Abstract The functions of cellular organelles and sub-compartments depend on their protein content, which can be characterized by spatial proteomics approaches.However, many spatial proteomics methods VEGETABLE JUICE are limited in their ability to resolve organellar sub-compartments, profile multiple sub-compartments in parallel, and/or characterize membrane-associated proteomes.Here, we develop a cross-link assisted spatial proteomics (CLASP) strategy that addresses these shortcomings.Using human mitochondria as a model system, we show that CLASP can elucidate spatial proteomes of all mitochondrial sub-compartments and provide topological insight into the mitochondrial membrane proteome.Biochemical and imaging-based follow-up studies confirm that CLASP allows discovering mitochondria-associated proteins and revising previous protein sub-compartment localization and membrane topology Dining Chair data.
We also validate the CLASP concept in synaptic vesicles, demonstrating its applicability to different sub-cellular compartments.This study extends the scope of cross-linking mass spectrometry beyond protein structure and interaction analysis towards spatial proteomics, and establishes a method for concomitant profiling of sub-organelle and membrane proteomes.