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bioRxiv · 10.64898/2026.04.01.715759

Importance of taking Single Amino Acid Variant and accessory proteome variability into account in Data Independent Acquisition Proteomics: illustrated with Legionella pneumophila analysis

Dupas, A.·Ibranosyan, M.·Ginevra, C.·Jarraud, S.·Lemoine, J.
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Abstract

Understanding allelic variability is crucial for elucidating intrinsic bacterial mechanisms and distinguishing phenotypic profiles. However, such variability poses a major challenge for the reliable identification of proteins in data-independent acquisition (DIA) proteomics. To address this, we developed an analytical workflow that integrates protein sequence variability to enhance proteome coverage. Fifteen Legionella pneumophila isolates were analyzed using DIA-NN, with spectral libraries generated either from a reference proteome or incorporating allelic variability. Our workflow includes protein clustering and subsequent protein inference from these clusters, allowing the accurate assignment of shared and variant-specific peptides. Integration of variability enabled the identification of a comparable number of proteins as the reference proteome while capturing between 28 and 77 % of variant-specific sequences in each isolate, all while maintaining a low false positive rate. These findings demonstrate that accounting for allelic variability substantially improves proteomic coverage and identification confidence, providing a more comprehensive view of the proteome. This approach facilitates a deeper understanding of biological mechanisms and enables precise bacterial proteotyping of Legionella pneumophila isolates.

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Dupas, A., Ibranosyan, M., Ginevra, C., Jarraud, S., Lemoine, J.. 2026-04-03. Importance of taking Single Amino Acid Variant and accessory proteome variability into account in Data Independent Acquisition Proteomics: illustrated with Legionella pneumophila analysis. https://doi.org/10.64898/2026.04.01.715759

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