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

Identification of new protein-coding potential in Leishmania donovani using a proteogenomics approach | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Identification of new protein-coding potential in Leishmania donovani using a proteogenomics approach Soumi Chowdhury, Shubhankar Pawar, Karthick Vasudevan, Nalini Mishra, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8719077/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 13 You are reading this latest preprint version Abstract 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. Leishmania donovani pseudogenes proteogenomics gene annotations protein coding genes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Leishmania donovani is a digenetic protozoan parasite belonging to the family Trypanosomatidae and is the principal causative agent of visceral leishmaniasis (VL), the most severe and often fatal form of leishmaniasis (Lukeš et al., 2007 ). Endemic across the Indian subcontinent, East Africa, and parts of the Mediterranean basin, VL is characterised by chronic fever, hepatosplenomegaly, severe anaemia, and progressive wasting, contributing to high mortality in untreated cases (Arenas et al., 2017 ). Transmission occurs through the bite of infected female Phlebotomus sand flies, primarily P. argentipes and P. orientalis , which inoculate non-dividing metacyclic promastigotes into the host dermis. Once phagocytosed by macrophages, the parasite differentiates into the amastigote form and replicates within the hostile, acidic phagolysosomal niche (Bates, 2007 ). The L. donovani strain BPK282A1 represents the primary reference genome for this pathogen and has played a central role in shaping current genomic understanding of visceral leishmaniasis. The genome was first released in 2011 (assembly GCA_000227135.1), providing a foundational but incomplete draft that contained several gaps and unresolved regions (Downing et al., 2011 ). Subsequent refinement using improved sequencing depth and assembly methods generated the updated version ASM22713v2/GCF_000227135.2, which significantly enhanced contiguity and accuracy by resolving gap regions and correcting structural inconsistencies (Iantorno et al., 2017 ). The reference genome of L. donovani strain BPK282A1 comprises approximately 32.44 Mb of sequence organised into 36 chromosomes and contains a total of 8,135 annotated genes. Among these, 7,969 are predicted to encode proteins, 166 correspond to non-protein-coding genes, and 54 are classified as pseudogenes. Comparative genomics indicates that L. donovani , L. infantum , and L. major share extensive synteny and high (> 99%) conservation of gene content, with only a small set of species-specific genes differentiating them (Freitas-Mesquita et al., 2021 ; Rochette et al., 2009 ; Tschoeke et al., 2014 ). However, genome organisation within the Leishmania genus exhibits structural plasticity, including chromosomal fusion events in New World species such as L. braziliensis and L. mexicana , which harbour 35 and 34 chromosomes, respectively (Kazemi, 2011 ). L. donovani exhibits complex genomic features such as gene dosage variations, frequent aneuploidy, polycistronic transcription, and post-transcriptional regulation, which collectively shape its developmental transitions and pathogenicity (Franssen et al., 2020 ). Despite the availability of its reference genome, a substantial portion of L. donovani genes remains annotated as “hypothetical,” lacking functional characterisation or experimental validation. Initial genome annotation also identified numerous “partial” or truncated gene models, many of which required structural correction (Downing et al., 2011 ; Jamdhade and Pawar et al., 2015 ; Zhang et al., 2014 ). Comparative analysis of related kinetoplastids, particularly L. infantum and L. major , enabled refinement of these partial annotations, culminating in the correction of over 300 gene models, including C-terminal extensions, merging of fragmented genes, tandem paralog resolution, and rectification of incorrect chromosomal assignments (Jamdhade and Pawar et al., 2015 ; Trinidad-Barnech et al., 2025 ). This highlights the persistent need for experimental evidence to validate predicted gene structures. Proteomic and proteogenomic analyses have played a crucial role in addressing these annotation gaps. Early proteome mapping of L. donovani (prior to genome availability) relied on similarity-based searches using genomes of L. infantum , L. major , and L. braziliensis , leading to the identification of more than 22,000 unique peptides and 3,711 putative proteins across promastigote and amastigote stages (Pawar et al., 2012 ). Following the release of the genome sequence, reanalysis of the dataset enabled direct mapping to the L. donovani proteome, identifying 3,999 proteins (~ 50% of the predicted proteome) and providing high-quality peptide evidence for over 2,200 proteins expressed in both life stages (Nirujogi et al., 2014 ). Importantly, genome search-specific peptides (GSSPs) revealed 20 novel protein-coding genes and enabled correction of 40 existing annotations, including N-terminal and C-terminal extensions. Beyond proteome mapping, genomic variation contributes significantly to L. donovani biology. Comparative genomic analysis of atypical Sri Lankan L. donovani strains causing cutaneous (CL-SL) versus visceral (VL-SL) disease revealed that differences in virulence correlate with single-nucleotide polymorphisms, gene copy number variations, and modulation of key virulence-associated gene families such as the A2 genes, rather than gross gene deletions or pseudogene formation (McCall & Matlashewski, 2012 ; Samarasinghe et al., 2018 ; Zhang et al., 2014 ). These findings emphasise the functional impact of subtle genomic alterations on tissue tropism and clinical outcomes. Proteogenomics provides an integrated framework to refine gene predictions, uncover novel coding regions, validate translational start/stop sites, detect frameshifts, characterise pseudogenes, and reveal conserved or lineage-specific protein families of biological significance. Applying such approaches to L. donovani is essential for strengthening its genome annotation, especially given the high proportion of hypothetical proteins and the unique features of Leishmania gene expression that complicate classical computational predictions. Our recent study demonstrated the strength of integrating high-resolution mass spectrometry data with genome-wide six-frame translation to refine parasite gene models. Using a comprehensive proteogenomic pipeline, we identified multiple GSSPs that provided direct experimental evidence for previously unannotated open reading frames (ORFs), corrections to existing gene boundaries, and validation of hypothetical proteins in L. braziliensis (Shenoy and Chowdhury et al., 2025 ). This work highlighted the substantial coding potential that remains uncharacterized in kinetoplastid genomes and underscored how peptide-level evidence can resolve fragmented annotations, extend gene termini, and uncover novel protein-coding loci. The success of this strategy in L. braziliensis provided a strong rationale for applying a similar proteogenomics framework to L. donovani , a species with a larger disease burden and a high proportion of hypothetical or partially annotated genes. On the other hand, in our recent proteogenomic study on Leishmania guyanensis , we refined the genome annotation by integrating mass spectrometry data with a six-frame translated genome database. This approach identified 653 GSSPs, leading to the discovery of 65 novel protein-coding genes and the correction of 62 existing gene models, including N- and C-terminal extensions. These findings underscore the value of proteogenomics in uncovering cryptic coding regions and improving the accuracy of L. guyanensis genome annotations (Pawar et al., 2026 ). In the present study, we conducted a comprehensive proteogenomic analysis of L. donovani Cutaneous Leishmania (CL) from Sri Lanka (SL) using publicly available high-resolution mass spectrometry datasets and a custom-built six-frame translated genome database. Our analysis identified numerous genome search-specific peptides, enabling the discovery of previously unannotated protein-coding regions and refinement of existing gene models. By integrating peptide-level evidence with updated genomic resources, we provide an enhanced annotation of the L. donovani genome, contributing to improved understanding of parasite biology, host-pathogen interactions, tissue tropism, and potential therapeutic targets. 2. Materials and Methods 2.1 Generation of protein database and mass spectrometry data analysis The L. donovani CL_SL whole genome sequence was downloaded from TriTrypDB, and the corresponding whole-genome FASTA file was used as the reference for proteogenomic analysis (Zhang et al., 2014 ). Using in-house Python scripts, we generated a six-frame translated database from the complete genome, with a minimum translated ORF length cutoff of 10 amino acids. This unbiased six-frame translation approach followed previously published proteogenomic workflows (Nirujogi et al., 2014 ; Pawar et al., 2012 ; Shenoy and Chowdhury et al., 2025 ). For peptide identification, we used our previously published L. donovani proteomic dataset (Nirujogi and Pawar et al., 2014 ), which was re-analysed against the newly constructed six-frame translated database to identify genome search-specific peptides and refine gene annotations. 2.2 Proteogenomic analysis : The L. donovani promastigote and amastigote proteomic dataset (Nirujogi and Pawar et al., 2014 ) was searched against the custom-built six-frame translated L. donovani genome database generated in this study. Database-dependent searches were performed using the Sequest HT search engine, implemented in Proteome Discoverer (version 2.4). Enzymatic digestion parameters were set to trypsin with a maximum allowance of one missed cleavage. Precursor ion mass tolerance was set at 20 ppm, and fragment ion tolerance was set at 0.1 Da. Carbamidomethylation of cysteine residues was specified as a fixed modification, while methionine oxidation and N-terminal acetylation were defined as variable modifications. Peptide-spectrum matches obtained from Sequest HT searches were filtered using a stringent 1% false discovery rate (FDR). High-confidence unique peptides that passed the FDR threshold were retained and used for subsequent proteogenomic analysis, including identification of genome search-specific peptides and the refinement of gene models (Shenoy and Chowdhury et al., 2025 ). All high-confidence unique peptides obtained from database searches were first aligned against the annotated L. donovani CL_SL protein database downloaded from TriTrypDB. Peptides that matched the annotated proteome were excluded from further analysis. Peptides that failed to map to the annotated protein database but showed a unique match to the six-frame translated L. donovani genome were classified as GSSPs. In this study, GSSPs are defined as MS-derived peptides that align exclusively to the translated genomic sequence and are absent from existing protein annotations. Such peptides provide direct experimental evidence for previously unrecognised protein-coding loci or highlight potential inaccuracies in current gene models, such as truncated ORFs, misannotated boundaries, or missing exons. Each GSSP was subsequently mapped back to its genomic coordinates to determine its exact positional context relative to existing annotations. This enabled the identification of novel coding regions, the extension of annotated ORFs, the merging of fragmented gene models, and the correction of erroneous start/stop codons. Only high-confidence GSSPs supported by unambiguous genomic mapping were considered for gene model refinement. 2.3 Bioinformatics analysis: GSSPs that uniquely matched the six-frame translated L. donovani genome were subjected to additional validation using BLAST-based searches (BLASTp and tBLASTn). These analyses enabled us to determine the precise genomic loci corresponding to each peptide and to identify conserved orthologous proteins in closely related Leishmania species (Shenoy and Chowdhury et al., 2025 ). Based on this integrative approach, the proteogenomic evidence was classified into two major categories: (i) novel protein-coding genes, representing previously unannotated ORFs not present in the reference L. donovani annotation, and (ii) refinements to annotated genes, including N-terminal and C-terminal extensions, correction of truncated models, and merging of fragmented ORFs. To further assess the functional relevance of these newly identified or corrected proteins, conserved domain architectures were predicted using the SMART database (Letunic & Bork, 2025 ). The corresponding domain illustrations and structural annotations are provided in Supplementary Figs. 5 . 