Targeted LC-MS/MS quantifies bacteriophage LUZ19 virions with high accuracy and precision, overcoming plaque assay limitations.

The global rise of antimicrobial resistance has revitalized interest in phage therapy as a potent alternative to conventional antibiotics. However, a significant hurdle in clinical development is the accurate and consistent quantitation of therapeutic bacteriophages. Current plaque-based enumeration methods are laborious, highly dependent on the host bacterium, and exhibit high variability, especially when attempting to differentiate and quantify individual phages within complex phage cocktails. This gap necessitates advanced, structure-based analytical strategies for reliable and scalable phage characterization.

Researchers developed a targeted proteomic liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for host-independent quantitation of the Pseudomonas aeruginosa podovirus LUZ19. Proteomic characterization was initially performed on an LTQ Orbitrap XL to assess sequence coverage and identify specific, sensitive surrogate peptide candidates. Following this, quantitative analysis was conducted on a QTRAP 7500+ using optimized multiple reaction monitoring (MRM) transitions. The selected tryptic peptide, EVAELDGQELAR, was evaluated for abundance, stability, and chromatographic performance. Phage concentrations derived from digested lysate samples were then correlated with concentrations determined by the standard double layer agar assay.

Proteomic characterization of bacteriophage LUZ19 identified multiple structural proteins, achieving 55% sequence coverage for the major head protein (YP_001671977.1). Out of fifteen detected and evaluated peptides, EVAELDGQELAR was selected as the optimal surrogate peptide for quantitation due to its superior performance characteristics. The developed LC-MS/MS assay demonstrated robust quantitative capabilities: > Back-calculated concentrations met accuracy criteria across a validated range of 0.008 to 80 pg/mL, with bias spanning -8.2 to 8.2%. Intra-day precision ranged from 0.5 to 9.8%, while inter-day precision was consistently between 6.3 and 9.7%. This method yielded an estimated three copies of the major head protein per virion when correlated with traditional plaque assay results, providing a direct structural link to virion count.