Mapping the Proteomes of Bacteriophages E215 and LUZ19 Using Liquid Chromatography Tandem Mass Spectrometry

Antimicrobial resistance is a global public health concern, and the World Health Organization has identified extensively-drug-resistant Pseudomonas aeruginosa as an urgent threat. The emergence of such pathogens has severely restricted the scope of therapeutic options for clinicians, and has reignited interest in the use of bacteriophage therapy to treat extensively-drug-resistant pathogens. Bacteriophages are viruses which infect and replicate in bacterial hosts, and lytic phages can kill host bacterial cells. Here, we adapted a bottom-up proteomics method used in serum proteomics for mapping the proteomes of two phages which have shown the ability to infect and destroy P. aeruginosa, E215 and LUZ19. This method employed surfactant-aided digestion/on-pellet digestion (SOD) with sodium dodecyl sulfate to denature the phage particles and form protein pellets; these pellets were subjected to disulfide reduction and alkylation, followed by trypsin digestion. The resulting mixtures were separated using a gradient LC approach and analyzed by high-resolution tandem mass spectrometry. For E215, 23 proteins, including ten previously hypothetical proteins from genomic sequencing, were identified. For LUZ19, only four proteins, including two hypothetic proteins from genomics, were identified. However, for both E215 and LUZ19, tryptic peptides from the major head protein of each phage were identified, including one candidate surrogate peptide from each phage to be explored in future quantification studies by LC-MS/MS. These results illustrate that surfactant-aided digestion/on-pellet digestion is an effective approach for proteomic investigations of bacteriophages.