3. Results 3.1. Overview of the proteomic data To refine the genome annotation of L. donovani CL_SL strain, we employed a proteogenomic approach using high-resolution tandem mass spectrometry (MS/MS) datasets generated from the amastigote life stage. These experimental MS/MS spectra were systematically analysed against both the annotated L. donovani protein database and a six-frame translated genomic database to enable comprehensive peptide discovery. The six-frame translation of the L. donovani genome yielded 362893 predicted ORFs across all reading frames, providing a broad search space for peptide mapping. Database-dependent searches of the L. donovani LC-MS/MS datasets, performed under stringent filtering criteria including a 1% false discovery rate threshold, identified 33210 unique peptides. Of these, 32852 peptides mapped to previously annotated proteins in the L. donovani CL reference genome, thereby supporting existing gene models. The remaining 358 peptides did not map to the annotated protein database but aligned uniquely to the six-frame translated genome; these were classified as GSSPs. All GSSPs were subsequently examined in detail to determine whether they corresponded to novel protein-coding regions, extensions of existing gene models, or corrections to misannotated loci. This detailed proteogenomic analysis uncovered multiple unannotated genomic features, highlighting opportunities to improve and refine the current L. donovani genome assembly. The full workflow of the proteogenomic strategy used in this study, including peptide identification, six-frame genome mapping, and integration with comparative genomics, is summarised in Fig. 1 . 3.2. Identification of protein-coding genes in the promastigote life stages of the L. donovani genome: 3.2.1. Identification of novel protein-coding genes in the promastigote life stages of L. donovani genome: A representative example of a novel gene identified in the promastigote stage is Cytochrome c oxidase subunit VIb, a key component of Complex IV of the mitochondrial electron transport chain. In the current L. donovani genome annotation, the genomic region corresponding to LdCL_320037500 on chromosome 32 (positive strand) is labelled as a hypothetical protein (Rab5-interacting protein). However, our proteogenomic analysis identified multiple peptides that mapped to an unannotated ORF upstream of this locus, providing strong evidence for the presence of a previously unrecognised protein-coding gene. This ORF exhibited high-confidence peptide support specifically from the promastigote life stage. The example has been represented in Figs. 2 A and 2 B. The newly identified ORF encodes a protein beginning with the conserved N-terminal sequence MQSAKKRELCYKTRDAFHKCLDTLPEDPEKECAAQKKLFEQSCPKSWVSYFEKQREREVI…, which shows near-complete identity with the orthologous protein in L. tropica (LTRL590_320038000). The strong conservation across the full length of the alignment, including the C-terminal motif LQLQVEQYKGR, supports the functional validity of this gene and confirms that it corresponds to cytochrome c oxidase subunit VIb, a mitochondrial protein essential for respiratory chain activity. Because this protein is completely absent from the current L. donovani annotation, the mapped peptides and ortholog conservation provide definitive evidence that this region contains a bona fide novel mitochondrial gene. The identification of this novel subunit of Complex IV demonstrates the effectiveness of proteogenomic analysis in uncovering hidden components of essential metabolic pathways and correcting incomplete annotations in L. donovani . Another representative example of a novel gene identified in the promastigote stage involves a hypothetical protein containing an N-terminal glutamine amidase (Nt_Gln_amidase) structural domain, a family of enzymes implicated in the processing or hydrolysis of N-terminal glutamine residues in diverse organisms. In the current L. donovani genome annotation, the genomic region corresponding to LdCL_040017800 on chromosome 4 (negative strand) is annotated as a leucine-rich repeat casein kinase I homolog. However, our proteogenomic analysis identified peptides that mapped to an upstream, unannotated ORF, providing strong evidence that this region encodes an independent protein absent from the current annotation. The novel ORF begins with the highly conserved N-terminal sequence MFRRCARRLGHQLIKEPMGHEETTFSRGDRTRNNALQHIYGLIGFCGVGCVLALFSFVSG…, followed by QRRVLTTITADGTITKGTCPTKWWNF, together forming a promastigote-expressed protein with clear orthologous conservation, as shown by alignment with L. gerbilli (LGELEM452_040017600). The near-identical conservation across the full N-terminal region underscores that this ORF represents a protein rather than a misannotated fragment of the neighbouring kinase gene. Functional annotation revealed the presence of an Nt_Gln_amidase structural domain, a domain characteristic of enzymes that catalyse the removal or modification of N-terminal glutamine or glutamate residues. Such catalytic domains are frequently involved in N-terminal processing, protein maturation, and regulated proteolysis, functions of potential significance in parasite development and adaptation during the promastigote stage. Because this ORF is completely absent from the current annotation of chromosome 4, peptide-level evidence combined with structural domain prediction strongly supports the existence of Novel gene 2, encoding a previously unrecognised Nt_Gln_amidase-family protein in L. donovani . The discovery of this novel enzymatic protein highlights the importance of proteogenomics in resolving unannotated or misclassified regions of the L. donovani genome and in uncovering potentially functional metabolic components. The complete set of GSSPs, along with their genomic coordinates, is provided in Supplementary Table 1 , and the graphical representation of this novel ORF, together with the corresponding MS/MS spectra of representative GSSPs, is shown in Supplementary Fig. 1 . 3.2.2. Identification of N-terminal extension in the promastigote life stages of L. donovani genome: A representative example of an N-terminal extension identified in L. donovani involves the ATP12 chaperone, a mitochondrial protein required for the assembly and stabilisation of the F₁ sector of the F₁F₀ ATP synthase complex. In the current genome annotation, the protein is represented by LdCL_180008400, located on chromosome 18 (positive strand), with the annotated coding sequence beginning downstream at MSRMNSKQLEEVMRKFEEQENESSR. However, our proteogenomic analysis uncovered multiple high-confidence GSSPs, including the peptide “NVPSEPGAHAELSTAELER” (5 PSMs) that mapped to the upstream locus LdCL_180008300, which had been annotated as a protein containing a DUF4475 domain. These peptides provide compelling evidence that translation initiates upstream of the currently annotated start site. The example has been represented in Figs. 3 A and 3 B. The upstream region begins with the extended N-terminal sequence MRPFTLAKGAAPRQLLAVAIAAARMSVSSSTPAEPSTPAGASQATAATGPTVAPKKPRRR…, which is strongly conserved in the orthologous gene from L. gerbilli (LGELEM452_180008600). The mapped GSSPs confirm active translation of this segment, demonstrating that the existing annotation truncates the true N-terminus of the ATP12 chaperone. Incorporating this upstream region adds additional amino acids to the N-terminus, increasing the full-length protein from the annotated 85 amino acids to an extended length of 180 amino acids, consistent with orthologous ATP12 proteins in related trypanosomatids. The extended N-terminal region contains a stretch of hydrophobic and basic residues characteristic of mitochondrial targeting sequences, suggesting that the annotation correction restores the accurate subcellular targeting information for this essential chaperone. Given that ATP12 is required for the proper folding of the α-subunit of mitochondrial ATP synthase, misannotation of its N-terminus may obscure critical functional domains involved in mitochondrial import and assembly. This example highlights how peptide-supported proteogenomic can correct truncated gene models and generate functionally relevant annotations for essential mitochondrial chaperones in L. donovani . Another clear example of an N-terminal extension identified in the promastigote stage involves the Guide RNA (gRNA) binding protein, a key component of the mitochondrial RNA editing machinery in kinetoplastids. In the current L. donovani genome annotation, this protein is represented by LdCL_080016400, located on chromosome 8 (positive strand). The annotated coding sequence begins downstream at MKKLHQE, which substantially truncates the N-terminal region of the protein. However, our proteogenomic analysis identified multiple high-confidence GSSPs uniquely mapping to the upstream locus LdCL_080016300, which had been annotated as hypothetical. Among these, the peptide “HLSPGELALPQHPR” provided strong evidence for active translation of an upstream N-terminal segment that is missing from the current annotation. This upstream region, beginning with MRGLGVLGLRGVPTRRPWLCDVARRSGDISMCSTASTSYRSQATSAGGTPPLPPPTSSPV, is highly conserved across other Leishmania species, including L. tropica (LTRL590_080015100), confirming that this extended N-terminal sequence represents the authentic start of the Guide RNA binding protein. Incorporation of this upstream sequence increases the length of the protein by amino acids, yielding a biologically accurate representation of the protein compared to the currently annotated isoform. The extended N-terminal domain is enriched in arginine, serine, and glycine residues, features that are characteristic of RNA-binding proteins involved in mitochondrial RNA editing and gRNA stabilisation. These residues frequently contribute to RNA interaction surfaces, targeting the kinetoplast and assembly of ribonucleoprotein complexes. Refining the annotation of this Guide RNA binding protein is therefore critical for understanding the organisation and regulation of the RNA editing machinery in L. donovani . This example demonstrates how proteogenomic evidence corrects truncated gene models and improves annotation of essential RNA-processing proteins during the promastigote stage. The complete set of GSSPs, along with their genomic coordinates, is provided in Supplementary Table 2 , and the graphical representation of this novel ORF, together with the corresponding MS/MS spectra of representative GSSPs, is shown in Supplementary Fig. 2 . 3.3. Identification of protein-coding genes in the amastigote life stages of the L. donovani genome: 3.2.1. Identification of novel protein-coding genes in the amastigote life stages of the L. donovani genome: GSSPs obtained from six-frame translated database searches were classified as novel genes when they mapped to intergenic regions of the L. donovani genome lacking any previously annotated protein-coding sequences. Such peptide evidence provides strong support for the existence of unrecognised protein-coding regions within the L. donovani genome. In the amastigote-stage dataset analysed in this study, several GSSPs uniquely aligned to an intergenic region on chromosome 27, offering direct peptide-level validation for a previously unannotated ORF. The complete set of GSSPs, along with their genomic coordinates, is provided in Supplementary Table 3 , and the graphical representation of this novel ORF, together with the corresponding MS/MS spectra of representative GSSPs, is shown in Supplementary Fig. 3 . An illustrative example is the unannotated ORF identified between the genes LdCL_270031900 (encoding a heat shock protein DNAJ) and LdCL_270032100 (encoding an aldo-keto reductase) on chromosome 27 (positive strand). This locus, previously lacking any assigned protein-coding gene, was supported by multiple GSSPs in our proteogenomic analysis. Sequence comparison revealed strong conservation with an orthologous protein from L. tarentolae (LtaPh_3029641), annotated as glycosomal glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The peptide evidence mapped directly to regions conserved across species, confirming active translation of this ORF during the amastigote stage. These findings indicate that this genomic region encodes a functional, previously unannotated glycosomal GAPDH in L. donovani . GAPDH is a glycolytic enzyme with critical roles in glycosomal metabolism, energy production, and redox balance, processes essential for the survival of parasites in the intracellular environment. The identification of peptide-supported translation within this intergenic region demonstrates the presence of a protein-coding gene that was missed in existing genome annotations. Figure 4 illustrates the genomic context of this newly identified gene, the alignment with orthologs from other Leishmania species, and the representative MS/MS spectrum validating one of the supporting peptides. The discovery of such novel genes underscores the value of proteogenomics in refining L. donovani genome annotation and in revealing cryptic protein-coding potential that may contribute to parasite adaptation and pathogenicity. 