Protein painting for structural and binding site analysis via intracellular lysine reactivity profiling with o-phthalaldehyde

Zheng, Zhenxiang1; Zeng, Ya1,2; Lai, Kunjia1; Liao, Bin1; Li, Pengfei1; Tan, Chris Soon Heng1

2024-04-24

10.1039/d4sc00032c

CHEMICAL SCIENCE   影响因子和分区

2041-6520

2041-6539

The three-dimensional structure and the molecular interaction of proteins determine their roles in many cellular processes. Chemical protein painting with protein mass spectrometry can identify changes in structural conformations and molecular interactions of proteins including their binding sites. Nevertheless, most current protein painting techniques identify protein targets and binding sites of drugs in vitro using a cell lysate or purified protein. Here, we tested 11 membrane-permeable lysine-reactive chemical probes for intracellular covalent labeling of endogenous proteins, which reveals ortho-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.

SCI ; EI

英语

第一 ; 通讯

National Natural Science Foundation of China[JCY20200109140814408] ; Shenzhen Innovation of Science and Technology Commission[2021YFA1302603] ; National Key Research and Development Program of China["22074060","22150610470"]

Chemistry

Chemistry, Multidisciplinary

WOS:001190804900001

ROYAL SOC CHEMISTRY

Web of Science

1.Southern Univ Sci & Technol, Coll Sci, Dept Chem, Shenzhen 518055, Guangdong, Peoples R China
2.Hong Kong Baptist Univ, Dept Chem, Kowloon, Hong Kong, Peoples R China

Zheng, Zhenxiang,Zeng, Ya,Lai, Kunjia,et al. Protein painting for structural and binding site analysis via intracellular lysine reactivity profiling with o-phthalaldehyde[J]. CHEMICAL SCIENCE,2024,15(16).

Zheng, Zhenxiang,Zeng, Ya,Lai, Kunjia,Liao, Bin,Li, Pengfei,&Tan, Chris Soon Heng.(2024).Protein painting for structural and binding site analysis via intracellular lysine reactivity profiling with o-phthalaldehyde.CHEMICAL SCIENCE,15(16).

Zheng, Zhenxiang,et al."Protein painting for structural and binding site analysis via intracellular lysine reactivity profiling with o-phthalaldehyde".CHEMICAL SCIENCE 15.16(2024).