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 15N 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 15N (input phage) and 14N (progeny phage) capsid proteins. Quantitation was achieved by comparing peak areas of target peptides from the metabolically labeled 15N bacteriophage peptide internal standard with that of the wild-type 14N 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 15N reference phage spiked at a concentration of 1.0 × 10 9 plaque forming units (PFU) ml -1. Amplification with 15N 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. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.