3.2.2. Identification of N-terminal extension in the amastigote life stages of L. donovani genome: In addition to identifying novel genes, our proteogenomic analysis enabled refinement of existing gene models in L. donovani . Several GSSPs mapped upstream of annotated start codons, providing direct peptide-level evidence for N-terminal extensions that were not captured in the current genome annotation. These findings reveal that multiple L. donovani proteins are longer than presently annotated. The full list of GSSPs supporting N-terminal extensions, along with their genomic coordinates and peptide sequences, is provided in Supplementary Table 4 , and detailed visual representations, including MS/MS spectra, are shown in Supplementary Fig. 4 . A representative example involves the exosome complex exonuclease RRP40, an essential component of the RNA exosome responsible for RNA processing and degradation. In the L. donovani genome, the currently annotated gene for this protein is LdCL_040006400, located on chromosome 4 (positive strand), which begins downstream with the sequence MTQPGAVVAIGGGLRLLAQPAPTPTADG . However, our proteogenomic analysis identified multiple high-confidence GSSPs, most notably the peptide “GITELAPLR” that mapped to an upstream region encoded by LdCL_040006300, a locus previously annotated as hypothetical. These peptides provide explicit evidence that translation initiates upstream of the LdCL_040006400 annotation. The upstream region, beginning with MSTVSTSSSPSRGITELAPLRGHVCLPGEPV , shows strong conservation with the orthologous RRP40 protein from L. mexicana (LmxM.04.0120), confirming that this N-terminal segment is genuine and evolutionarily preserved. Inclusion of this extended region increases the length of the RRP40 protein by 32 amino acids, correcting the truncated annotation and producing a more accurate gene model for this essential exonuclease. The example has been represented in Fig. 5 . This extended N-terminal region is enriched in serine and charged residues, features commonly associated with regulatory functions, protein-protein interactions, RNA substrate recognition, and post-translational modification sites. Given the central role of the exosome in RNA metabolism and quality control, accurate annotation of RRP40 is critical for understanding RNA regulatory mechanisms in L. donovani . This example underscores the power of proteogenomics in resolving incomplete gene models and refining the functional annotation of essential components of the parasite’s RNA processing machinery. 4. Discussion The life cycle of L. donovani involves a transition between two highly distinct stages: the sand fly midgut, where the parasite exists as a motile extracellular promastigote, and the mammalian macrophage phagolysosome, where it survives as an intracellular amastigote. These developmental transitions are accompanied by extensive biochemical, metabolic, and structural reprogramming, driven predominantly by post-transcriptional regulation rather than transcriptional control (Gossage et al., 2003 ; Sadlova et al., 2017 ). Although a well-established reference genome for L. donovani exists (Downing et al., 2011 ). Persistent limitations, including mis-predicted start codons, incomplete ORFs, and hypothetically annotated genes, continue to hamper a clear understanding of parasite biology. Such inaccuracies are expected in kinetoplastids, where polycistronic transcription, aneuploidy, and widespread gene dosage variation complicate genome annotation (Cosentino et al., 2021 ). Proteogenomics provides direct peptide-level evidence to address these challenges, refining gene models and revealing previously unannotated coding regions (Nirujogi et al., 2014 ; Pawar et al., 2012 ; Shenoy and Chowdhury et al., 2025 ). In this study, stage-specific proteogenomic mapping in L. donovani identified 50 high-confidence GSSPs, including 13 promastigote-specific, 24 amastigote-specific, and 13 peptides common to both stages for the N-terminal extensions. This distribution strongly reflects the divergent physiological environments in which the two forms of Leishmania reside. Promastigotes, living in the nutrient-variable and physically dynamic sand fly gut, rely on pathways that support motility, flagellar assembly, membrane trafficking, RNA processing, and environmental sensing (Alcolea et al., 2019 ). In contrast, amastigotes face oxidative bursts, acidic pH, nutrient restriction, and host immune pressure inside macrophages and therefore upregulate stress-adaptation mechanisms, redox regulation, and metabolic reconfiguration for long-term persistence (Pham et al., 2005 ; Van Assche et al., 2011 ). The stage specificity of peptide-supported proteins discovered here is fully consistent with these well-defined biological adaptations. One of the major insights from this proteogenomic analysis is the discovery of several novel protein-coding genes supported by unique peptides that map to previously unannotated or intergenic genomic regions. These novel ORFs were detected in both life stages. Promastigote-specific novel genes are likely involved in mitochondrial RNA editing, flagellar homeostasis, protein transport, and regulatory pathways necessary for vector colonisation. These observations are in line with previous studies showing that promastigotes maintain highly active RNA metabolism and surface remodelling machinery to support growth and attachment within the sand fly (Clos et al., 2022 ; Sinha et al., 2018 ). In contrast, the amastigote-specific novel genes mapped to regions encoding proteins implicated in oxidative stress handling, pH adaptation, iron/thiol metabolism, and intracellular survival, pathways essential for coping with the hostile environment of the phagolysosome (Requena et al., 2015 ). Several novel amastigote proteins showed strong conservation with orthologs in L. infantum and L. major , suggesting they are not random translation products but correspond to bona fide coding regions missed during automated genome annotation. Similar outcomes have been documented in proteogenomic studies of L. braziliensis and L. major , where novel ORFs redefined the coding capacity of kinetoplastid genomes (Nirujogi et al., 2014 ; Pawar et al., 2014 ; Shenoy and Chowdhury et al., 2025 ). A second significant outcome of this study is the identification of 50 N-terminal extensions, revealing widespread inaccuracies in start-codon predictions in the current L. donovani genome. Many extensions restore essential regions that were missing in the original annotations. For instance, promastigote-derived N-terminal peptides corrected the structures of guide RNA-binding proteins and RNA helicases, both core components of the mitochondrial RNA editing machinery unique to kinetoplastids (Neboháčová et al., 2009 ). The identification of N-terminal extensions in SEC23, a COPII vesicle trafficking protein, suggests that critical targeting or regulatory sequences were absent in the reference genome. Given the importance of ER-to-Golgi transport for promastigote surface remodelling and secretion, restoring these N-terminal regions enhances functional interpretation (Kim et al., 2021 ). The discovery of long, highly conserved extensions in these proteins reinforces the notion that kinetoplastid genomes frequently misannotate start sites due to their compact, intron-poor structure and support on trans-splicing rather than promoter-driven transcription (Trinidad-Barnech et al., 2025 ). Amastigote-stage N-terminal extensions provide equally compelling biological insights. Peptides detected uniquely in amastigotes extended the coding regions of proteins associated with redox homeostasis, chaperone activity, temperature adaptation, antioxidant metabolism, and intracellular survival pathways. Amastigotes must neutralise reactive oxygen and nitrogen species generated by host macrophages, and our peptide evidence identifies extensions that restore predicted targeting motifs, mitochondrial transit peptides, and regulatory domains that are essential for these functions (Adán-Jiménez et al., 2024 ; Henard et al., 2014 ). Restoring these regions in redox-related proteins aligns with transcriptomic and proteomic datasets, highlighting thiol-based detoxification and metabolic reprogramming in amastigote biology (Requena et al., 2015 ). Together, these N-terminal extensions reveal that many amastigote proteins were previously truncated due to incorrect start-site annotation, and that peptide validation is crucial for reconstructing complete protein structures. The 13 peptides shared across both stages corresponded primarily to essential housekeeping functions, including RNA-binding proteins, exosomal machinery, and core metabolic enzymes indispensable throughout the parasite life cycle. These proteins are required for fundamental processes irrespective of environmental context, which explains their stable expression in both promastigotes and amastigotes (Krobitsch et al., 1998 ). The presence of shared peptides also validates the robustness of the proteogenomic workflow used here. Taken together, the identification of novel genes, N-terminal extensions, and stage-specific proteins underscores that current L. donovani genome annotations remain incomplete, despite the availability of a high-quality reference genome. Misannotations in kinetoplastid genomes are pervasive due to the absence of conventional promoter structures and their reliance on trans-splicing and post-transcriptional control (Kostygov et al., 2024 ). By providing direct peptide evidence, our study refines gene boundaries, restores missing regulatory and targeting signals, and uncovers previously unrecognised coding regions. Similar to our preceding L. braziliensis proteogenomic study, the frequency and functional importance of N-terminal extensions seen here emphasise systemic start-site inaccuracies in Leishmania genomes. These proteogenomically validated corrections enable more precise interpretation of parasite biology, particularly regarding stage-specific survival strategies, virulence-associated pathways, and host-parasite interactions. Ultimately, the refined annotations generated in this study offer a strong resource for future genetic, biochemical, and therapeutic investigations into visceral leishmaniasis. 5. Conclusion This study presents a comprehensive, stage-specific proteogenomic reannotation of the L. donovani genome, providing experimental evidence that markedly improves the accuracy and completeness of existing gene models. By integrating high-resolution MS/MS data with searches against a six-frame translated genome, we detected 50 genome GSSPs in the two stages that supported N-terminal extensions, in addition to evidence for 15 previously unannotated protein-coding regions. These GSSPs enabled the discovery of several novel protein-coding genes and revealed numerous N-terminal extensions, correcting misassigned start codons and completing truncated ORFs. Such peptide-supported refinements resolve long-standing gaps in genome annotation and explore proteins involved in RNA metabolism, vesicular trafficking, stress adaptation, redox regulation, drug resistance, and intracellular survival, pathways fundamental to the pathogenicity of visceral leishmaniasis. The improvements generated here complement existing genomic and transcriptomic datasets, providing a high-confidence gene set that enhances downstream functional, evolutionary, and translational studies. By revealing hidden coding potential and refining structural boundaries, this work strengthens the foundation for understanding stage-specific biology in L. donovani , including the contrasting adaptations required for promastigote survival in the sand fly vector and amastigote persistence within mammalian macrophages. Although limited to two major life stages, these findings highlight the value of peptide-based evidence in correcting kinetoplastid genome annotations and underscore the need for broader lifecycle proteomic coverage to uncover additional regulatory layers. Overall, this work demonstrates that even high-quality kinetoplastid genomes without any sequencing errors (gaps) can have issues with genome annotations and require experimental validation to reveal their full coding site. By providing peptide-supported corrections across both life stages, our study offers an improved and functionally relevant gene set for L. donovani , enabling more accurate analyses of virulence factors, host-parasite interactions, stage differentiation mechanisms, and potential therapeutic targets. The refined annotations and stage-specific insights generated here serve as an important resource for future genomic, proteomic, and functional studies aimed at advancing our understanding of visceral leishmaniasis and supporting the development of novel interventions. Declarations Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements We would like to acknowledge the infrastructure support provided by the Institute of Bioinformatics for undertaking this study and analysis. Funding source No funding. 