Improved detection and consistency of RNA-interacting proteomes using DIA SILAC

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.