Abstract
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
Understanding the dynamics of endogenous protein–protein interactions in complex networks is pivotal in deciphering disease mechanisms
We have established a modified RIME assay to monitor the dynamics of chromatin-associated complexes using a quantitative multiplexed workflow
The workflow starts with a twostep fixation procedure using disuccinimidyl glutarate (DSG) and formaldehyde (FA) that has been previously applied in combination with Chromatin Immunoprecipitation (ChIP) assays to capture transient interactions more efficiently[12,18]
Summary
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). To gain insight into the complex interactions between such regulators, the combination of Chromatin Immunoprecipitation (ChIP) with mass spectrometry has been used to study the composition of chromatin-associated complexes[10,11,12] In line with this strategy we have previously developed RIME (Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins)[13], a method which has several advantages for the analysis of protein interactomes[14]. Our data demonstrate that the qPLEX-RIME method combines multiplexity with quantitative accuracy and increased sensitivity, to enable the in-depth characterisation of dynamic changes in chromatin-associated protein complexes in vitro and in vivo
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