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Supplementary Files Supplementaryfigure1NovelPromastigote.pdf Supplementaryfigure2NterminalPromastigote.pdf Supplementaryfigure3NovelAmastigote.pdf Supplementaryfigure4NterminalAmastigote.pdf SupplementaryFigure5.pptx SupplementaryTable1PromastigoteNovelgenes.xlsx SupplementaryTable2PromastigoteNterminalextensions.xlsx SupplementaryTable3AmastigoteNovelgene.xlsx SupplementaryTable4AmastigoteNterminalextension.xlsx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 26 Mar, 2026 Reviews received at journal 19 Mar, 2026 Reviews received at journal 18 Mar, 2026 Reviews received at journal 12 Mar, 2026 Reviewers agreed at journal 05 Mar, 2026 Reviewers agreed at journal 03 Mar, 2026 Reviewers agreed at journal 03 Mar, 2026 Reviews received at journal 24 Feb, 2026 Reviewers agreed at journal 09 Feb, 2026 Reviewers invited by journal 09 Feb, 2026 Editor assigned by journal 03 Feb, 2026 Submission checks completed at journal 30 Jan, 2026 First submitted to journal 28 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8719077","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":589042685,"identity":"7f307b80-18ee-458c-898b-819ee866471a","order_by":0,"name":"Soumi Chowdhury","email":"","orcid":"","institution":"Institute of Bioinformatics","correspondingAuthor":false,"prefix":"","firstName":"Soumi","middleName":"","lastName":"Chowdhury","suffix":""},{"id":589042686,"identity":"4af82d29-c0af-4049-8e41-795846dfe50c","order_by":1,"name":"Shubhankar Pawar","email":"","orcid":"","institution":"Institute of Bioinformatics","correspondingAuthor":false,"prefix":"","firstName":"Shubhankar","middleName":"","lastName":"Pawar","suffix":""},{"id":589042687,"identity":"d02ed0a3-d585-42b0-9698-7abcf0fef221","order_by":2,"name":"Karthick Vasudevan","email":"","orcid":"","institution":"Institute of Bioinformatics","correspondingAuthor":false,"prefix":"","firstName":"Karthick","middleName":"","lastName":"Vasudevan","suffix":""},{"id":589042688,"identity":"1a0433c8-ae23-423a-976a-82d7dda3283e","order_by":3,"name":"Nalini Mishra","email":"","orcid":"","institution":"Regional Medical Research Centre","correspondingAuthor":false,"prefix":"","firstName":"Nalini","middleName":"","lastName":"Mishra","suffix":""},{"id":589042689,"identity":"7f067c5c-e259-4de7-9151-8e6184c1e38b","order_by":4,"name":"Mahendra Jamdhade","email":"","orcid":"","institution":"Translational Health Science and Technology Institute","correspondingAuthor":false,"prefix":"","firstName":"Mahendra","middleName":"","lastName":"Jamdhade","suffix":""},{"id":589042690,"identity":"97b70794-3438-48de-9625-12cb3cf971ea","order_by":5,"name":"Harsh Pawar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABC0lEQVRIiWNgGAWjYBACAyBmZqiQSGBjYGxgqACLARmEtZyBajkDEmIjRgtjG0MCmAfRQsBh5uxnH38unGeRx8fe3PbgQMW9aP75zY0ffzDY5Ms7YNdi2ZNuJj1zm0QxG8/BdoMDZ4pzZxxjbJbmYUiz3HgAh8MOpLEx826TSGwDIumPbQm5DccY25gZGA4bGOLwksH5Z8yfeedAtEgcBGqZD9TC+AOflhtpDNK8DUhaNgC1MPAAtcjj8L7BjWds0jzHwH5pkzhwJiF347FEoF8M0gwMcGk5n8b8maemLk++vf2ZxIGKhNx5h48//PijwsZAHofDcAEDUMiQpgUISLVlFIyCUTAKhi0AAAIjW1cCKdigAAAAAElFTkSuQmCC","orcid":"","institution":"Institute of Bioinformatics","correspondingAuthor":true,"prefix":"","firstName":"Harsh","middleName":"","lastName":"Pawar","suffix":""}],"badges":[],"createdAt":"2026-01-28 10:11:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8719077/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8719077/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102747532,"identity":"f43c54a4-050d-4041-b62f-0681e26b0f2a","added_by":"auto","created_at":"2026-02-16 09:04:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":231571,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProteogenomic workflow for the identification of life-stage-specific peptides and novel ORFs in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eL. donovani\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8719077/v1/cc193dcede76faccc3714967.png"},{"id":102748015,"identity":"26a81253-83e1-4a32-b755-7fd0dd424dd5","added_by":"auto","created_at":"2026-02-16 09:05:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":510468,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Genomic region on chromosome 32 (positive strand) of \u003cem\u003eL. donovani\u003c/em\u003e CL-SL showing the current annotated genes LdCL_30037500 (hypothetical protein) and LdCL_320037600 (Rab5-interacting protein, Rab5ip) represented as red boxes. Proteogenomic mapping of promastigote-stage mass spectrometry data identified multiple peptides mapping to the intergenic region between these annotated genes. Gene prediction analysis revealed a novel open reading frame (ORF) (magenta box) supported by peptide evidence, predicted to encode cytochrome c oxidase subunit VIb. Genomic coordinates are indicated along the chromosome, and peptide-mapped regions are highlighted beneath the ORF.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Pairwise sequence alignment of the predicted novel protein with its corresponding sequence (LTRI590_320038000). Peptides identified by mass spectrometry are highlighted in red, providing direct translational evidence for the predicted ORF. Conserved amino acid residues are indicated by asterisks, demonstrating high sequence identity and supporting the validity of the newly identified protein-coding gene.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e Representative MS/MS spectrum of the peptide \u003cstrong\u003eCLDTLPEDPEKECAAQK\u003c/strong\u003e, identified from promastigote-stage proteomic data. The annotated b- and y-ion series confirm the peptide sequence, providing experimental mass spectrometric evidence for translation of the novel ORF.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Genomic region on chromosome 04 (negative strand) of \u003cem\u003eL. donovani\u003c/em\u003e CL-SL showing the currently annotated genes LdCL_040017800 (leucine-rich repeat protein) and LdCL_040017900 (casein kinase I), represented as red boxes. Proteogenomic mapping of promastigote-stage mass spectrometry data revealed peptide evidence mapping to the intergenic region between these annotated genes. Gene prediction analysis identified a novel open reading frame (ORF) (magenta box) supported by peptide evidence, corresponding to a previously unannotated hypothetical protein. Genomic coordinates are indicated along the chromosome, and peptide-mapped regions are shown beneath the ORF.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Pairwise sequence alignment of the predicted novel protein with its corresponding sequence (LGELEM452_040017600). Peptides identified by mass spectrometry are highlighted in red, providing translational evidence for the predicted ORF. Conserved amino acid residues are indicated by asterisks, demonstrating strong sequence conservation and supporting the validity of the newly identified protein-coding gene.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e Representative MS/MS spectrum of the peptide \u003cstrong\u003eEPMGHEETTFSR\u003c/strong\u003e, identified from promastigote-stage proteomic data. The annotated b- and y-ion series confirm the peptide sequence, providing direct experimental evidence for translation of the novel ORF.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8719077/v1/064f4ffecc45ea1db72e064f.png"},{"id":102747780,"identity":"0d7422ca-47b3-4f20-8743-384e12b16770","added_by":"auto","created_at":"2026-02-16 09:05:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":975701,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Genomic region on chromosome 18 (negative strand) of \u003cem\u003eL. donovani\u003c/em\u003e CL-SL showing the currently annotated genes LdCL_180008300 (DUF4475), LdCL_180008400 (ATP12 chaperone), and LdCL_180008500 (GPI-anchor transamidase subunit 8, GPI8), represented as red and grey boxes. Proteogenomic mapping of promastigote-stage mass spectrometry data revealed peptide evidence upstream of the annotated ATP12 chaperone gene, indicating the presence of an N-terminal extension. Gene prediction analysis supported an extended open reading frame (ORF) (magenta box) incorporating this upstream region. Genomic coordinates are indicated along the chromosome, and peptide-mapped regions are shown beneath the extended ORF.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Pairwise sequence alignment of the extended ATP12 chaperone protein (N-terminal extension 9) with its corresponding orthologous sequence (LGELEM452_180008600). Peptides identified by mass spectrometry are highlighted in red, providing direct translational evidence for the N-terminal extension. Conserved amino acid residues are indicated by asterisks, demonstrating strong sequence conservation across the full-length protein and supporting the revised gene model.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e Representative MS/MS spectrum of the peptide \u003cstrong\u003eNVPEPGAHAELSTAELER\u003c/strong\u003e, identified from promastigote-stage proteomic data. The annotated b- and y-ion series confirm the peptide sequence, providing experimental mass spectrometric evidence for translation of the extended N-terminal region of the ATP12 chaperone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Genomic region on chromosome 08 (positive strand) of \u003cem\u003eL. donovani\u003c/em\u003e CL-SL showing the currently annotated genes LdCL_080016300 (thiopurine S-methyltransferase, TPMT), LdCL_080016400 (guide RNA binding protein), and LdCL_080016500 (hypothetical protein), represented as red and grey boxes. Proteogenomic mapping of promastigote-stage mass spectrometry data revealed peptide evidence upstream of the annotated guide RNA binding protein gene, indicating the presence of an N-terminal extension. Gene prediction analysis supported an extended open reading frame (ORF) (magenta box) incorporating this upstream region. Genomic coordinates are indicated along the chromosome, and peptide-mapped regions are shown beneath the extended ORF.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Pairwise sequence alignment of the extended guide RNA binding protein (N-terminal extension 1) with its corresponding orthologous sequence (LTRI590_080015100). Peptides identified by mass spectrometry are highlighted in red, providing direct translational evidence for the N-terminal extension. Conserved amino acid residues are indicated by asterisks, demonstrating strong sequence conservation across the full-length protein and supporting the revised gene model.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e Representative MS/MS spectrum of the peptide HLSPGELALPQHPR, identified from promastigote-stage proteomic data. The annotated b- and y-ion series confirm the peptide sequence, providing experimental mass spectrometric evidence for translation of the extended N-terminal region of the guide RNA binding protein.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8719077/v1/ccb446692708b67a106ae821.png"},{"id":102620595,"identity":"aa9f1be8-0e17-4c35-89d5-2da359778d16","added_by":"auto","created_at":"2026-02-13 16:43:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":412580,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of a novel glycosomal glyceraldehyde-3-phosphate dehydrogenase supported by peptide evidence in the amastigote stage of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eL. donovani\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Genomic region on chromosome 27 (positive strand) of \u003cem\u003eL. donovani\u003c/em\u003e CL-SL showing the currently annotated genes LdCL_270031900 (heat shock protein DNAJ) and LdCL_270032000 (aldo-keto reductase), represented as red boxes. Proteogenomic mapping of amastigote-stage mass spectrometry data revealed peptide evidence mapping to the intergenic region between these annotated genes. Gene prediction analysis identified a novel open reading frame (ORF) (magenta box) supported by peptide evidence, predicted to encode a glycosomal glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Genomic coordinates are indicated along the chromosome, and peptide-mapped regions are shown beneath the ORF.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Pairwise sequence alignment of the predicted novel protein (Novel_5) with its corresponding orthologous sequence (LtaPh_3029641). Peptides identified by mass spectrometry are highlighted in red, providing translational evidence for the predicted ORF. Conserved amino acid residues are indicated by asterisks, demonstrating significant sequence conservation and supporting the functional annotation of the newly identified glycosomal enzyme.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e Representative MS/MS spectrum of the peptide \u003cstrong\u003eATLDVALGLQLVR\u003c/strong\u003e, identified from amastigote-stage proteomic data. The annotated b- and y-ion series confirm the peptide sequence, providing experimental mass spectrometric evidence for translation of the novel ORF.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8719077/v1/6b591134018fcaf874b8e20e.png"},{"id":102620601,"identity":"f254f237-0b4b-4a4b-96aa-c7b65e0fc818","added_by":"auto","created_at":"2026-02-13 16:43:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":426960,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of an amastigote-stage-specific N-terminal extension of the exosome complex exonuclease RRP40 supported by peptide evidence in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eL. donovani\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Genomic region on chromosome 04 (positive strand) of \u003cem\u003eL. donovani\u003c/em\u003e CL-SL showing the currently annotated genes LdCL_040006300 and LdCL_040006500 (both annotated as hypothetical proteins) flanking the annotated gene LdCL_040006400, encoding the exosome complex exonuclease RRP40, represented as red and grey boxes. Proteogenomic mapping of amastigote-stage mass spectrometry data revealed peptide evidence upstream of the annotated RRP40 gene, indicating the presence of an N-terminal extension. Gene prediction analysis supported an extended open reading frame (ORF) (magenta box) incorporating this upstream region. Genomic coordinates are indicated along the chromosome, and peptide-mapped regions are shown beneath the extended ORF.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Pairwise sequence alignment of the extended RRP40 protein (N-terminal extension 2) with its corresponding orthologous sequence (LmxM.04.0120). Peptides identified by mass spectrometry are highlighted in red, providing direct translational evidence for the N-terminal extension. Conserved amino acid residues are indicated by asterisks, demonstrating strong sequence conservation across the full-length protein and supporting the revised gene model.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e Representative MS/MS spectrum of the peptide \u003cstrong\u003eGITELAPLR\u003c/strong\u003e, identified from amastigote-stage proteomic data. The annotated b- and y-ion series confirm the peptide sequence, providing experimental mass spectrometric evidence for translation of the extended N-terminal region of the exosome complex exonuclease RRP40.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8719077/v1/739cc08297b15c61538fcaed.png"},{"id":102750939,"identity":"94952075-ed50-4643-97a2-afa4d450e843","added_by":"auto","created_at":"2026-02-16 09:22:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3337111,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8719077/v1/e9385676-15de-497a-b156-af0bca870b6b.pdf"},{"id":102620597,"identity":"cdb22589-f919-4aa2-910d-a917996c6df2","added_by":"auto","created_at":"2026-02-13 16:43:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":606440,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigure1NovelPromastigote.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8719077/v1/65f4984d5317afcf30ebfd64.pdf"},{"id":102620593,"identity":"672f9b67-7326-418c-9de6-0a521228a094","added_by":"auto","created_at":"2026-02-13 16:43:44","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2965956,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigure2NterminalPromastigote.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8719077/v1/0e950c5e2da824f488d03e33.pdf"},{"id":102747386,"identity":"b70656cb-b471-4292-bd58-329debe7fca1","added_by":"auto","created_at":"2026-02-16 09:04:41","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":814011,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigure3NovelAmastigote.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8719077/v1/a0031862d3dd58373d052106.pdf"},{"id":102620603,"identity":"9ae90954-cefe-40db-a26d-126b619cfff9","added_by":"auto","created_at":"2026-02-13 16:43:45","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":5014392,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigure4NterminalAmastigote.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8719077/v1/be61291a5d54735788f5b282.pdf"},{"id":102620604,"identity":"973b552d-c06b-4429-8bc2-1af5d50bbaf6","added_by":"auto","created_at":"2026-02-13 16:43:45","extension":"pptx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":230821,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure5.pptx","url":"https://assets-eu.researchsquare.com/files/rs-8719077/v1/0461aeac7ebd4718fe15929c.pptx"},{"id":102620598,"identity":"89dd5c8e-bbcd-4655-ae99-07733e1ca7ba","added_by":"auto","created_at":"2026-02-13 16:43:44","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":11663,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1PromastigoteNovelgenes.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8719077/v1/a1b664c7a05840fec6f07168.xlsx"},{"id":102620602,"identity":"6fbc6ae2-d7cd-49f6-8508-acba3c4cbec4","added_by":"auto","created_at":"2026-02-13 16:43:45","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":16512,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2PromastigoteNterminalextensions.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8719077/v1/1b779542129cfdd9df0fc865.xlsx"},{"id":102620600,"identity":"0fc9a4c1-6e76-46e9-b84b-a593bfd06e9d","added_by":"auto","created_at":"2026-02-13 16:43:44","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":12079,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable3AmastigoteNovelgene.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8719077/v1/44fa8b8804315094ec93b031.xlsx"},{"id":102748145,"identity":"cf311f53-2a08-4f04-8f3e-ec842515638b","added_by":"auto","created_at":"2026-02-16 09:06:06","extension":"xlsx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":20016,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable4AmastigoteNterminalextension.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8719077/v1/874954161d003829bace7785.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Identification of new protein-coding potential in Leishmania donovani using a proteogenomics approach","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cem\u003eLeishmania donovani\u003c/em\u003e is a digenetic protozoan parasite belonging to the family Trypanosomatidae and is the principal causative agent of visceral leishmaniasis (VL), the most severe and often fatal form of leishmaniasis (Lukeš et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Endemic across the Indian subcontinent, East Africa, and parts of the Mediterranean basin, VL is characterised by chronic fever, hepatosplenomegaly, severe anaemia, and progressive wasting, contributing to high mortality in untreated cases (Arenas et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Transmission occurs through the bite of infected female \u003cem\u003ePhlebotomus\u003c/em\u003e sand flies, primarily \u003cem\u003eP. argentipes\u003c/em\u003e and \u003cem\u003eP. orientalis\u003c/em\u003e, which inoculate non-dividing metacyclic promastigotes into the host dermis. Once phagocytosed by macrophages, the parasite differentiates into the amastigote form and replicates within the hostile, acidic phagolysosomal niche (Bates, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eL. donovani\u003c/em\u003e strain BPK282A1 represents the primary reference genome for this pathogen and has played a central role in shaping current genomic understanding of visceral leishmaniasis. The genome was first released in 2011 (assembly GCA_000227135.1), providing a foundational but incomplete draft that contained several gaps and unresolved regions (Downing et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Subsequent refinement using improved sequencing depth and assembly methods generated the updated version ASM22713v2/GCF_000227135.2, which significantly enhanced contiguity and accuracy by resolving gap regions and correcting structural inconsistencies (Iantorno et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe reference genome of \u003cem\u003eL. donovani\u003c/em\u003e strain BPK282A1 comprises approximately 32.44 Mb of sequence organised into 36 chromosomes and contains a total of 8,135 annotated genes. Among these, 7,969 are predicted to encode proteins, 166 correspond to non-protein-coding genes, and 54 are classified as pseudogenes. Comparative genomics indicates that \u003cem\u003eL. donovani\u003c/em\u003e, \u003cem\u003eL. infantum\u003c/em\u003e, and \u003cem\u003eL. major\u003c/em\u003e share extensive synteny and high (\u0026gt;\u0026thinsp;99%) conservation of gene content, with only a small set of species-specific genes differentiating them (Freitas-Mesquita et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Rochette et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Tschoeke et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). However, genome organisation within the \u003cem\u003eLeishmania\u003c/em\u003e genus exhibits structural plasticity, including chromosomal fusion events in New World species such as \u003cem\u003eL. braziliensis\u003c/em\u003e and \u003cem\u003eL. mexicana\u003c/em\u003e, which harbour 35 and 34 chromosomes, respectively (Kazemi, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). \u003cem\u003eL. donovani\u003c/em\u003e exhibits complex genomic features such as gene dosage variations, frequent aneuploidy, polycistronic transcription, and post-transcriptional regulation, which collectively shape its developmental transitions and pathogenicity (Franssen et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite the availability of its reference genome, a substantial portion of \u003cem\u003eL. donovani\u003c/em\u003e genes remains annotated as \u0026ldquo;hypothetical,\u0026rdquo; lacking functional characterisation or experimental validation. Initial genome annotation also identified numerous \u0026ldquo;partial\u0026rdquo; or truncated gene models, many of which required structural correction (Downing et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Jamdhade and Pawar et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Comparative analysis of related kinetoplastids, particularly \u003cem\u003eL. infantum\u003c/em\u003e and \u003cem\u003eL. major\u003c/em\u003e, enabled refinement of these partial annotations, culminating in the correction of over 300 gene models, including C-terminal extensions, merging of fragmented genes, tandem paralog resolution, and rectification of incorrect chromosomal assignments (Jamdhade and Pawar et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Trinidad-Barnech et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This highlights the persistent need for experimental evidence to validate predicted gene structures.\u003c/p\u003e \u003cp\u003eProteomic and proteogenomic analyses have played a crucial role in addressing these annotation gaps. Early proteome mapping of \u003cem\u003eL. donovani\u003c/em\u003e (prior to genome availability) relied on similarity-based searches using genomes of \u003cem\u003eL. infantum\u003c/em\u003e, \u003cem\u003eL. major\u003c/em\u003e, and \u003cem\u003eL. braziliensis\u003c/em\u003e, leading to the identification of more than 22,000 unique peptides and 3,711 putative proteins across promastigote and amastigote stages (Pawar et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Following the release of the genome sequence, reanalysis of the dataset enabled direct mapping to the \u003cem\u003eL. donovani\u003c/em\u003e proteome, identifying 3,999 proteins (~\u0026thinsp;50% of the predicted proteome) and providing high-quality peptide evidence for over 2,200 proteins expressed in both life stages (Nirujogi et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Importantly, genome search-specific peptides (GSSPs) revealed 20 novel protein-coding genes and enabled correction of 40 existing annotations, including N-terminal and C-terminal extensions.\u003c/p\u003e \u003cp\u003eBeyond proteome mapping, genomic variation contributes significantly to \u003cem\u003eL. donovani\u003c/em\u003e biology. Comparative genomic analysis of atypical Sri Lankan \u003cem\u003eL. donovani\u003c/em\u003e strains causing cutaneous (CL-SL) versus visceral (VL-SL) disease revealed that differences in virulence correlate with single-nucleotide polymorphisms, gene copy number variations, and modulation of key virulence-associated gene families such as the A2 genes, rather than gross gene deletions or pseudogene formation (McCall \u0026amp; Matlashewski, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Samarasinghe et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). These findings emphasise the functional impact of subtle genomic alterations on tissue tropism and clinical outcomes. Proteogenomics provides an integrated framework to refine gene predictions, uncover novel coding regions, validate translational start/stop sites, detect frameshifts, characterise pseudogenes, and reveal conserved or lineage-specific protein families of biological significance. Applying such approaches to \u003cem\u003eL. donovani\u003c/em\u003e is essential for strengthening its genome annotation, especially given the high proportion of hypothetical proteins and the unique features of \u003cem\u003eLeishmania\u003c/em\u003e gene expression that complicate classical computational predictions.\u003c/p\u003e \u003cp\u003eOur recent study demonstrated the strength of integrating high-resolution mass spectrometry data with genome-wide six-frame translation to refine parasite gene models. Using a comprehensive proteogenomic pipeline, we identified multiple GSSPs that provided direct experimental evidence for previously unannotated open reading frames (ORFs), corrections to existing gene boundaries, and validation of hypothetical proteins in \u003cem\u003eL. braziliensis\u003c/em\u003e (Shenoy and Chowdhury et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This work highlighted the substantial coding potential that remains uncharacterized in kinetoplastid genomes and underscored how peptide-level evidence can resolve fragmented annotations, extend gene termini, and uncover novel protein-coding loci. The success of this strategy in \u003cem\u003eL. braziliensis\u003c/em\u003e provided a strong rationale for applying a similar proteogenomics framework to \u003cem\u003eL. donovani\u003c/em\u003e, a species with a larger disease burden and a high proportion of hypothetical or partially annotated genes. On the other hand, in our recent proteogenomic study on \u003cem\u003eLeishmania guyanensis\u003c/em\u003e, we refined the genome annotation by integrating mass spectrometry data with a six-frame translated genome database. This approach identified 653 GSSPs, leading to the discovery of 65 novel protein-coding genes and the correction of 62 existing gene models, including N- and C-terminal extensions. These findings underscore the value of proteogenomics in uncovering cryptic coding regions and improving the accuracy of \u003cem\u003eL. guyanensis\u003c/em\u003e genome annotations (Pawar et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2026\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the present study, we conducted a comprehensive proteogenomic analysis of \u003cem\u003eL. donovani\u003c/em\u003e Cutaneous \u003cem\u003eLeishmania\u003c/em\u003e (CL) from Sri Lanka (SL) using publicly available high-resolution mass spectrometry datasets and a custom-built six-frame translated genome database. Our analysis identified numerous genome search-specific peptides, enabling the discovery of previously unannotated protein-coding regions and refinement of existing gene models. By integrating peptide-level evidence with updated genomic resources, we provide an enhanced annotation of the \u003cem\u003eL. donovani\u003c/em\u003e genome, contributing to improved understanding of parasite biology, host-pathogen interactions, tissue tropism, and potential therapeutic targets.