{"id":"doi:10.1074/mcp.m111.012849","kind":"article","title":"Viable Staphylococcus aureus Quantitation using 15N Metabolically Labeled Bacteriophage Amplification Coupled with a Multiple Reaction Monitoring Proteomic Workflow","authors":["Carrie L. Pierce","Jon C. Rees","Facundo M. Fernández","John Robert Barr"],"author_count":4,"year":2011,"published":"2011-10-05","venue":"Molecular & Cellular Proteomics","cited_by":21,"doi":"10.1074/mcp.m111.012849","arxiv_id":null,"nct_id":null,"source_url":"https://doi.org/10.1074/mcp.m111.012849","archive_url":"https://space-kitty.com/papers/doi:10.1074/mcp.m111.012849","updated":null,"pmid":"21972246","pmcid":null,"openalex_id":"W2126962070","pdf_url":"https://www.mcponline.org/content/mcprot/11/1/M111.012849.full.pdf","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 × 104 colony forming units (CFU) ml−1 to 2.0 × 106 CFU ml−1, with the 15N reference phage spiked at a concentration of 1.0 × 109 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 × 104 CFU ml−1), accuracy, and precision for quantitation of S. aureus. 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 × 104 colony forming units (CFU) ml−1 to 2.0 × 106 CFU ml−1, with the 15N reference phage spiked at a concentration of 1.0 × 109 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 × 104 CFU ml−1), accuracy, and precision for quantitation of S. aureus. Staphylococcus aureus (S. aureus) is a versatile pathogenic bacterium (1Schmidt F. Scharf S.S. Hildebrandt P. Burian M. Bernhardt J. Dhople V. Kalinka J. Gutjahr M. Hammer E. Völker U. Time-resolved quantitative proteome profiling of host-pathogen interactions: the response of Staphylococcus aureus RN1HG to internalisation by human airway epithelial cells.Proteomics. 2010; 10: 2801-2811Crossref PubMed Scopus (45) Google Scholar) responsible for a significant number of healthcare-associated and community-acquired infections (2Kurlenda J. Grinholc M. Current diagnostic tools for methicillin-resistant Staphylococcus aureus infections.Mol. Diagn. Ther. 2010; 14: 73-80Crossref PubMed Google Scholar). It causes a broad spectrum of infections ranging from acute to chronic disease (3van den Berg S. Bowden M.G. Bosma T. Buist G. van Dijl J.M. van Wamel W.J. de Vogel C.P. van Belkum A. Bakker-Woudenberg I.A. A multiplex assay for the quantification of antibody responses in Staphylococcus aureus infections in mice.J. Immunol. Methods. 2011; 365: 142-148Crossref PubMed Scopus (18) Google Scholar, 4François P. Scherl A. Hochstrasser D. Schrenzel J. Proteomic approaches to study Staphylococcus aureus pathogenesis.J. Proteomics. 2010; 73: 701-708Crossref PubMed Scopus (41) Google Scholar) and is a common etiological agent of opportunistic infections. An increasing prevalence of antibiotic resistant S. aureus strains are emerging, posing an even greater threat to the general public worldwide (5Durai R. Ng P.C. Hoque H. Methicillin-resistant Staphylococcus aureus: an update.Aorn. J. 2010; 91 (quiz 607–609): 599-606Crossref PubMed Scopus (44) Google Scholar). For these reasons, the development and improvement of diagnostic methods that allow rapid identification and quantitation of this bacterium are highly critical (2Kurlenda J. Grinholc M. Current diagnostic tools for methicillin-resistant Staphylococcus aureus infections.Mol. Diagn. Ther. 2010; 14: 73-80Crossref PubMed Google Scholar). Several methods are available for definitive identification of S. aureus. Traditional identification through plate-culture requires 2–3 days, can require subculturing or biochemical analysis (6Oliveira K. Procop G.W. Wilson D. Coull J. Stender H. Rapid identification of Staphylococcus aureus directly from blood cultures by fluorescence in situ hybridization with peptide nucleic cid probes.J. Clin. Microbiol. 2002; 40: 247-251Crossref PubMed Scopus (153) Google Scholar), and necessitates blood sample volumes difficult to obtain in pediatric patients (7Wada M. Lkhagvadorj E. Bian L. Wang C. Chiba Y. Nagata S. Shimizu T. Yamashiro Y. Asahara T. Nomoto K. Quantitative reverse transcription-PCR assay for the rapid detection of methicillin-resistant Staphylococcus aureus.J. Appl. Microbiol. 2010; 108: 779-788Crossref PubMed Scopus (50) Google Scholar). Molecular methods, such as polymerase chain reaction, reverse transcription-polymerase chain reaction, and quantitative reverse transcription-polymerase chain reaction (PCR, RT-PCR, and qRT-PCR) are primarily based on particular genes specific to S. aureus (2Kurlenda J. Grinholc M. Current diagnostic tools for methicillin-resistant Staphylococcus aureus infections.Mol. Diagn. Ther. 2010; 14: 73-80Crossref PubMed Google Scholar) and offer the most sensitive measurements in the least amount of time, with the least amount of sample, but can be prone to ambiguous results that can only be resolved through sample cultivation (5Durai R. Ng P.C. Hoque H. Methicillin-resistant Staphylococcus aureus: an update.Aorn. J. 2010; 91 (quiz 607–609): 599-606Crossref PubMed Scopus (44) Google Scholar). Additionally PCR is unable to distinguish dead bacteria from live cells, and the required reagents are relatively costly. Gene probe assays are promising in that they offer simple, rapid, and sensitive measurements and are lower in cost than PCR techniques (2Kurlenda J. Grinholc M. Current diagnostic tools for methicillin-resistant Staphylococcus aureus infections.Mol. Diagn. Ther. 2010; 14: 73-80Crossref PubMed Google Scholar, 6Oliveira K. Procop G.W. Wilson D. Coull J. Stender H. Rapid identification of Staphylococcus aureus directly from blood cultures by fluorescence in situ hybridization with peptide nucleic cid probes.J. Clin. Microbiol. 