Proteins à la carte: riboproteogenomic exploration of bacterial N-terminal proteoform expression.

In recent years, it has become evident that the true complexity of bacterial proteomes remains underestimated. Gene annotation tools are known to propagate biases and overlook certain classes of truly expressed proteins, particularly proteoforms-protein isoforms arising from a single gene. Recent (re-)annotation efforts heavily rely on ribosome profiling by providing a direct readout of translation to fully describe bacterial proteomes. In this study, we employ a robust riboproteogenomic pipeline to conduct a systematic census of expressed N-terminal proteoform pairs, representing two isoforms encoded by a single gene raised by annotated and alternative translation initiation, in IMPORTANCE: With the emerging theme of genes within genes comprising the existence of alternative open reading frames (ORFs) generated by translation initiation at in-frame start codons, mechanisms that control the relative utilization of annotated and alternative TIS need to be unraveled and our molecular understanding of resulting proteoforms broadened. Utilizing complementary ribosome profiling strategies to map ORF boundaries, we uncovered dual-encoding ORFs generated by in-frame TIS usage in