Identification of new protein-coding potential in Leishmania braziliensis using a proteogenomics approach.

American tegumentary leishmaniasis (ATL) is primarily caused by Leishmania (Viannia) species such as Leishmania braziliensis, Leishmania panamensis, and Leishmania guyanensis, which show complex genomic organisation and stage-specific adaptations underlying their pathogenicity. Despite the availability of its reference genome, limitations in gene annotation persist due to the presence of hypothetical proteins, pseudogenes, and unrecognised coding regions. In this study, we used a proteogenomic approach integrating publicly available high-resolution mass spectrometry data with a custom six-frame translated genome database to refine the genome annotation of L. braziliensis strain MHOM/BR/75/M2904. Utilising stringent database-dependent searches with a 1 % false discovery rate, we identified many unique peptides, of which 1034 were genome search-specific peptides (GSSPs) mapping exclusively to unannotated genomic regions. These GSSPs facilitated the discovery of 56 novel protein-coding genes and the correction of 228 existing gene models, including N- and C-terminal extensions. Notably, several novel genes encode proteins with conserved domains such as membrane attack complex/perforin (MACPF), kinesin K39, and peptidase S9/S15, suggesting functional relevance in parasite biology. Our findings demonstrate the power of proteogenomics to uncover cryptic protein-coding regions and improve genome annotations beyond conventional predictions. This refined annotation enhances our understanding of L.braziliensis biology, providing a more accurate proteomic landscape that can inform studies on parasite virulence, host interaction, and potential therapeutic targets. The study underscores the importance of integrating proteomic evidence with genomic data to capture the full coding potential of kinetoplastid parasites, paving the way for improved diagnostics and interventions against leishmaniasis.