Capture, Release, and Identification of Newly Synthesized Proteins for Improved Profiling of Functional Translatomes.

ABSTRACT: New protein synthesis is regulated both at the level of mRNA transcription and translation. RNA-Seq is effective at measuring levels of mRNA expression, but techniques to monitor mRNA translation are much more limited. Previously, we reported results from O-propargyl-puromycin (OPP) labeling of proteins undergoing active translation in a 2-h time frame, followed by biotinylation using click chemistry, affinity purification, and on-bead digestion to identify nascent proteins by mass spectrometry (OPP-ID). As with any on-bead digestion protocol, the problem of nonspecific binders complicated the rigorous categorization of nascent proteins by OPP-ID. Here, we incorporate a chemically cleavable linker, Dde biotin-azide, into the protocol (OPP-ID_CL) to provide specific release of mod

SUBMITTER: Phillips NJ 

PROVIDER: S-EPMC9971285 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature