Translational Quantitative Proteomic Assay for Bacteriophages: Opening a Frontier in Phage Pharmaceutical Development

Abstract

Bacteriophages are a promising therapeutic class against drug-resistant bacterial infections but require analytical methods meeting modern drug development standards. Plaque-based enumeration, the gold standard, is laborious, host-dependent, and highly variable. Targeted mass spectrometry of virion structural proteins offers an orthogonal, host-independent, scalable alternative. Here, we describe a targeted proteomic LC-MS/MS assay for the Pseudomonas aeruginosa podovirus LUZ19. Peptide mapping identified multiple structural proteins, providing 55% sequence coverage of a LUZ19 major capsid protein, from which the signature peptide EVAELDGQELAR was selected for quantitation. Using multiple reaction monitoring, calibrators and quality controls were evaluated across three independent runs, with calibrator bias of -7.0 to 4.6%, intra-day precision of 0.5 to 9.3%, and inter-day precision of 6.6 to 12.0%. Peptide concentrations were related to plaque-assay titers by weighted Deming regression, yielding an empirical copy number of 1,314 (95% CI: 1,098 to 1,529) peptide copies per PFU and a phage-equivalent concentration. This proof-of-concept study establishes the analytical foundation for a fully validated assay.

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