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Uzbas, F. ; O’Neill, A.

Spatial centrosome proteomic profiling of human iPSC-derived neural cells.

Bio Protoc. 13:e4812 (2023)
Verlagsversion DOI PMC
Creative Commons Lizenzvertrag
Open Access Green möglich sobald Postprint bei der ZB eingereicht worden ist.
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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Publikationstyp Artikel: Journalartikel
Dokumenttyp Wissenschaftlicher Artikel
Korrespondenzautor
Schlagwörter Centrosome ; Human Ipsc-derived Neurons ; Interactome ; Mass Spectrometry ; Maxquant ; Microtubule Organization ; Perseus ; Spatial Proteomics; Inheritance
e-ISSN 2331-8325
Zeitschrift Bio-Protocol
Quellenangaben Band: 13, Heft: 17, Seiten: , Artikelnummer: e4812 Supplement: ,
Verlag bio-protocol.org
Verlagsort Sunnyvale, CA
Nichtpatentliteratur Publikationen
Begutachtungsstatus Peer reviewed
Institut(e) Institute of Stem Cell Research (ISF)
Förderungen Neurological Foundation of New Zealand
EU
ERC