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 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 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.
CITATION STYLE
Zhu, Y., Akkaya, K. C., Ruta, J., Yokoyama, N., Wang, C., Ruwolt, M., … Liu, F. (2024). Cross-link assisted spatial proteomics to map sub-organelle proteomes and membrane protein topologies. Nature Communications, 15(1). https://doi.org/10.1038/s41467-024-47569-x
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