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Generation of protein database and mass spectrometry data analysis\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eL. donovani\u003c/em\u003e CL_SL whole genome sequence was downloaded from TriTrypDB, and the corresponding whole-genome FASTA file was used as the reference for proteogenomic analysis (Zhang et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Using in-house Python scripts, we generated a six-frame translated database from the complete genome, with a minimum translated ORF length cutoff of 10 amino acids. This unbiased six-frame translation approach followed previously published proteogenomic workflows (Nirujogi et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Pawar et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Shenoy and Chowdhury et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). For peptide identification, we used our previously published \u003cem\u003eL. donovani\u003c/em\u003e proteomic dataset (Nirujogi and Pawar et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), which was re-analysed against the newly constructed six-frame translated database to identify genome search-specific peptides and refine gene annotations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 \u003cem\u003eProteogenomic analysis\u003c/em\u003e:\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eL. donovani\u003c/em\u003e promastigote and amastigote proteomic dataset (Nirujogi and Pawar et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) was searched against the custom-built six-frame translated \u003cem\u003eL. donovani\u003c/em\u003e genome database generated in this study. Database-dependent searches were performed using the Sequest HT search engine, implemented in Proteome Discoverer (version 2.4). Enzymatic digestion parameters were set to trypsin with a maximum allowance of one missed cleavage. Precursor ion mass tolerance was set at 20 ppm, and fragment ion tolerance was set at 0.1 Da. Carbamidomethylation of cysteine residues was specified as a fixed modification, while methionine oxidation and N-terminal acetylation were defined as variable modifications. Peptide-spectrum matches obtained from Sequest HT searches were filtered using a stringent 1% false discovery rate (FDR). High-confidence unique peptides that passed the FDR threshold were retained and used for subsequent proteogenomic analysis, including identification of genome search-specific peptides and the refinement of gene models (Shenoy and Chowdhury et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAll high-confidence unique peptides obtained from database searches were first aligned against the annotated \u003cem\u003eL. donovani\u003c/em\u003e CL_SL protein database downloaded from TriTrypDB. Peptides that matched the annotated proteome were excluded from further analysis. Peptides that failed to map to the annotated protein database but showed a unique match to the six-frame translated \u003cem\u003eL. donovani\u003c/em\u003e genome were classified as GSSPs. In this study, GSSPs are defined as MS-derived peptides that align exclusively to the translated genomic sequence and are absent from existing protein annotations. Such peptides provide direct experimental evidence for previously unrecognised protein-coding loci or highlight potential inaccuracies in current gene models, such as truncated ORFs, misannotated boundaries, or missing exons.\u003c/p\u003e \u003cp\u003eEach GSSP was subsequently mapped back to its genomic coordinates to determine its exact positional context relative to existing annotations. This enabled the identification of novel coding regions, the extension of annotated ORFs, the merging of fragmented gene models, and the correction of erroneous start/stop codons. Only high-confidence GSSPs supported by unambiguous genomic mapping were considered for gene model refinement.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Bioinformatics analysis:\u003c/h2\u003e \u003cp\u003eGSSPs that uniquely matched the six-frame translated \u003cem\u003eL. donovani\u003c/em\u003e genome were subjected to additional validation using BLAST-based searches (BLASTp and tBLASTn). These analyses enabled us to determine the precise genomic loci corresponding to each peptide and to identify conserved orthologous proteins in closely related \u003cem\u003eLeishmania\u003c/em\u003e species (Shenoy and Chowdhury et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Based on this integrative approach, the proteogenomic evidence was classified into two major categories: (i) novel protein-coding genes, representing previously unannotated ORFs not present in the reference \u003cem\u003eL. donovani\u003c/em\u003e annotation, and (ii) refinements to annotated genes, including N-terminal and C-terminal extensions, correction of truncated models, and merging of fragmented ORFs.\u003c/p\u003e \u003cp\u003eTo further assess the functional relevance of these newly identified or corrected proteins, conserved domain architectures were predicted using the SMART database (Letunic \u0026amp; Bork, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The corresponding domain illustrations and structural annotations are provided in \u003cb\u003eSupplementary Figs.\u0026nbsp;5\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Overview of the proteomic data\u003c/h2\u003e \u003cp\u003eTo refine the genome annotation of \u003cem\u003eL. donovani\u003c/em\u003e CL_SL strain, we employed a proteogenomic approach using high-resolution tandem mass spectrometry (MS/MS) datasets generated from the amastigote life stage. These experimental MS/MS spectra were systematically analysed against both the annotated \u003cem\u003eL. donovani\u003c/em\u003e protein database and a six-frame translated genomic database to enable comprehensive peptide discovery. The six-frame translation of the \u003cem\u003eL. donovani\u003c/em\u003e genome yielded 362893 predicted ORFs across all reading frames, providing a broad search space for peptide mapping. Database-dependent searches of the \u003cem\u003eL. donovani\u003c/em\u003e LC-MS/MS datasets, performed under stringent filtering criteria including a 1% false discovery rate threshold, identified 33210 unique peptides. Of these, 32852 peptides mapped to previously annotated proteins in the \u003cem\u003eL. donovani\u003c/em\u003e CL reference genome, thereby supporting existing gene models. The remaining 358 peptides did not map to the annotated protein database but aligned uniquely to the six-frame translated genome; these were classified as GSSPs. All GSSPs were subsequently examined in detail to determine whether they corresponded to novel protein-coding regions, extensions of existing gene models, or corrections to misannotated loci. This detailed proteogenomic analysis uncovered multiple unannotated genomic features, highlighting opportunities to improve and refine the current \u003cem\u003eL. donovani\u003c/em\u003e genome assembly. The full workflow of the proteogenomic strategy used in this study, including peptide identification, six-frame genome mapping, and integration with comparative genomics, is summarised in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Identification of protein-coding genes in the promastigote life stages of the L. donovani genome:\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1. Identification of novel protein-coding genes in the promastigote life stages of L. donovani genome:\u003c/h2\u003e \u003cp\u003eA representative example of a novel gene identified in the promastigote stage is Cytochrome c oxidase subunit VIb, a key component of Complex IV of the mitochondrial electron transport chain. In the current \u003cem\u003eL. donovani\u003c/em\u003e genome annotation, the genomic region corresponding to LdCL_320037500 on chromosome 32 (positive strand) is labelled as a hypothetical protein (Rab5-interacting protein). However, our proteogenomic analysis identified multiple peptides that mapped to an unannotated ORF upstream of this locus, providing strong evidence for the presence of a previously unrecognised protein-coding gene. This ORF exhibited high-confidence peptide support specifically from the promastigote life stage. The example has been represented in Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB.\u003c/p\u003e \u003cp\u003eThe newly identified ORF encodes a protein beginning with the conserved N-terminal sequence MQSAKKRELCYKTRDAFHKCLDTLPEDPEKECAAQKKLFEQSCPKSWVSYFEKQREREVI\u0026hellip;, which shows near-complete identity with the orthologous protein in \u003cem\u003eL. tropica\u003c/em\u003e (LTRL590_320038000). The strong conservation across the full length of the alignment, including the C-terminal motif LQLQVEQYKGR, supports the functional validity of this gene and confirms that it corresponds to cytochrome c oxidase subunit VIb, a mitochondrial protein essential for respiratory chain activity. Because this protein is completely absent from the current \u003cem\u003eL. donovani\u003c/em\u003e annotation, the mapped peptides and ortholog conservation provide definitive evidence that this region contains a bona fide novel mitochondrial gene. The identification of this novel subunit of Complex IV demonstrates the effectiveness of proteogenomic analysis in uncovering hidden components of essential metabolic pathways and correcting incomplete annotations in \u003cem\u003eL. donovani\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAnother representative example of a novel gene identified in the promastigote stage involves a hypothetical protein containing an N-terminal glutamine amidase (Nt_Gln_amidase) structural domain, a family of enzymes implicated in the processing or hydrolysis of N-terminal glutamine residues in diverse organisms. In the current \u003cem\u003eL. donovani\u003c/em\u003e genome annotation, the genomic region corresponding to LdCL_040017800 on chromosome 4 (negative strand) is annotated as a leucine-rich repeat casein kinase I homolog. However, our proteogenomic analysis identified peptides that mapped to an upstream, unannotated ORF, providing strong evidence that this region encodes an independent protein absent from the current annotation.\u003c/p\u003e \u003cp\u003eThe novel ORF begins with the highly conserved N-terminal sequence MFRRCARRLGHQLIKEPMGHEETTFSRGDRTRNNALQHIYGLIGFCGVGCVLALFSFVSG\u0026hellip;, followed by QRRVLTTITADGTITKGTCPTKWWNF, together forming a promastigote-expressed protein with clear orthologous conservation, as shown by alignment with \u003cem\u003eL. gerbilli\u003c/em\u003e (LGELEM452_040017600). The near-identical conservation across the full N-terminal region underscores that this ORF represents a protein rather than a misannotated fragment of the neighbouring kinase gene. Functional annotation revealed the presence of an Nt_Gln_amidase structural domain, a domain characteristic of enzymes that catalyse the removal or modification of N-terminal glutamine or glutamate residues. Such catalytic domains are frequently involved in N-terminal processing, protein maturation, and regulated proteolysis, functions of potential significance in parasite development and adaptation during the promastigote stage. Because this ORF is completely absent from the current annotation of chromosome 4, peptide-level evidence combined with structural domain prediction strongly supports the existence of Novel gene 2, encoding a previously unrecognised Nt_Gln_amidase-family protein in \u003cem\u003eL. donovani\u003c/em\u003e. The discovery of this novel enzymatic protein highlights the importance of proteogenomics in resolving unannotated or misclassified regions of the \u003cem\u003eL. donovani\u003c/em\u003e genome and in uncovering potentially functional metabolic components. The complete set of GSSPs, along with their genomic coordinates, is provided in \u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e, and the graphical representation of this novel ORF, together with the corresponding MS/MS spectra of representative GSSPs, is shown in \u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2. Identification of N-terminal extension in the promastigote life stages of L. donovani genome:\u003c/h2\u003e \u003cp\u003eA representative example of an N-terminal extension identified in \u003cem\u003eL. donovani\u003c/em\u003e involves the ATP12 chaperone, a mitochondrial protein required for the assembly and stabilisation of the F₁ sector of the F₁F₀ ATP synthase complex. In the current genome annotation, the protein is represented by LdCL_180008400, located on chromosome 18 (positive strand), with the annotated coding sequence beginning downstream at MSRMNSKQLEEVMRKFEEQENESSR. However, our proteogenomic analysis uncovered multiple high-confidence GSSPs, including the peptide \u0026ldquo;NVPSEPGAHAELSTAELER\u0026rdquo; (5 PSMs) that mapped to the upstream locus LdCL_180008300, which had been annotated as a protein containing a DUF4475 domain. These peptides provide compelling evidence that translation initiates upstream of the currently annotated start site. The example has been represented in Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eB.