2002; 40: 247-251Crossref PubMed Scopus (153) Google Scholar). Several rapid methods (culture-based and molecular-based screening methods) for S. aureus are also available, allowing diagnostics within hours of collection time; however these tests can be costly and the majority yield qualitative, not quantitative results (8Fuchs B. Süss R. Schiller J. An update of mass in 2010; PubMed Scopus Google Scholar, M. C. B. 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PubMed Google Scholar). phage amplification detection amplification forming of reaction forming standard amplification forming of reaction forming standard techniques are highly but the phage produced through be from the input phage to the For this standard techniques of input the detection of the detection to analysis increasing detection and increasing the of these a rapid 15N approach for analysis of phage as a of detecting S. aureus via mass of Staphylococcus aureus bacteriophage amplification coupled with mass 2011; PubMed Scopus Google Scholar). this 15N method for detecting viable S. aureus via amplified phage capsid is unable to S. aureus concentration of the in quantitative phage amplification detection colony forming units of multiple reaction monitoring plaque forming units standard phage amplification detection colony forming units of multiple reaction monitoring plaque forming units standard a approach that and liquid reaction monitoring tandem mass to bacteriophage peptides from a phage capsid when are of the of viable host S. aureus bacteria that 15N LC-MS/MS method offers accuracy, and sensitivity for high-throughput quantitation of viable S. aureus. this is the study 15N with LC-MS/MS to target cultures of S. aureus and bacteriophage were from the were in a standard 14N bacteriophage were by of with of wild-type bacteriophage × 106 in a the a and the at Following incubation, the was into a by with with a and with a was for at to the and the was through an to phage was a plaque assay M. 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A 15N was to that the method an in phage the of S. aureus. this 1.0 × 109 ml−1 of 15N labeled bacteriophage and 2.0 × CFU ml−1 of S. aureus were Following of the labeled bacteriophage, one was and analyzed the was subjected to a 2-h at ion for this for 15N quantitative response to the bacteriophage quantitative is Following the 2-h incubation, significant were in 15N to the h ion a in was for 14N quantitative the only of the 14N phage peptides is through amplification of the bacteriophage by a viable of S. these results that the 15N LC-MS/MS method can be to the of S. aureus. to for of the 14N phage peptides and to for a h S. aureus sample was into sample This sample was to for 15N with the that was not subjected to the h phage amplification Following sample this was and analyzed by 5.0 × 104 CFU of 14N phage peptides in of the and that were not to the analysis of 15N labeled bacteriophage in is to distinguish bacteriophage labeled from bacteriophage by mass of a phage in the mass spectrometric the and can be by by the 15N labeled phage as an internal standard and a standard the number of bacteria can be the bacteria in a concentration of 15N bacteriophage be to at least one 15N bacteriophage is to S. aureus For this to be a of the of bacteriophage to and a concentration of bacteriophage bacteriophage as a PubMed Scopus Google Scholar). number of that a can be from the is the of by and is the was to be × CFU ml−1 the of For the 15N bacteriophage concentration of × 109 ml−1 in this the that be in the is for a of the can be that of bacteria at least one bacteriophage for a sample of S. aureus at a concentration of the × CFU ml−1. of bacteria are × CFU ml−1, bacteria can be to accurate as the concentration of viable S. aureus approaches × CFU ml−1, the number of live bacteria be of dead S. aureus cells, in with viable S. aureus cells, in a sample, and in 15N bacteriophage the lower than For the of this quantitative study the was 5.0 × 104 CFU ml−1 to 2.0 × 106 CFU ml−1, lower than × CFU ml−1, to that of or bacteria at least one bacteriophage even in the of a concentration of dead S. aureus and that bacteriophage and bacteria concentration that be for a phage A. C. J. M.G. the phage a with for phage 2002; PubMed Scopus Google Scholar). that at phage bacteria are of bacteria × 104 to × CFU were at 1.0 × 109 ml−1 bacteriophage, and to for Following incubation, sample was and the on were and that the bacteriophage concentration the cultures with 1.0 × 109 ml−1 bacteriophage that S. aureus were within the of phage Following these method 6-point calibration ranging from 5.0 × 104 CFU ml−1 to 2.0 × 106 CFU ml−1 were on with LC-MS/MS for metabolically 15N reference phage was spiked at a concentration of 1.0 × 109 ml−1. unlabeled and labeled for