Understanding the dynamics of endogenous protein–protein interactions in complex networks is pivotal in deciphering disease mechanisms. To enable the in-depth analysis of protein interactions in chromatin-associated protein complexes, we have previously developed a method termed RIME (Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins). Here, we present a quantitative multiplexed method (qPLEX-RIME), which integrates RIME with isobaric labelling and tribrid mass spectrometry for the study of protein interactome dynamics in a quantitative fashion with increased sensitivity. Using the qPLEX-RIME method, we delineate the temporal changes of the Estrogen Receptor alpha (ERα) interactome in breast cancer cells treated with 4-hydroxytamoxifen. Furthermore, we identify endogenous ERα-associated proteins in human Patient-Derived Xenograft tumours and in primary human breast cancer clinical tissue. Our results demonstrate that the combination of RIME with isobaric labelling offers a powerful tool for the in-depth and quantitative characterisation of protein interactome dynamics, which is applicable to clinical samples.

Highlights d IL6/STAT3 signaling drives metastasis in ER + breast cancer mouse models d IL6/STAT3 establishes shared ER-FOXA1-STAT3 enhancers independent of FOXA1 d STAT3 co-opts shared enhancers to drive a distinct gene program independent of ER d JAK inhibitor ruxolitinib represses IL6/STAT3 activity and in vivo invasion Authors

Divinylpyrimidine (DVP) linkers enable access to highly stable and functional antibody–drug conjugates.