High-resolution differentiation and identification of the clinically relevant pathogens <i>Haemophilus influenzae</i> and <i>Haemophilus aegyptius</i>: combined whole-cell MALDI-TOF MS and nano-LC-MS/MS proteotyping for defining protein biomarkers

Precise differentiation between closely related species is crucial for effective clinical diagnosis and treatment. <i>Haemophilus influenzae</i> and <i>Haemophilus aegyptius</i> are phenotypically similar but differ in pathogenic potential, with <i>H. influenzae</i> involved in respiratory and invasive infections, and <i>H. aegyptius</i> mainly associated with ocular disease and, rarely, severe systemic outcomes. Traditional phenotypic methods and gene-based analyses often struggle to reliably distinguish them due to high sequence similarity and shifting taxonomic boundaries. In this study, we present a high-resolution proteotyping method that combines whole-cell MALDI-TOF MS, nano-LC-MS/MS, and comparative genomics to identify and verify species-specific protein biomarkers across a broad mass range (<i>m/z</i> 3,000-40,000). Using sequenced reference strains and extensive <i>in silico</i> analyses, we found 31 unique protein biomarkers that allow reliable differentiation between <i>H. influenzae</i> and <i>H. aegyptius</i>. These markers were confirmed through LC-MS/MS peptide mapping and comparative genomics, showing consistent sequence differences and phylogenetic clustering. Our results demonstrate that combining advanced proteomic profiling with genome-based validation effectively addresses the limitations of conventional identification methods. This approach enhances diagnostic accuracy for closely related pathogens and facilitates the ongoing refinement of spectral and genomic reference databases, which are essential for precise microbial identification in clinical microbiology.IMPORTANCESpecies-level resolution of clinically relevant pathogens remains a challenge in clinical diagnostics. <i>Haemophilus influenzae</i> and <i>Haemophilus aegyptius</i> are particularly problematic because of their high genetic similarity, overlapping phenotypes, and distinct clinical implications. Conventional techniques often fail to reliably distinguish them, leading to frequent misclassification. Although diagnostic issues within the <i>H. influenzae</i> complex are well recognized, differentiation between <i>H. influenzae</i> and <i>H. aegyptius</i> remains underexplored. Accurate and rapid identification is essential, as these opportunistic pathogens differ in disease manifestations and epidemiology, and misidentification can result in suboptimal treatment. Our study presents a robust high-resolution nano-LC-MS/MS proteotyping approach, combined with whole-cell MALDI-TOF MS and comparative genomics, to confidently classify <i>H. influenzae</i> and <i>H. aegyptius</i> at species and strain levels. By identifying species-unique peptide biomarkers, this method fills a diagnostic gap and improves species-level microbial diagnostics, understanding of pathogenicity, and epidemiological surveillance.