Protocol for label-free ribosome-associated protein enrichment from mammalian cells by RAPIDASH

Area of Science:

• Molecular Biology
• Proteomics
• Gene Expression

Background:

• Ribosome-associated proteins (RAPs) are crucial regulators of gene expression.
• Understanding RAPs' roles requires effective isolation methods for studying mRNA translation.

Purpose of the Study:

• To introduce a novel, tag-free method for isolating RAPs bound to ribosomes.
• To enable mass spectrometry-based proteomics for identifying RAPs.

Main Methods:

• The RAPIDASH (ribosome-associated protein identification by affinity to sulfhydryl-charged resin) method was developed.
• It involves cell lysis, sucrose cushion ultracentrifugation, and chromatography on sulfhydryl-charged resin.
• The protocol is optimized for mammalian samples but adaptable for others.

Main Results:

• RAPIDASH allows for the tag-free isolation of RAP-bound ribosomes.
• This facilitates downstream mass spectrometry-based proteomic analysis.
• The method provides a new tool for studying translational control.

Conclusions:

• RAPIDASH is an effective technique for isolating RAPs from mammalian ribosomes.
• This method advances the study of gene expression regulation via mRNA translation.
• The protocol is adaptable, offering broad applicability in biological research.