Cysteine reactive chloroalkane probe enables HaloTag ligation for downstream chemical proteomics analysis

Abstract Chemical proteomics is a powerful method to track proteins labelled by reactive small molecules in living cells on proteome-wide scale. The strategy relies on reactivity and specificity of bioorthogonal ‘click reactions’. Although a variety of bioorthogonal reactions have been developed to facilitate chemical proteomics, their reactivity and specificity might not be comparable with enzymatic reactions. Here we describe an iodoacetamide chloroalkane cysteine reactive probe that is, upon the reaction with nucleophilic cysteine of thioredoxin (TrxA), efficiently and specifically conjugated with HaloTag protein. The TrxA-HaloTag conjugate is utilized for downstream chemoproteomics analysis including in-gel shift assay and mass spectrometry-based proteomics. The TrxA-HaloTag conjugation in whole cell lysate allows fast and efficient pull-down of labelled protein on anti-HaloTag nanobeads resulting in low background after mass spectrometric analysis. The main advantage of the system is its high efficiency and complete biorthogonality due to enzymatic reactivity that is characteristic for HaloTag ligation. The study demonstrates the utility of chloroalkane small compound probes for chemoproteomics applications.