Identification of Cytosolic Protein Targets of Catechol Estrogens in Breast Cancer Cells Using a Click Chemistry-Based Workflow

Catechol estrogens (CEs) are known
to be toxic metabolites and
the initiators of the oncogenesis of breast cancers via forming covalent
adducts with DNAs. CEs shall also react with proteins, but their cellular
protein targets remain unexplored. Here, we reported the identification
of protein targets of CEs in the soluble cytosol of estrogen-sensitive
breast cancer cells by multiple comparative proteomics using liquid
chromatography–tandem mass spectrometry (LC–MS/MS) coupled
with an improved click chemistry-based workflow. Multiple comparative
proteomics composed of an experimental pair (probe versus solvent)
and two control pairs (solvent versus solvent and probe versus solvent
without enrichment) were studied using stable isotope dimethyl labeling.
The use of 4-hydroxyethynylestradiol (4OHEE2) probe with an amide-free
linker coupled with on-bead digestion and redigestion of the proteins
cleaved from the beads was shown to greatly improve the recovery and
identification of CE-adducted peptides. A total of 310 protein targets
and 40 adduction sites were repeatedly (n ≥
2) identified with D/H (probe/solvent) ratio >4 versus only one
identified
with D/H >4 from the two control pairs, suggesting that our workflow
imposes only a very low background. Meanwhile, multiple comparative
D/H ratios revealed that CEs may downregulate many target proteins
involved in the metabolism or detoxification, suggesting a negative
correlation between CE-induced adduction and expression of proteins
acting on the alleviation of stress-induced cellular damages. The
reported method and data will provide opportunities to study the progression
of estrogen metabolism-derived diseases and biomarkers.