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Abstract:
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.
