MiPRISMA: Interactome profiling of small peptides and young microproteins translated from human short open reading frames

All species continuously evolve small open reading frames (sORFs) that can be templated for protein synthesis and may provide raw material for evolutionary adaptation. We analyzed the evolutionary origins of 7,264 recently cataloged human sORFs and found that most were evolutionary young and emerged de novo. We additionally discovered 221 yet uncataloged peptides smaller than 16 amino acids, for which we found evidence of translation in human and rodent tissues. To investigate the potential bioactivity of microproteins and peptides translated from young and very small ORFs we established MiPRISMA: a mass spectrometry-based interactome screen with sequence motif resolution. We assessed 266 candidates and implicated several in essential cellular processes including splicing, translational regulation, and endocytosis, a subset of which we validate in cellular assays. MiPRISMA provides a scalable platform to characterize small and evolutionarily young proteins, shedding light on a largely unexplored territory of the putative human proteome.