Chemical Bias in Cysteine-Reactive Probe Profiling: In Vivo Perfusion versus Postmortem Lysis and Cells

Abstract Chemoproteomic probes are typically screened in postmortem lysates or cell cultures, assuming these models accurately reflect native protein reactivity. However, the impact of the reaction medium, intact tissues versus homogenate, remains poorly defined. Here we utilize iodoacetamide-alkyne to benchmark probe performance across three distinct reaction environments, in vivo perfusion, postmortem lysate, and live cell culture. Contrary to the assumption that lysate offers comprehensive access, we show that homogenization systematically suppresses the labeling of ATP-dependent ligases and integral membrane proteins, likely due to the rapid collapse of active-site energetics and membrane depolarization. Postmortem lysate showed greater nuclear access and enrichment of oxidoreductase/stress-response signatures, consistent with processing effects, whereas the cell model overlapped least with the tissue and displayed culture-specific pathway biases. Furthermore, we address the confounding variable of probe distribution by demonstrating that perfusion achieves equivalent saturation to lysate dosing, confirming that observed differences stems from native-state reactivity rather than accessibility. These findings provide a critical correction factor for synthetic chemists: probes validated solely in lysate may suffer from predictable false negatives against metabolically active targets.