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 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.

Moonlighting Proteins and Cardiopathy in the Spatial Response of MCF‐7 Breast Cancer Cells to Tamoxifen

“…Although a restoration of the ERα complex probably linked to the half‐life of OHT was recently observed at the latest time point of 24 h by interactome dynamics studies, a longer stimulation up to 36 h was adopted by others for the investigation of late‐response proteins, in agreement with results on late‐response genes at 24 h by transcriptomic approaches …”

Complement C5a impairs phagosomal maturation in the neutrophil through phosphoproteomic remodelling

“…In total, 4859 proteins and 2712 phosphoproteins were identified in peripheral blood neutrophils 76 obtained from four healthy volunteers. C5a-induced suppression of phagocytosis in these donors was 77 confirmed ( Figure S2A), and technical reproducibility was high (Figures S2B-E) with the magnitude 78 of phosphorylation changes within the previously reported range (Papachristou et al, 2018). Changes 79 in the human proteome were minimal (2 % of total proteome with S. aureus treatment) whereas 80 phosphoprotein expression varied markedly (31.6 % of total phosphoproteome with S. aureus 81 treatment, Table S1).…”

“…Heatmaps and volcano plots were generated as shown in Results. Statistical analyses were performed in RStudio (RStudio Team, 2016) using the qPLEXanalyzer (Papachristou et al, 2018) package, and plots were produced using the ggplot2 (Wickham, 2016) package.…”

Network analysis of ChIP-seq data by VULCAN identifies GRHL2 as a key co-regulator of ERa in luminal breast cancer

“…We undertook a complementary, unbiased, experimental approach combining RIME [9] with TMT [33] , called qPLEXRIME [34] , to identify interactors of ER within the ERchromatin complex. We generated ER qPLEXRIME data from MCF7 cells treated with estradiol at both 45 and 90 minutes and compared this to the VULCAN dataset ( Suppl.…”

“…Validation of the ERGRHL2 interaction by qPLEXRIME and coIP qPLEXRIME [34] analysis of GRHL2 in both the estrogenfree and estrogenic conditions (Supplementary File 4) showed high levels of transcriptionrelated protein interactors including HDAC1 (pvalue = 6.4 x 10 9 ), TIF1A (pvalue = 6.4 x 10 9 ), PRMT (pvalue = 6.4 x 10 9 ) and GTF3C2 (pvalue = 4.6 x 10 9 ). Pvalues given for estrogenfree and estrogenic conditions conditions were comparable.…”