A quantitative mass spectrometry-based approach to monitor the dynamics of endogenous chromatin-associated protein complexes

ABSTRACT: Understanding the dynamics of endogenous protein-protein interactions in complex networks is pivotal in deciphering the mechanisms underlying diseases such as cancer. 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 delineated the temporal changes of the Estrogen Receptor alpha (ERα) interactome in breast cancer cells treated with 4-hydrotamoxifen and we successfully identified endogenous ERα-associated proteins in human Patient Derived Xenograft (PDX) 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 characterization of protein interactome dynamics which is applicable to clinical samples.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Breast, Breast Cancer Cell Line

DISEASE(S): Luminal Breast Carcinoma

SUBMITTER: Evangelia Papachristou  

LAB HEAD: Jason Carroll

PROVIDER: PXD007968 | Pride | 2018-06-08

REPOSITORIES: Pride