title: Viable Staphylococcus aureus quantitation using ¹⁵N metabolically labeled bacteriophage amplification coupled with a multiple reaction monitoring proteomic workflow.
abstract: A multiple reaction monitoring liquid chromatography method with tandem mass spectrometric detection for quantitation of Staphylococcus aureus via phage amplification detection is described. This phage amplification detection method enables rapid and accurate quantitation of viable S. aureus by detecting an amplified capsid protein from a specific phage. A known amount of metabolically labeled (15)N reference bacteriophage, utilized as the input phage and as the internal standard for quantitation, was spiked into S. aureus samples. Following a 2-h incubation, the sample was subjected to a 3-min rapid trypsin digest and analyzed by high-throughput liquid chromatography tandem mass spectrometric detection targeting peptides unique to both the (15)N (input phage) and (14)N (progeny phage) capsid proteins. Quantitation was achieved by comparing peak areas of target peptides from the metabolically labeled (15)N bacteriophage peptide internal standard with that of the wild-type (14)N peptides that were produced by phage amplification and subsequent digestion when the host bacteria was present. This approach is based on the fact that a labeled species differs from the unlabeled one in terms of its mass but exhibits almost identical chemical properties such as ion yields and retention times. A 6-point calibration curve for S. aureus concentration was constructed with standards ranging from 5.0 × 10(4) colony forming units (CFU) ml(-1) to 2.0 × 10(6) CFU ml(-1), with the (15)N reference phage spiked at a concentration of 1.0 × 10(9) plaque forming units (PFU) ml(-1). Amplification with (15)N bacteriophage coupled with LC-MS/MS detection offers speed (3 h total analysis time), sensitivity (LOD: < 5.0 × 10(4) CFU ml(-1)), accuracy, and precision for quantitation of S. aureus.
authors.affiliation: National Center for Environmental Health, Centers for Disease Control and Prevention, 4770 Buford Highway, MS F-50, Atlanta, GA 30341; School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332.
authors.forename: Carrie L
authors.initials: CL
authors.lastname: Pierce
authors.affiliation: National Center for Environmental Health, Centers for Disease Control and Prevention, 4770 Buford Highway, MS F-50, Atlanta, GA 30341.
authors.forename: Jon C
authors.initials: JC
authors.lastname: Rees
authors.affiliation: School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332.
authors.forename: Facundo M
authors.initials: FM
authors.lastname: Fernández
authors.affiliation: National Center for Environmental Health, Centers for Disease Control and Prevention, 4770 Buford Highway, MS F-50, Atlanta, GA 30341. Electronic address: JBarr@cdc.gov.
authors.forename: John R
authors.initials: JR
authors.lastname: Barr
mesh_terms: D001435:Bacteriophages / Q000502:physiology*; D002853:Chromatography, Liquid; D015169:Colony Count, Microbial; D007553:Isotope Labeling; D009587:Nitrogen Isotopes; D040901:Proteomics; D013211:Staphylococcus aureus / Q000302:isolation & purification* / Q000821:virology; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D009587:Nitrogen Isotopes