Characterizing Proteins of Unknown Function: Orphan Cytochrome P450 Enzymes as a Paradigm

Strategies for elucidating substrates and reactions of proteins, as exemplified by P450s. One strategy involves the use of transgenic animals, either devoid of an ortholog of the protein under consideration or with the protein over-expressed. Body fluids or tissues can be utilized for LC-MS analysis. Alternatively, the expressed protein can be used in vitro with tissue extracts from wild type (WT) and knock-out (KO) animals (or human) tissues. See Figure 3 for an ¹⁸O₂ labeling strategy. LC-MS analysis is done using an appropriate program, e.g. principal component analysis (PCA), DoGEX (for ¹⁸O data), MZmine, etc. Differences are used to provide leads, which may require extensive characterization. Ultimately the relevance of any findings must be considered in the context of known pathways and in repeated experiments.