Subcellular Interactomes Revealed by Merging APEX with Cross-Linking Mass Spectrometry

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.