Endogenous HiBiT-tagging combined with affinity complementation: A new strategy for small open reading frame-encoded polypeptide detection in bacteria

• HiBiT-tagging and blotting for endogenous SEP detection. • Validation of riboproteogenomics-discovered bacterial SEPs using HiBiT blotting. • Complementary proteome extraction methods for improved SEP detection. • Integration of proteomics and protein blotting for comprehensive SEP discovery. Bacterial genome annotations are continuously refined with the advent of novel techniques. Ribosome profiling, or Ribo-seq, utilizing next-generation sequencing to link genomic regions with translational activity, has uncovered numerous small open reading frames (sORFs) - arbitrarily defined as ORFs no longer than 300 base pairs - as a generally under-annotated class of genomic elements in both eukaryotic and prokaryotic genomes. While sORFs can function as regulatory elements, they may also translate into small proteins (equal to or shorter than 100 amino acids), classified as sORF-encoded polypeptides (SEPs). The inherent limitations of ribosome profiling necessitate the experimental validation of predicted (s)ORFs at the protein expression level. However, the small size and unique biochemical characteristics of SEPs pose significant challenges for traditional protein detection methods, like mass spectrometry and immunoblotting. In this study, we introduce HiBiT blotting, a luminescent, complementation-based protein detection method, as a highly sensitive alternative to antibody-based immunoblotting for investigating SEP expression in Salmonella enterica (serovar) Typhimurium ( S. Typhimurium) at endogenous levels. We demonstrate its complementarity to mass spectrometry as an expression validation tool. Additionally, employing a biochemical fractionation approach, we determined the subcellular localization of validated S. Typhimurium SEPs, initiating exploration into the functional aspects of these SEPs, and proposed enrichment strategies that may facilitate future experimental validation of Ribo-seq-predicted sORFs.