Identification of Protein Targets of Catechol Estrogens in Cancer Cells by Affinity Enrichment and Modification-induced Gel Staining Coupled with LC-MS/MS

Abstract

Catechol estrogens (CEs) are toxic metabolites which form stable covalent conjugation with cellular proteins via transformation to catechol quinones and CEs-conjugation has been linked to cancer formation Identification and characterization of cellular targets of CEs are essential to better understand CEs adduction-induced disease mechanism but challenging goals We previously used click reaction coupled with dimethyl labeling to identify protein targets of CEs in rat liver microsomes However the identification of conjugation sites is still difficult by the previous method Moreover preparation and treatment of culture cells related to CEs-related diseases have not yet developed In this thesis an improved enrichment method was developed and applied to identify CE targets in MCF-7 breast cancer cells Moreover a modification-induced gel staining method was developed to detect the conjugation of high abundant proteins 4OHEE2 (4-hydroxyethynylestradiol) was used as a CEs probe to capture protein targets by covalent conjugation The CEs-conjugated proteins were further enriched by streptavidin beads through a new cleavable biotin linker short of amide functional group The resulting peptides were found to minimize linker fragmentation and improve protein identification by LC-MSMS-based proteomics CEs-conjugated peptides were further enriched by borate gel to assist the assignment of multiple conjugation sites on cytosolic nuclear and histone proteins We expect to explore functional roles of some identified protein targets and study their usefulness as disease markers or drug targets

Date of Award	2019
Original language	English
Supervisor	Shu-Hui Chen (Supervisor)