Proteomic profiling of centrosomes across multiple mammalian cell and tissue types by an affinity capture method

  • Dev Cell. 2023 Nov 6;58(21):2393-2410.e9. doi: 10.1016/j.devcel.2023.09.008.
Sarah Carden  1 ,  Elisa Vitiello  2 ,  Ivan Rosa E Silva  3 ,  James Holder  2 ,  Valentina Quarantotti  1 ,  Kamal Kishore  1 ,  Valar Nila Roamio Franklin  1 ,  Clive D'Santos  1 ,  Takashi Ochi  4 ,  Mark van Breugel  5 ,  Fanni Gergely  6
Affiliations
  • 1. CRUK Cambridge Institute, Li Ka Shing Centre, University of Cambridge, Cambridge, UK.
  • 2. Department of Biochemistry, University of Oxford, Oxford, UK.
  • 3. MRC Laboratory of Molecular Biology, Cambridge, UK.
  • 4. MRC Laboratory of Molecular Biology, Cambridge, UK; The Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, University of Leeds, Leeds, UK. Electronic address: [email protected].
  • 5. MRC Laboratory of Molecular Biology, Cambridge, UK; School of Biological & Behavioural Sciences, Queen Mary University of London, London, UK. Electronic address: [email protected].
  • 6. CRUK Cambridge Institute, Li Ka Shing Centre, University of Cambridge, Cambridge, UK; Department of Biochemistry, University of Oxford, Oxford, UK. Electronic address: [email protected].
PMID: 37852252 DOI: 10.1016/j.devcel.2023.09.008
Abstract

Centrosomes are the major microtubule-organizing centers in Animals and play fundamental roles in many cellular processes. Understanding how their composition varies across diverse cell types and how it is altered in disease are major unresolved questions, yet currently available centrosome isolation protocols are cumbersome and time-consuming, and they lack scalability. Here, we report the development of centrosome affinity capture (CAPture)-mass spectrometry (MS), a powerful one-step purification method to obtain high-resolution centrosome proteomes from mammalian cells. Utilizing a synthetic peptide derived from CCDC61 protein, CAPture specifically isolates intact centrosomes. Importantly, as a bead-based affinity method, it enables rapid sample processing and multiplexing unlike conventional approaches. Our study demonstrates the power of CAPture-MS to elucidate cell-type-dependent heterogeneity in centrosome composition, dissect hierarchical interactions, and identify previously unknown centrosome components. Overall, CAPture-MS represents a transformative tool to unveil temporal, regulatory, cell-type- and tissue-specific changes in centrosome proteomes in health and disease.

Keywords
Basal body; appendage; centriole; centrosomal proteins; centrosome; cilia; mass spectrometry; proteome; tissue-specific.
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