\u003c/p\u003e \u003cp\u003eThe upstream region begins with the extended N-terminal sequence MRPFTLAKGAAPRQLLAVAIAAARMSVSSSTPAEPSTPAGASQATAATGPTVAPKKPRRR\u0026hellip;, which is strongly conserved in the orthologous gene from \u003cem\u003eL. gerbilli\u003c/em\u003e (LGELEM452_180008600). The mapped GSSPs confirm active translation of this segment, demonstrating that the existing annotation truncates the true N-terminus of the ATP12 chaperone. Incorporating this upstream region adds additional amino acids to the N-terminus, increasing the full-length protein from the annotated 85 amino acids to an extended length of 180 amino acids, consistent with orthologous ATP12 proteins in related trypanosomatids. The extended N-terminal region contains a stretch of hydrophobic and basic residues characteristic of mitochondrial targeting sequences, suggesting that the annotation correction restores the accurate subcellular targeting information for this essential chaperone. Given that ATP12 is required for the proper folding of the α-subunit of mitochondrial ATP synthase, misannotation of its N-terminus may obscure critical functional domains involved in mitochondrial import and assembly. This example highlights how peptide-supported proteogenomic can correct truncated gene models and generate functionally relevant annotations for essential mitochondrial chaperones in \u003cem\u003eL. donovani\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAnother clear example of an N-terminal extension identified in the promastigote stage involves the Guide RNA (gRNA) binding protein, a key component of the mitochondrial RNA editing machinery in kinetoplastids. In the current \u003cem\u003eL. donovani\u003c/em\u003e genome annotation, this protein is represented by LdCL_080016400, located on chromosome 8 (positive strand). The annotated coding sequence begins downstream at MKKLHQE, which substantially truncates the N-terminal region of the protein. However, our proteogenomic analysis identified multiple high-confidence GSSPs uniquely mapping to the upstream locus LdCL_080016300, which had been annotated as hypothetical. Among these, the peptide \u0026ldquo;HLSPGELALPQHPR\u0026rdquo; provided strong evidence for active translation of an upstream N-terminal segment that is missing from the current annotation.\u003c/p\u003e \u003cp\u003eThis upstream region, beginning with MRGLGVLGLRGVPTRRPWLCDVARRSGDISMCSTASTSYRSQATSAGGTPPLPPPTSSPV, is highly conserved across other \u003cem\u003eLeishmania\u003c/em\u003e species, including \u003cem\u003eL. tropica\u003c/em\u003e (LTRL590_080015100), confirming that this extended N-terminal sequence represents the authentic start of the Guide RNA binding protein. Incorporation of this upstream sequence increases the length of the protein by amino acids, yielding a biologically accurate representation of the protein compared to the currently annotated isoform. The extended N-terminal domain is enriched in arginine, serine, and glycine residues, features that are characteristic of RNA-binding proteins involved in mitochondrial RNA editing and gRNA stabilisation. These residues frequently contribute to RNA interaction surfaces, targeting the kinetoplast and assembly of ribonucleoprotein complexes. Refining the annotation of this Guide RNA binding protein is therefore critical for understanding the organisation and regulation of the RNA editing machinery in \u003cem\u003eL. donovani\u003c/em\u003e. This example demonstrates how proteogenomic evidence corrects truncated gene models and improves annotation of essential RNA-processing proteins during the promastigote stage. The complete set of GSSPs, along with their genomic coordinates, is provided in \u003cb\u003eSupplementary Table\u0026nbsp;2\u003c/b\u003e, and the graphical representation of this novel ORF, together with the corresponding MS/MS spectra of representative GSSPs, is shown in \u003cb\u003eSupplementary Fig.\u0026nbsp;2\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Identification of protein-coding genes in the amastigote life stages of the L. donovani genome:\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1. Identification of novel protein-coding genes in the amastigote life stages of the L. donovani genome:\u003c/h2\u003e \u003cp\u003eGSSPs obtained from six-frame translated database searches were classified as novel genes when they mapped to intergenic regions of the \u003cem\u003eL. donovani\u003c/em\u003e genome lacking any previously annotated protein-coding sequences. Such peptide evidence provides strong support for the existence of unrecognised protein-coding regions within the \u003cem\u003eL. donovani\u003c/em\u003e genome. In the amastigote-stage dataset analysed in this study, several GSSPs uniquely aligned to an intergenic region on chromosome 27, offering direct peptide-level validation for a previously unannotated ORF. The complete set of GSSPs, along with their genomic coordinates, is provided in \u003cb\u003eSupplementary Table\u0026nbsp;3\u003c/b\u003e, and the graphical representation of this novel ORF, together with the corresponding MS/MS spectra of representative GSSPs, is shown in \u003cb\u003eSupplementary Fig.\u0026nbsp;3\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eAn illustrative example is the unannotated ORF identified between the genes LdCL_270031900 (encoding a heat shock protein DNAJ) and LdCL_270032100 (encoding an aldo-keto reductase) on chromosome 27 (positive strand). This locus, previously lacking any assigned protein-coding gene, was supported by multiple GSSPs in our proteogenomic analysis. Sequence comparison revealed strong conservation with an orthologous protein from \u003cem\u003eL. tarentolae\u003c/em\u003e (LtaPh_3029641), annotated as glycosomal glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The peptide evidence mapped directly to regions conserved across species, confirming active translation of this ORF during the amastigote stage. These findings indicate that this genomic region encodes a functional, previously unannotated glycosomal GAPDH in \u003cem\u003eL. donovani\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eGAPDH is a glycolytic enzyme with critical roles in glycosomal metabolism, energy production, and redox balance, processes essential for the survival of parasites in the intracellular environment. The identification of peptide-supported translation within this intergenic region demonstrates the presence of a protein-coding gene that was missed in existing genome annotations. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003e illustrates the genomic context of this newly identified gene, the alignment with orthologs from other \u003cem\u003eLeishmania\u003c/em\u003e species, and the representative MS/MS spectrum validating one of the supporting peptides. The discovery of such novel genes underscores the value of proteogenomics in refining \u003cem\u003eL. donovani\u003c/em\u003e genome annotation and in revealing cryptic protein-coding potential that may contribute to parasite adaptation and pathogenicity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2. Identification of N-terminal extension in the amastigote life stages of L. donovani genome:\u003c/h2\u003e \u003cp\u003eIn addition to identifying novel genes, our proteogenomic analysis enabled refinement of existing gene models in \u003cem\u003eL. donovani\u003c/em\u003e. Several GSSPs mapped upstream of annotated start codons, providing direct peptide-level evidence for N-terminal extensions that were not captured in the current genome annotation. These findings reveal that multiple \u003cem\u003eL. donovani\u003c/em\u003e proteins are longer than presently annotated. The full list of GSSPs supporting N-terminal extensions, along with their genomic coordinates and peptide sequences, is provided in \u003cb\u003eSupplementary Table\u0026nbsp;4\u003c/b\u003e, and detailed visual representations, including MS/MS spectra, are shown in \u003cb\u003eSupplementary Fig.\u0026nbsp;4\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eA representative example involves the exosome complex exonuclease RRP40, an essential component of the RNA exosome responsible for RNA processing and degradation. In the \u003cem\u003eL. donovani\u003c/em\u003e genome, the currently annotated gene for this protein is LdCL_040006400, located on chromosome 4 (positive strand), which begins downstream with the sequence \u003cem\u003eMTQPGAVVAIGGGLRLLAQPAPTPTADG\u003c/em\u003e. However, our proteogenomic analysis identified multiple high-confidence GSSPs, most notably the peptide \u0026ldquo;GITELAPLR\u0026rdquo; that mapped to an upstream region encoded by LdCL_040006300, a locus previously annotated as hypothetical. These peptides provide explicit evidence that translation initiates upstream of the LdCL_040006400 annotation. The upstream region, beginning with \u003cem\u003eMSTVSTSSSPSRGITELAPLRGHVCLPGEPV\u003c/em\u003e, shows strong conservation with the orthologous RRP40 protein from \u003cem\u003eL. mexicana\u003c/em\u003e (LmxM.04.0120), confirming that this N-terminal segment is genuine and evolutionarily preserved. Inclusion of this extended region increases the length of the RRP40 protein by 32 amino acids, correcting the truncated annotation and producing a more accurate gene model for this essential exonuclease. The example has been represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThis extended N-terminal region is enriched in serine and charged residues, features commonly associated with regulatory functions, protein-protein interactions, RNA substrate recognition, and post-translational modification sites. Given the central role of the exosome in RNA metabolism and quality control, accurate annotation of RRP40 is critical for understanding RNA regulatory mechanisms in \u003cem\u003eL. donovani\u003c/em\u003e. This example underscores the power of proteogenomics in resolving incomplete gene models and refining the functional annotation of essential components of the parasite\u0026rsquo;s RNA processing machinery.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe life cycle of \u003cem\u003eL. donovani\u003c/em\u003e involves a transition between two highly distinct stages: the sand fly midgut, where the parasite exists as a motile extracellular promastigote, and the mammalian macrophage phagolysosome, where it survives as an intracellular amastigote. These developmental transitions are accompanied by extensive biochemical, metabolic, and structural reprogramming, driven predominantly by post-transcriptional regulation rather than transcriptional control (Gossage et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Sadlova et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Although a well-established reference genome for \u003cem\u003eL. donovani\u003c/em\u003e exists (Downing et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Persistent limitations, including mis-predicted start codons, incomplete ORFs, and hypothetically annotated genes, continue to hamper a clear understanding of parasite biology. Such inaccuracies are expected in kinetoplastids, where polycistronic transcription, aneuploidy, and widespread gene dosage variation complicate genome annotation (Cosentino et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Proteogenomics provides direct peptide-level evidence to address these challenges, refining gene models and revealing previously unannotated coding regions (Nirujogi et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Pawar et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Shenoy and Chowdhury et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, stage-specific proteogenomic mapping in \u003cem\u003eL. donovani\u003c/em\u003e identified 50 high-confidence GSSPs, including 13 promastigote-specific, 24 amastigote-specific, and 13 peptides common to both stages for the N-terminal extensions. This distribution strongly reflects the divergent physiological environments in which the two forms of \u003cem\u003eLeishmania\u003c/em\u003e reside. Promastigotes, living in the nutrient-variable and physically dynamic sand fly gut, rely on pathways that support motility, flagellar assembly, membrane trafficking, RNA processing, and environmental sensing (Alcolea et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In contrast, amastigotes face oxidative bursts, acidic pH, nutrient restriction, and host immune pressure inside macrophages and therefore upregulate stress-adaptation mechanisms, redox regulation, and metabolic reconfiguration for long-term persistence (Pham et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Van Assche et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The stage specificity of peptide-supported proteins discovered here is fully consistent with these well-defined biological adaptations.