2026/01/01 by Rubaba R Abanti, Dongqing Wu, Pavel Kielkowski · 1 voice
Biochemistry, Genetics and Molecular Biology · Chemistry · #Biotin and Related Studies #Click Chemistry and Applications #Molecular Sensors and Ion Detection
paper · pdf · doi:10.1039/d6cb00004e
openalex publication_date 2026/01/01 · openalex created_date 2026/04/10 · openalex updated_date 2026/06/22
Chemical proteomics is a powerful method to track proteins labelled by reactive small molecules in living cells on a proteome-wide scale. The strategy relies on the 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 reaction with the nucleophilic cysteine of thioredoxin (TrxA), efficiently and specifically conjugated with the 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 proteins on anti-HaloTag nanobeads, resulting in low background after mass spectrometric analysis. The main advantage of the system is its high efficiency and complete bioorthogonality due to enzymatic reactivity that is characteristic for HaloTag ligation. This study demonstrates the utility of chloroalkane small compound probes for chemoproteomics applications.