Abstract
Subcellular protein-protein interactions (PPIs) are essential to understanding the mechanism of diverse cellular signaling events and the pathogenesis of diseases. Herein, we report an integrated APEX proximity labeling and chemical cross-linking coupled with mass spectrometry (CXMS) platform named APEX-CXMS for spatially resolved subcellular interactome profiling in a high-throughput manner. APEX proximity labeling rapidly captures subcellular proteomes, and the highly reactive chemical cross-linkers can capture weak and dynamic interactions globally without extra genetic manipulation. APEX-CXMS was first applied to mitochondria and identified 653 pairs of interprotein cross-links. Six pairs of new interactions were selected and verified by coimmunoprecipitation, the mammalian two-hybrid system, and surface plasmon resonance method. Besides, our approach was further applied to the nucleus, capturing 336 pairs of interprotein cross-links with approximately 94% nuclear specificity. APEX-CXMS thus provides a simple, fast, and general alternative to map diverse subcellular PPIs.
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