Interactome Profiling (RIME)

identify protein interactions by mass spec

 

RIME Services Overview

RIME Service
 

RIME (Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins) is a powerful technique ideally suited for the identification of transcriptional co-factors and chromatin associated proteins.

The RIME methodology was originally described in this Cell Reports publication.

The RIME Service includes:

  1. Nuclear isolation and sonication.
  2. Immunoprecipitation.
  3. Purification of proteins and tryptic digestion.
  4. Mass spectrometry.
  5. Data analysis.
RIME Method

What does RIME do?

With RIME, mass spectrometry is used, along with immunoprecipitation of a target protein, to detect endogenous-interacting proteins in formaldehyde cross-linked cells.

Cross-linking advantages:

  1. Preserves bona fide interactions allowing for more stringent washes and minimal detection of non-specific interactions.
  2. Captures low-affinity interactions that are typically lost during washing.
  3. Captures adjacent DNA binding proteins that participate in gene regulation but do not directly interact with the targeted protein.

Learn More About RIME

Benefits of RIME include:

  1. Identify transcriptional co-factors/co-regulators.
  2. Identify proteins complexed with epigenetic modifiers.
  3. Detect low-affinity interactions.
  4. Map protein interaction networks.
  5. Validate identified proteins by ChIP-Seq and map global co-occupancy with the RIME target protein.
  6. Understand how co-factors participate in differential gene regulation.
  7. Identify the prominent co-occurrence of transcription factor binding at adjacent sites.

What our customers are saying about us:

"We used Active Motif's RIME services several times and found their protocol to be reliable and reproducible. Expected interactors were detected robustly and specifically."

Till Adhikary, PhD
Phillips University
Marburg, Germany

"I have ordered RIME/ChIP-MS services. Since the target antibody was first tested by the Antibody Validation Service, I felt confident in proceeding with the main test. The technical staff explained the data and were willing to repeatedly answer my questions. This service provided me with valuable information and helpful support from all the staff members at Active Motif."

Naoko Hattori, PhD
Staff Scientist
Division of Epigenomics,
National Cancer Center Research Institute,
Tokyo, Japan

   
 

RIME Services Data

RIME Venn Data

Figure 1: Venn Diagram of detected proteins in RIME treated MCF7 cells
RIME was performed to identify proteins that interact with the RNA polymerase machinery. Immunoprecipitations were performed with an antibody against POLR2A and two different amounts of cross-linked chromatin from MCF7 cells. Immunoprecipitated proteins were measured by mass spectrometry. The Venn diagram shows strong overlap of the detected proteins even in experiments using a 10 fold difference in starting material.


RIME POLR2A Coverage

Figure 2. Detection of target protein POLR2A and potential proteins of interest
The upper panel shows the percent coverage in duplicate RIME reactions performed for POLR2A using 500ug of sample chromatin. Each experiment is assigned a grade to describe the performance of the antibody a specific sample, with >40% indicating a successful assay with very good coverage (<10% suggests that the antibody did not work well in RIME). In the bottom panel, the top 13 proteins of interest that were found to interact with POLR2A in this experiment are listed, after removing proteins present in the IgG negative control.


RIME GO data

Figure 3: RIME identifies most prevalent molecular pathways among enriched proteins
PANTHER Functional Classification was performed for the list of proteins identified as interacting with POLR2A. The top eight gene ontology terms are identified and ranked by p-value.


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.

ChIP-Seq shows co-localization of TOP2A and BRD4 binding sites.
ChIP-Seq shows co-localization of TOP2A and BRD4 binding sites.

Figures 4 & 5: ChIP-Seq shows co-localization of TOP2A and BRD4 binding sites.
ChIP-Seq was performed to map binding sites for the RIME-identified BRD4 interacting protein, TOP2A. TOP2A and BRD4 binding sites are shown and confirm co-localization at many sites across the genome. The top panel shows sites on chromosome 1 and the bottom panel shows sites on chromosome 3.

References


 

RIME Services Publications

Search our database of customer publications that have used our RIME services.


 

 

 

RIME Services Documents

 

 

 

RIME Services Sample Submission Portal

Our online sample submission portal allows you to easily upload your service project samples and track your project status. Follow the sample submission instructions in the portal to ensure that all your samples arrive at Active Motif in the best possible condition and properly associated with your project.

 

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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:

  1. Preserves bona fide interactions allowing for more stringent washes and minimizing the detection of non-specific interactions.
  2. Captures low-affinity interactions that are typically lost during washing.
  3. 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:

  1. Identify transcriptional co-factors/co-regulators.
  2. Identify proteins complexed with epigenetic modifiers.
  3. Detect low-affinity interactions.
  4. Map protein interaction networks.
  5. Validate identified proteins by ChIP-Seq and map global co-occupancy with the RIME target protein.
  6. Understand how co-factors participate in differential gene regulation.
  7. Identify the prominent co-occurrence of transcription factor binding at adjacent sites.
RIME Venn Data

Figure 1: Venn Diagram of detected proteins in RIME treated MCF7 cells
RIME was performed to identify proteins that interact with the RNA polymerase machinery. Immunoprecipitations were performed with an antibody against POLR2A and two different amounts of cross-linked chromatin from MCF7 cells. Immunoprecipitated proteins were measured by mass spectrometry. The Venn diagram shows strong overlap of the detected proteins even in experiments using a 10 fold difference in starting material.


RIME POLR2A Coverage

Figure 2. Detection of target protein POLR2A and potential proteins of interest
The upper panel shows the percent coverage in duplicate RIME reactions performed for POLR2A using 500ug of sample chromatin. Each experiment is assigned a grade to describe the performance of the antibody a specific sample, with >40% indicating a successful assay with very good coverage (<10% suggests that the antibody did not work well in RIME). In the bottom panel, the top 13 proteins of interest that were found to interact with POLR2A in this experiment are listed, after removing proteins present in the IgG negative control.


RIME GO data

Figure 3: RIME identifies most prevalent molecular pathways among enriched proteins
PANTHER Functional Classification was performed for the list of proteins identified as interacting with POLR2A. The top eight gene ontology terms are identified and ranked by p-value.

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.

ChIP-Seq shows co-localization of TOP2A and BRD4 binding sites.

 

ChIP-Seq shows co-localization of TOP2A and BRD4 binding sites.
Figures 1 & 2: ChIP-Seq shows co-localization of TOP2A and BRD4 binding sites.

ChIP-Seq was performed to map binding sites for the RIME-identified BRD4 interacting protein, TOP2A. TOP2A and BRD4 binding sites are shown and confirm co-localization at many sites across the genome. The top panel shows sites on chromosome 1 and the bottom panel shows sites on chromosome 3.

References

Search our database of customer publications that have used our RIME services.