Multidimensional LC–MS/MS analysis of liver proteins in rat, mouse and human microsomal and S9 fractions

•UHPLC–MS/MS for the identification of liver proteins with high sequence coverage.•Protein and peptide ion exchange fractionation combined with reverse-phase UHPLC.•Cross-species analysis of rat, mouse and human liver microsomes and S9 fractions.•MSA analysis revealed homology between “unique” proteins from each species.

Liver plays a key role in metabolism and detoxification, therefore analysis of its proteome is relevant for toxicology and drug discovery studies. To optimize for high proteome coverage, protein and peptide-level ion exchange fractionation were assessed using rat liver microsomes and S9 fractions. 2D-(SCX-RP)-LC–MS/MS analysis with peptide fractionation was subsequently employed for rat, mouse and human samples, yielding between 1400 and 1939 identified proteins, 58% of which were shared between species, and with relatively high sequence coverage. This rich dataset is specifically interesting for the toxicology community, and could serve as an excellent source for targeted assay development.

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