Translational Quantitative Proteomic Assay for Bacteriophages: Opening a Frontier in Phage Pharmaceutical Development
- 1University at Buffalo
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.
Related
- A mechanistic quantitative systems pharmacology framework enriched by multiscale data enables model-informed translational development of mesenchymal stem cell therapy in lupus nephritis.
- Quantitative assessment of the influence of tapered dies and compaction position on the propensity of lamination due to air entrapment during pharmaceutical compression
- DIA Quantitative Proteomic Analysis Reveals Early Proteomic Responses to Honeybee-Derived Melittin in Murine Breast Cancer Cells.
- Quantitative proteomic landscape reveals the role of CSPG5 in the recurrence of glioma.
- TMT-based quantitative proteomic analysis of vitreous humor in proliferative diabetic retinopathy.
BibTeX
@misc{nguyen2026translational,
title = {{Translational Quantitative Proteomic Assay for Bacteriophages: Opening a Frontier in Phage Pharmaceutical Development}},
author = {T. D. Nguyen and C. E. Gould and J. T. Sanborn and J. Tutin and Y. Pan and H. Gao and D. Ruszaj and D. Angevine and J. Bussa and D. Atakora and L. Chen and D. R. Roach and T. D. Wood and N. M. Smith},
howpublished = {bioRxiv},
year = {2026},
month = may,
doi = {10.64898/2026.05.27.728049},
url = {https://www.biorxiv.org/content/10.64898/2026.05.27.728049v2},
note = {CC BY},
}
RIS
TY - UNPB
TI - Translational Quantitative Proteomic Assay for Bacteriophages: Opening a Frontier in Phage Pharmaceutical Development
AU - T. D. Nguyen
AU - C. E. Gould
AU - J. T. Sanborn
AU - J. Tutin
AU - Y. Pan
AU - H. Gao
AU - D. Ruszaj
AU - D. Angevine
AU - J. Bussa
AU - D. Atakora
AU - L. Chen
AU - D. R. Roach
AU - T. D. Wood
AU - N. M. Smith
PY - 2026
DA - 2026/05/27
PB - bioRxiv
DO - 10.64898/2026.05.27.728049
UR - https://www.biorxiv.org/content/10.64898/2026.05.27.728049v2
AB - 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.
ER -