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Proteomic profiling of centrosomes across multiple cell and tissue types by a new affinity capture method

ABSTRACT: Centrosomes are small cytoplasmic organelles that play fundamental roles in a range of cellular processes. Nearly all vertebrate cell types contain centrosomes, but whether centrosome composition and function differ between cell types, tissues and pathologies remains an outstanding question. Until now, probing centrosome composition has relied on proteomic profiling of centrosomes obtained by consecutive sucrose density gradient centrifugations, requiring up to one billion cells, thus rendering studies with multiple conditions cumbersome. Here we describe centrosome affinity capture (CAPture)-mass spectrometry (MS), a method that achieves high-coverage proteomes from 20-30 million cells. Utilising a biotin-labelled peptide derived from the CCDC61 protein, CAPture isolates intact centrosomes in a manner dependent on Ninein, a conserved centrosome component. CAPture-MS performs well across several untransformed and primary cell lines, thereby enabling comparisons of high-resolution centrosome proteomes. Using distal appendage proteins as an example, we demonstrate the utility of CAPture-MS for dissecting hierarchical interactions within the centrosome. Overall CAPture-MS represents a powerful tool to unveil temporal, regulatory, cell type- and tissue-specific changes in centrosome proteomes in health and disease.

INSTRUMENT(S): Q Exactive HF, Q Exactive

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Spleen

SUBMITTER: Evangelia Papachristou

LAB HEAD: Fanni Gergely

PROVIDER: PXD040308 | Pride | 2023-10-18

REPOSITORIES: Pride

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Proteomic profiling of centrosomes across multiple cell and tissue types by a new affinity capture method

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Proteomic profiling of centrosomes across multiple cell and tissue types by a new affinity capture method

Project description:Centrosomes are the major microtubule-organising 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 aremajor unresolved questions, yet currently available centrosome isolation protocols are cumbersome, time-consuming and lack scalability. Here, we report the development of centrosome affinity capture (CAPture)-mass spectrometry (MS), a powerful one-step method to obtain high-resolution centrosome proteomes from untransformed and primary cell lines. Utilising 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 typedependent heterogeneity in centrosome composition, dissect hierarchical interactions and identify novel 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.

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Gene expression in mitotic tissues of Drosophila larvae without centrosomes or too many centrosomes

2012-08-20 | GSE35240 | GEO

Gene expression in mitotic tissues of Drosophila larvae without centrosomes or too many centrosomes

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