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Open Access Green as soon as Postprint is submitted to ZB.
Spatial centrosome proteomic profiling of human iPSC-derived neural cells.
Bio Protoc. 13:e4812 (2023)
The centrosome governs many pan-cellular processes including cell division, migration, and cilium formation. However, very little is known about its cell type-specific protein composition and the sub-organellar domains where these protein interactions take place. Here, we outline a protocol for the spatial interrogation of the centrosome proteome in human cells, such as those differentiated from induced pluripotent stem cells (iPSCs), through co-immunoprecipitation of protein complexes around selected baits that are known to reside at different structural parts of the centrosome, followed by mass spectrometry. The protocol describes expansion and differentiation of human iPSCs to dorsal forebrain neural progenitors and cortical projection neurons, harvesting and lysis of cells for protein isolation, co-immunoprecipitation with antibodies against selected bait proteins, preparation for mass spectrometry, processing the mass spectrometry output files using MaxQuant software, and statistical analysis using Perseus software to identify the enriched proteins by each bait. Given the large number of cells needed for the isolation of centrosome proteins, this protocol can be scaled up or down by modifying the number of bait proteins and can also be carried out in batches. It can potentially be adapted for other cell types, organelles, and species as well.
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Publication type
Article: Journal article
Document type
Scientific Article
Keywords
Centrosome ; Human Ipsc-derived Neurons ; Interactome ; Mass Spectrometry ; Maxquant ; Microtubule Organization ; Perseus ; Spatial Proteomics; Inheritance
e-ISSN
2331-8325
Journal
Bio-Protocol
Quellenangaben
Volume: 13,
Issue: 17,
Article Number: e4812
Publisher
bio-protocol.org
Publishing Place
Sunnyvale, CA
Non-patent literature
Publications
Reviewing status
Peer reviewed
Institute(s)
Institute of Stem Cell Research (ISF)
Grants
Neurological Foundation of New Zealand
EU
ERC
EU
ERC