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 in the oncogenesis of breast cancers via forming covalent adducts with DNAs. CEs shall also react with proteins but their cellular protein targets remain un-explored. 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 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 re-digestion of the peptides cleaved from the beads was shown to greatly improve the recovery and identification of CEs-adducted peptides. A total of 310 protein targets and 35 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 down-regulate many target proteins involved in the metabolism or detoxification, suggesting a negative correlation between CEs-induced adduction and expression of proteins acting on the alleviation of stress-induced cellular damages. The reported method and data shall provide opportunities to study the progression of estrogen metabolism-derived diseases and biomarkers.