Activity-based protein profiling (ABPP) has emerged as a post-genomic technology for characterizing functional proteins in complex biological systems. This approach applies chemical activity-based probes that monitor the functional activity of enzymes under physiological conditions, thereby providing high-content proteomic information beyond the reach of standard gene- and protein-expression profiling. We have previously developed a suite of activity-based probes (ABPs) that display broad coverage across mouse and human phase I metabolizing enzymes. Each probe contains three moieties: (1) a reactive group that forms a covalent bond to a functional P450 through a mechanism-based reaction, (2) a binding group, such as a polyaromatic hydrocarbon, that targets a probe towards P450s, and (3) a click chemistry compatible alkyne handle for attachment of an enrichment moiety or fluorescent reporter group. In this study, we utilized a multiplexed suite of three P450 ABPs to measure P450 enzyme activity from gestational days 87-140 in human fetal lung, liver, and thymus tissues. This data was compared to global protein abundance measurements by proteomics, and transcript levels by RT-PCR. Of particular interest to us are the key xenobiotic detoxifying P450 families 1, 2, and 3. Though RT-PCR shows transcript for these key families, the level of protein expression and probe-determined functional activity are far lower; thus, revealing that transcript does not directly correlate with activity.  However, we did find considerable activity at a range of gestational times for the P450 3A subfamily.  In total, this research shows xenobiotic degradation capabilities by the fetus at early gestational time points.  This work is ongoing as we expand to adolescent ontogeny and phase II metabolic enzymes.

Activity-based protein profiling of cytochrome P450 enzyme ontogeny in the developing human fetus

248th National Meeting (2014)

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