In Situ Click Reaction Coupled with Quantitative Proteomics for Identifying Protein Targets of Catechol Estrogens

Catechol estrogens (CEs) are metabolic
electrophiles that actively
undergo covalent interaction with cellular proteins, influencing molecular
function. There is no feasible method to identify their binders in
a living system. Herein, we developed a click chemistry-based approach
using ethinylestradiol (EE2) as the precursor probe coupled with quantitative
proteomics to identify protein targets of CEs and classify their binding
strengths. Using in situ metabolic conversion and click reaction in
liver microsomes, CEs-protein complex was captured by the probe, digested
by trypsin, stable isotope labeled via reductive amination, and analyzed
by liquid chromatography–mass spectrometry (LC–MS).
A total of 334 liver proteins were repeatedly identified (n ≥ 2); 274 identified proteins were classified as
strong binders based on precursor mass mapping. The binding strength
was further scaled by D/H ratio (activity probe/solvent): 259 strong
binders had D/H > 5.25; 46 weak binders had 5.25 > D/H >
1; 5 nonspecific
binders (keratins) had D/H < 1. These results were confirmed using
spiked covalent control (strong binder) and noncovalent control (weak
binder), as well as in vitro testing of cytochrome c (D/H = 5.9),
which showed covalent conjugation with CEs. Many identified strong
binders, such as glutathione transferase, catechol-O-methyl transferase,
superoxide dismutase, catalase, glutathione peroxidase, and cytochrome
c, are involved in cellular redox processes or detoxification activities.
CE conjugation was shown to suppress the superoxide oxidase activity
of cytochrome c, suggesting that CEs modification may alter the redox
action of cellular proteins. Due to structural similarity and inert
alkyne group, EE2 probe is very likely to capture protein targets
of CEs in general. Thus, this strategy can be adopted to explore the
biological impact of CEs modification in living systems.