proteins in HEK293T cells, HIT-MAP recovers 576 high-confidence protein-protein interactions, identifies canonical complexes, and resolves cross-modality protein communities. Integrative analysis identifies CCDC12 as a previously uncharacterized component of the Bact spliceosomal complex, supported by AP-MS interaction data, tag-free size-exclusion chromatography-mass spectrometry (SEC-MS) co-fractionation, and Perturb-seq transcriptional signatures. HIT-MAP lowers technical and economic barriers to generating coordinated multimodal protein maps.