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

Leishmania donovani , the causative agent of visceral leishmaniasis, possesses a highly plastic genome and relies extensively on post-transcriptional regulation, posing challenges for accurate genome annotation. Despite the availability of a high-quality reference genome, many protein-coding genes remain incomplete or misannotated. In this study, we utilised a proteogenomic strategy integrating high-resolution tandem mass spectrometry (MS/MS) data with a custom six-frame translated genome database to refine the genome annotation of L. donovani . Re-analysis of previously published promastigote and amastigote proteomic datasets identified 50 N-terminal extensions across both developmental stages and revealed 15 novel protein-coding regions absent from the current annotation. Several of the newly identified or extended proteins displayed conserved orthologs across related Leishmania species and contained functional domains implicated in essential cellular processes, including metabolism, vesicular trafficking, and intracellular survival. Collectively, our findings demonstrate that proteogenomic integration significantly improves the accuracy of L. donovani genome annotation by resolving truncated gene models and uncovering hidden coding potential. The refined proteome presented here provides a valuable resource for future functional studies and enhances our understanding of parasite biology, host adaptation, and pathogenicity.