Pairing Data Independent Acquisition and High-Resolution Full Scan for Fast Urinary Tract Infection Diagnosis

Abstract

BackgroundRapid and accurate identification of urinary tract infection (UTI) pathogens is critical for effective treatment and combating antimicrobial resistance. Conventional culture-based diagnostics are slow, and standard tandem mass spectrometry workflows are resource-intensive. MethodsWe present a proof-of-concept workflow that integrates high-resolution data-independent acquisition (DIA) MS/MS on the Thermo Scientific Orbitrap Astral with MS1-only spectra from the Orbitrap Exploris 480. DIA data establish a reference panel of pathogen-specific peptides, which are then identified in MS1 spectra from urine samples. Machine learning models trained on these matched MS1 features were used to classify eight common uropathogens and non-infected controls across synthetic inoculations, pure cultures, and clinical patient samples. Model development employed a one-vs-all Random Forest (Ranger) framework with nested cross-validation for feature selection and hyperparameter tuning, followed by evaluation on an independent held-out external patient cohort. ResultsThe approach accurately distinguished bacterial species in both controlled inoculated samples and clinical patient samples. Using repeated nested cross-validation, the model achieved a mean Matthews Correlation Coefficient (MCC) of 0.88, indicating robust classification performance across resampled training partitions. Performance generalized to an independent patient cohort, achieving an MCC of 0.822, confirming the models ability to maintain predictive accuracy under external validation. ConclusionsThis proof-of-concept demonstrates that pairing DIA-derived peptide panels with MS1-only data acquired on a cost-effective instrument suitable for routine analysis, enables rapid, culture-free identification of UTI pathogens. The method provides a scalable, high-throughput platform suitable for clinical applications and establishes a foundation for broader biomarker discovery and potential quantitative workflows. Key Points- DIA-derived peptide panels enable pathogen detection using MS1-only measurements. - Machine learning classifies eight common uropathogens covering 84% of infections. - MS1-only workflows enable culture-free pathogen identification of 300 samples/day.