\u003c/p\u003e \u003cp\u003eOne of the major insights from this proteogenomic analysis is the discovery of several novel protein-coding genes supported by unique peptides that map to previously unannotated or intergenic genomic regions. These novel ORFs were detected in both life stages. Promastigote-specific novel genes are likely involved in mitochondrial RNA editing, flagellar homeostasis, protein transport, and regulatory pathways necessary for vector colonisation. These observations are in line with previous studies showing that promastigotes maintain highly active RNA metabolism and surface remodelling machinery to support growth and attachment within the sand fly (Clos et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Sinha et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In contrast, the amastigote-specific novel genes mapped to regions encoding proteins implicated in oxidative stress handling, pH adaptation, iron/thiol metabolism, and intracellular survival, pathways essential for coping with the hostile environment of the phagolysosome (Requena et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Several novel amastigote proteins showed strong conservation with orthologs in \u003cem\u003eL. infantum\u003c/em\u003e and \u003cem\u003eL. major\u003c/em\u003e, suggesting they are not random translation products but correspond to bona fide coding regions missed during automated genome annotation. Similar outcomes have been documented in proteogenomic studies of \u003cem\u003eL. braziliensis\u003c/em\u003e and \u003cem\u003eL. major\u003c/em\u003e, where novel ORFs redefined the coding capacity of kinetoplastid genomes (Nirujogi et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Pawar et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Shenoy and Chowdhury et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA second significant outcome of this study is the identification of 50 N-terminal extensions, revealing widespread inaccuracies in start-codon predictions in the current \u003cem\u003eL. donovani\u003c/em\u003e genome. Many extensions restore essential regions that were missing in the original annotations. For instance, promastigote-derived N-terminal peptides corrected the structures of guide RNA-binding proteins and RNA helicases, both core components of the mitochondrial RNA editing machinery unique to kinetoplastids (Neboh\u0026aacute;čov\u0026aacute; et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The identification of N-terminal extensions in SEC23, a COPII vesicle trafficking protein, suggests that critical targeting or regulatory sequences were absent in the reference genome. Given the importance of ER-to-Golgi transport for promastigote surface remodelling and secretion, restoring these N-terminal regions enhances functional interpretation (Kim et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The discovery of long, highly conserved extensions in these proteins reinforces the notion that kinetoplastid genomes frequently misannotate start sites due to their compact, intron-poor structure and support on trans-splicing rather than promoter-driven transcription (Trinidad-Barnech et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmastigote-stage N-terminal extensions provide equally compelling biological insights. Peptides detected uniquely in amastigotes extended the coding regions of proteins associated with redox homeostasis, chaperone activity, temperature adaptation, antioxidant metabolism, and intracellular survival pathways. Amastigotes must neutralise reactive oxygen and nitrogen species generated by host macrophages, and our peptide evidence identifies extensions that restore predicted targeting motifs, mitochondrial transit peptides, and regulatory domains that are essential for these functions (Ad\u0026aacute;n-Jim\u0026eacute;nez et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Henard et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Restoring these regions in redox-related proteins aligns with transcriptomic and proteomic datasets, highlighting thiol-based detoxification and metabolic reprogramming in amastigote biology (Requena et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Together, these N-terminal extensions reveal that many amastigote proteins were previously truncated due to incorrect start-site annotation, and that peptide validation is crucial for reconstructing complete protein structures.\u003c/p\u003e \u003cp\u003eThe 13 peptides shared across both stages corresponded primarily to essential housekeeping functions, including RNA-binding proteins, exosomal machinery, and core metabolic enzymes indispensable throughout the parasite life cycle. These proteins are required for fundamental processes irrespective of environmental context, which explains their stable expression in both promastigotes and amastigotes (Krobitsch et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). The presence of shared peptides also validates the robustness of the proteogenomic workflow used here.\u003c/p\u003e \u003cp\u003eTaken together, the identification of novel genes, N-terminal extensions, and stage-specific proteins underscores that current \u003cem\u003eL. donovani\u003c/em\u003e genome annotations remain incomplete, despite the availability of a high-quality reference genome. Misannotations in kinetoplastid genomes are pervasive due to the absence of conventional promoter structures and their reliance on trans-splicing and post-transcriptional control (Kostygov et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). By providing direct peptide evidence, our study refines gene boundaries, restores missing regulatory and targeting signals, and uncovers previously unrecognised coding regions. Similar to our preceding \u003cem\u003eL. braziliensis\u003c/em\u003e proteogenomic study, the frequency and functional importance of N-terminal extensions seen here emphasise systemic start-site inaccuracies in \u003cem\u003eLeishmania\u003c/em\u003e genomes. These proteogenomically validated corrections enable more precise interpretation of parasite biology, particularly regarding stage-specific survival strategies, virulence-associated pathways, and host-parasite interactions. Ultimately, the refined annotations generated in this study offer a strong resource for future genetic, biochemical, and therapeutic investigations into visceral leishmaniasis.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study presents a comprehensive, stage-specific proteogenomic reannotation of the \u003cem\u003eL. donovani\u003c/em\u003e genome, providing experimental evidence that markedly improves the accuracy and completeness of existing gene models. By integrating high-resolution MS/MS data with searches against a six-frame translated genome, we detected 50 genome GSSPs in the two stages that supported N-terminal extensions, in addition to evidence for 15 previously unannotated protein-coding regions. These GSSPs enabled the discovery of several novel protein-coding genes and revealed numerous N-terminal extensions, correcting misassigned start codons and completing truncated ORFs. Such peptide-supported refinements resolve long-standing gaps in genome annotation and explore proteins involved in RNA metabolism, vesicular trafficking, stress adaptation, redox regulation, drug resistance, and intracellular survival, pathways fundamental to the pathogenicity of visceral leishmaniasis.\u003c/p\u003e \u003cp\u003eThe improvements generated here complement existing genomic and transcriptomic datasets, providing a high-confidence gene set that enhances downstream functional, evolutionary, and translational studies. By revealing hidden coding potential and refining structural boundaries, this work strengthens the foundation for understanding stage-specific biology in \u003cem\u003eL. donovani\u003c/em\u003e, including the contrasting adaptations required for promastigote survival in the sand fly vector and amastigote persistence within mammalian macrophages. Although limited to two major life stages, these findings highlight the value of peptide-based evidence in correcting kinetoplastid genome annotations and underscore the need for broader lifecycle proteomic coverage to uncover additional regulatory layers.\u003c/p\u003e \u003cp\u003eOverall, this work demonstrates that even high-quality kinetoplastid genomes without any sequencing errors (gaps) can have issues with genome annotations and require experimental validation to reveal their full coding site. By providing peptide-supported corrections across both life stages, our study offers an improved and functionally relevant gene set for \u003cem\u003eL. donovani\u003c/em\u003e, enabling more accurate analyses of virulence factors, host-parasite interactions, stage differentiation mechanisms, and potential therapeutic targets. The refined annotations and stage-specific insights generated here serve as an important resource for future genomic, proteomic, and functional studies aimed at advancing our understanding of visceral leishmaniasis and supporting the development of novel interventions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to acknowledge the infrastructure support provided by the Institute of Bioinformatics for undertaking this study and analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding source\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot required.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAd\u0026aacute;n-Jim\u0026eacute;nez, J., S\u0026aacute;nchez-Salvador, A., Morato, E., Solana, J. 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Genome plasticity in cultured \u003cem\u003eLeishmania donovani\u003c/em\u003e: Comparison of early and late passages. \u003cem\u003eFrontiers in Microbiology\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e(JUL). https://doi.org/10.3389/fmicb.2018.01279\u003c/li\u003e\n \u003cli\u003eTrinidad-Barnech, J. M., Sotelo-Silveira, J., Do Porto, D. F., \u0026amp; Smircich, P. (2025). Expanding kinetoplastid genome annotation through protein structure comparison. \u003cem\u003ePLoS Pathogens\u003c/em\u003e, \u003cem\u003e21\u003c/em\u003e(4 APRIL). https://doi.org/10.1371/journal.ppat.1013120\u003c/li\u003e\n \u003cli\u003eTschoeke, D. A., Nunes, G. L., Jardim, R., Lima, J., Dumaresq, A. S. R., Gomes, M. R., Pereira, L. de M., Loureiro, D. R., Stoco, P. H., de Matos Guedes, H. L., de Miranda, A. B., Ruiz, J., Pitaluga, A., Silva, F. P., Probst, C. M., Dickens, N. J., Mottram, J. C., Grisard, E. C., \u0026amp; D\u0026aacute;vila, A. M. R. (2014). The comparative genomics and phylogenomics of \u003cem\u003eLeishmania amazonensis\u003c/em\u003e parasite. \u003cem\u003eEvolutionary Bioinformatics\u003c/em\u003e, \u003cem\u003e10\u003c/em\u003e, 131\u0026ndash;153. https://doi.org/10.4137/EBO.S13759\u003c/li\u003e\n \u003cli\u003eVan Assche, T., Deschacht, M., Da Luz, R. A. I., Maes, L., \u0026amp; Cos, P. (2011). \u003cem\u003eLeishmania\u003c/em\u003e-macrophage interactions: Insights into the redox biology. In \u003cem\u003eFree Radical Biology and Medicine\u003c/em\u003e (Vol. 51, Number 2, pp. 337\u0026ndash;351). https://doi.org/10.1016/j.freeradbiomed.2011.05.011\u003c/li\u003e\n \u003cli\u003eZhang, W. W., Ramasamy, G., McCall, L. I., Haydock, A., Ranasinghe, S., Abeygunasekara, P., Sirimanna, G., Wickremasinghe, R., Myler, P., \u0026amp; Matlashewski, G. (2014). Genetic Analysis of \u003cem\u003eLeishmania donovani\u003c/em\u003e Tropism Using a Naturally Attenuated Cutaneous Strain. \u003cem\u003ePLoS Pathogens\u003c/em\u003e, \u003cem\u003e10\u003c/em\u003e(7). https://doi.org/10.1371/journal.ppat.1004244\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":false,"email":"","identity":"current-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Current Microbiology","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false},"keywords":"Leishmania donovani, pseudogenes, proteogenomics, gene annotations, protein coding genes","lastPublishedDoi":"10.21203/rs.3.rs-8719077/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8719077/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eLeishmania donovani\u003c/em\u003e, 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 \u003cem\u003eL. donovani\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eRe-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 \u003cem\u003eLeishmania\u003c/em\u003e species and contained functional domains implicated in essential cellular processes, including metabolism, vesicular trafficking, and intracellular survival.\u003c/p\u003e \u003cp\u003eCollectively, our findings demonstrate that proteogenomic integration significantly improves the accuracy of \u003cem\u003eL. donovani\u003c/em\u003e 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.\u003c/p\u003e","manuscriptTitle":"Identification of new protein-coding potential in Leishmania donovani using a proteogenomics approach","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-13 16:43:39","doi":"10.21203/rs.3.rs-8719077/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-26T05:44:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-19T06:51:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-18T06:55:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-12T11:22:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"211933972623274087763548027093652995731","date":"2026-03-06T01:27:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"5370175326305367940766399012502195690","date":"2026-03-04T03:48:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"46409328494511414880682944649954034924","date":"2026-03-04T02:26:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-25T01:36:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"215627165141922214218900277494780276399","date":"2026-02-09T20:19:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-09T20:00:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-03T20:45:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-30T15:15:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Current Microbiology","date":"2026-01-28T08:21:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":false,"email":"","identity":"current-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Current Microbiology","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"636a21ea-51a7-4187-8acc-61c6e4680f9a","owner":[],"postedDate":"February 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-15T14:43:23+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-13 16:43:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8719077","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8719077","identity":"rs-8719077","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}