Protein painting for structural and binding site analysis <i>via</i> intracellular lysine reactivity profiling with <i>o</i> -phthalaldehyde
Zhenxiang Zheng, Ya Zeng, Kunjia Lai, Bin Liao, Pengfei Li, Chris Soon Heng Tan
Abstract
-phthalaldehyde (OPA) as the most reactive probe in the intracellular environment. An MS workflow and a new data analysis strategy termed RAPID (Reactive Amino acid Profiling by Inverse Detection) was developed to enhance detection sensitivity. RAPID with OPA successfully identified structural changes induced by the allosteric drug TEPP-46 on its target protein PKM2 and was applied to profile the conformation change of the proteome occurring in cells during thermal denaturation. The application of RAPID-OPA on cells treated with geldanamycin, selumetinib, and staurosporine successfully revealed their binding sites on target proteins. Thus, RAPID-OPA for cellular protein painting enables the identification of ligand-binding sites and detection of protein structural changes occurring in cells.