Interactome Profiling (RIME)
identify protein interactions by mass spec
RIME (Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins) is a recently developed technique ideally suited for the identification of transcriptional co-factors and chromatin associated proteins.
RIME was originally described in this Cell Reports publication.
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The RIME Service includes:
- Nuclear isolation and sonication.
- Purification of proteins and tryptic digestion.
- Mass spectrometry.
- Data analysis.
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The RIME methodology was originally described in this Cell Reports publication.
With RIME, mass spectrometry is used, along with immunoprecipitation of a target protein, to detect endogenous-interacting proteins in formaldehyde cross-linked cells.
Advantages of cell cross-linking include:
- Preserves bona fide interactions allowing for more stringent washes and minimizing the detection of non-specific interactions.
- Captures low-affinity interactions that are typically lost during washing.
- Captures adjacent DNA binding proteins that participate in gene regulation but do not function through direct interaction with the targeted protein.
Benefits of RIME include:
- Identify transcriptional co-factors/co-regulators.
- Identify proteins complexed with epigenetic modifiers.
- Detect low-affinity interactions.
- Map protein interaction networks.
- Validate identified proteins by ChIP-Seq and map global co-occupancy with the RIME target protein.
- Understand how co-factors participate in differential gene regulation.
- Identify the prominent co-occurrence of transcription factor binding at adjacent sites.
Figure 1: Venn Diagram of RIME treated MCF7 cells
Advanced Application: RIME and ChIP-Seq
RIME was performed using BRD4 as the target protein and from the list of interacting proteins. DNA Topoisomerase 2A was identified as an interactor, which is supported by literature demonstrating that ligand-dependent Topoisomerases have been shown to be recruited to enhancers†. Therefore, ChIP-Seq targeting BRD4 and TOP2A was performed to show co-localization. Expanding on RIME data with ChIP-Seq can help elucidate gene subsets that require different transcription factors or confirm co-localization of RIME-identified co-factors to the same genes.
Figures 1 & 2: ChIP-Seq shows co-localization of TOP2A and BRD4 binding sites.
- †Puc, J. et al. Ligand-Dependent Enhancer Activation Regulated by Topoisomerase-I Activity, Cell. 2015 Oct 8;163(2):520-2.