Ribo-ITP expands the translatome of limited input samples

In the last decade, an unexpectedly large number of translated regions (translons) have been discovered using ribosome profiling and proteomics. Translons can regulate mRNA translation and encode micropeptides that contribute to multiprotein complex formation, Ca ²⁺ regulation in muscle, and signaling during embryonic development. However, identification of translons has been limited to cell lines or large organs due to high input requirements for conventional ribosome profiling and mass spectrometry. Here, we address this challenge using Ribo-ITP on difficult-to-collect samples like microdissected hippocampal tissues and single pre-implantation embryos. Comparative analysis of more than a thousand ribosome profiling datasets across a wide range of cell types revealed distinct sample specific expression patterns of the detected translons. To test the translational capacity of the identified translons, we engineered a translon-dependent GFP reporter system and detected expression of translons initiating at near-cognate start codons in mouse embryonic stem cells (mESCs). Mutating the translons in mESCs identified a small proportion that negatively impacted growth. Taken together, we present a proof-of-concept study to identify non-canonical translation events from low input samples which can be applied to cell and tissue types inaccessible to conventional methods.