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Ribosome Profiling

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Applications of ribosome profiling
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Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics
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Improved detection and consistency of RNA-interacting proteomes using DIA SILAC.

Thomas C J Tan1, Christos Spanos1, David Tollervey1

  • 1Wellcome Centre for Cell Biology and Institute of Cell Biology, School of Biological Sciences, University of Edinburgh. Edinburgh EH9 3BF, Scotland, UK.

Nucleic Acids Research
|January 10, 2024
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Summary

This study introduces a more sensitive method for identifying RNA-binding proteins using data-independent acquisition (DIA) with SILAC labeling. This approach enhances protein detection and reduces costs compared to traditional methods.

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Area of Science:

  • Proteomics
  • Molecular Biology
  • Biochemistry

Background:

  • Characterizing the RNA-interacting proteome typically involves UV-crosslinking, RNA purification, and SILAC labeling followed by data-dependent acquisition (DDA).
  • The sensitivity limitations of DDA and low UV-crosslinking efficiency often restrict protein detection to abundant species, requiring extensive sample processing.

Purpose of the Study:

  • To evaluate the application of data-independent acquisition (DIA) coupled with SILAC labeling in a total RNA-associated protein purification (TRAPP) UV-crosslinking experiment.
  • To compare the efficacy of DIA-SILAC with conventional DDA-SILAC for RNA-interactome profiling.

Main Methods:

  • Utilized UV-crosslinking and SILAC labeling in a TRAPP experiment.
  • Employed data-independent acquisition (DIA) for proteomic analysis, contrasting it with data-dependent acquisition (DDA).
  • Applied the method to analyze the effects of arsenite treatment on the RNA-bound proteome of HEK293T cells.

Main Results:

  • DIA-SILAC demonstrated a 15% increase in protein detection and reduced inter-replicate variation compared to DDA-SILAC.
  • Single-shot sample analysis was feasible with DIA, significantly reducing machine time and costs.
  • DIA identified additional proteins, including those involved in cellular stress responses, which were missed by DDA.

Conclusions:

  • The DIA-SILAC approach offers improved protein detection and cost-efficiency for RNA-interactome studies compared to DDA-SILAC.
  • This method enhances the characterization of RNA-binding proteins and is adaptable to various RNA-binding protein enrichment techniques.
  • DIA-SILAC provides a more comprehensive and efficient means to study the dynamic changes in the RNA-bound proteome.