Analysis of the rat primary hepatocyte nuclear proteome through sub-cellular fractionation

Characterising primary hepatocytes and their de-differentiation in culture is vital for the refinement of current culture techniques and for the development of new and improved in vitro hepatocyte models. We have performed multiplexed iTRAQ proteomics on whole cell preparations and further employed nuclear fractionation to expand the coverage of this important organelle. We identify many proteins that change in abundance during culture of rat hepatocytes for 48h and map their molecular functions. 431 proteins were identified and quantified in whole cell homogenates, mapping to 69 molecular functions using the PANTHER (Protein ANalysis THrough Evolutionary Relationships) classification system. In whole cell homogenates liver-associated functions, such as oxidoreductase activity, were enriched compared with the reference rat proteome dataset but some functions, such as transcriptional activity, were under-represented. Nuclear fractionation resulted in the identification of an additional 156 proteins which mapped to 31 molecular functions. These proteins included some associated with hepatic differentiation, such as HNF4alpha and CCAAT/enhancer-binding protein beta and others with less well-defined roles. Hierarchical clustering of samples within each experiment showed segregation of fresh and cultured sample types and stringent statistical analysis demonstrated significant changes in 36% of proteins from the whole cell homogenates and 21% of proteins from the nuclear dataset (adjusted p valu