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

Ribosome-associated proteins (RAPs) enable modulation of gene expression at the level of mRNA translation in response to cellular needs. Here, we describe a method called ribosome-associated protein identification by affinity to sulfhydryl-charged resin (RAPIDASH) for tag-free isolation of RAP-bound ribosomes from mammalian samples for mass spectrometry-based proteomics. Samples are first lysed and then undergo sucrose cushion ultracentrifugation and subsequent chromatography using a sulfhydryl-charged resin. While RAPIDASH is optimized for mammalian samples, we expect that it can be adapted for non-mammalian samples. For complete details on the use and execution of this protocol, please refer to Susanto et al.¹.