quantitative were S. aureus for analysis the 5.0 × 104 CFU ml−1 to 2.0 × 106 CFU ml−1 for quantitative peptide for can be in the calibration were highly from to to highly and were in of were the and by analysis of the calibration constructed from quantitative analyzed on that precision and accuracy, with known of S. aureus were spiked at × CFU and × 106 CFU ml−1), subjected to the 2-h phage amplification and 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peptide yields were to achieved when in 14N or 15N peptide be specific peptides from of the protein were to digestion of the protein and of the digestion were digestion at 2-h digestion at and 3-min digestion at For S. aureus at 5.0 × CFU ml−1, were subjected to the 2-h phage amplification and analyzed by sample was analyzed in the of the digestion number of live S. aureus from of a standard of 5.0 × CFU ml−1 phage were to be 5.0 × CFU ml−1, × CFU ml−1, and × CFU ml−1 for the and digestion that digestion techniques precision and for the quantitative peptide at digest were with of at digest in of that was and for the peptides of the 3-min 15N method was to be that the rapid 3-min digest at was a rapid to the digestion methods that and h digestion times. the of the method and the on the peptides that digestion techniques digestion of the capsid protein of bacteriophage of of digestion for the and quantitative peptide For 15N 5.0 × CFU ml−1 of spiked S. aureus was subjected to phage and LC-MS/MS results for are and and ml−1 standard and standard for sample is standard standard × CFU ml−1 spiked S. 15N 15N 15N 15N × × × × × 104 × × 104 × × × × × 104 × × × × × 104 × CFU ml−1 spiked S. 15N 15N 15N 15N 15N × × × × × 104 × × 104 × × × × × 104 × × × × × 104 × CFU ml−1 spiked S. 15N 15N 15N 15N 15N × × × × × 104 × × 104 × × × × × 104 × × × × × 104 in a Rapid and accurate detection of pathogenic is to and of of 15N labeled bacteriophage in for greater in the detection of the phage the input and phage are by of 15N labeled bacteriophage for greater of input phage to be the of analysis and the of LC-MS/MS detection 15N labeled bacteriophage is in that the 15N labeled phage as an internal standard allowing rapid, and sensitive quantitation of S. aureus. rapid identification and accurate quantification of S. aureus required a rapid 15N phage amplification that S. aureus a rapid and digestion and high-throughput for rapid, and specific of method to with phage than the LC-MS/MS detection S. aureus to be S. aureus quantitation and standard Traditional digestion and by trypsin that from hours to rapid 3-min digest produced peptide yields that significant in peptide when with digest the of the digest were by LC-MS/MS for to sample and one for from to ion of the labeled and unlabeled peptide of the peptide with retention and the 15N to be as retention for the peptide precision for peptide quantitation of peptides from the phage capsid protein as a of S. aureus concentration and method was by monitoring ion for peptide for a total of ion for quantitation and of the phage protein of the of this approach S. bacteriophage and this quantitative be to the development and of labeled methods for rapid in to as an to assays for detection of the is in its most of is the of the phage to only in its allowing detection with a of of a approach the of by quantitative results with detection and for quantitation of bacteria based on phage amplification S. aureus and its This was as a and host be diagnostic be in a are to the 15N quantification method the of the bacteriophage to the bacteria of and be to target bacteria in and Traditional techniques require an by and subsequent of to obtain 15N method detection and accurate quantification of a target bacteria in the the of detection × 104 CFU of analysis directly from and samples. phage only in viable cells, the of bacteriophage as tools for and antibiotic and the of on a S. aureus or is with 15N the for with rapid and can be This of for or diagnostics in reference or labeled coupled with unique that are to be with into the of and of the is are common in and that this of the of phage cultivation and the cost of required into a that offers analysis is a to viable bacteria in a of sample the for an when with PCR and rapid LC-MS/MS are to sensitivity and than and offer detection and that and a and its in this for detection and quantification of to be a diagnostic for and in this to specific or not an or a by the or the for and and in this are of the and not the of the for and with","abstract_license":"CC-BY (publisher license, via OpenAlex)","license":"cc-by","is_open_access":true,"retracted":false,"peer_reviewed":null,"categories":["Advanced Proteomics Techniques and Applications"],"details":null,"provenance":"openalex","attribution":"Abstract of \"Viable Staphylococcus aureus Quantitation using 15N Metabolically Labeled Bacteriophage Amplification Coupled with a Multiple Reaction Monitoring Proteomic Workflow\" by Carrie L. Pierce, Jon C. Rees, Facundo M. Fernández et al., https://doi.org/10.1074/mcp.m111.012849, used under its Creative Commons Attribution license (CC-BY); markup removed, otherwise unmodified.","topics":[{"slug":"algorithms-complexity","name":"Algorithms & Complexity Breakthroughs","domain_slug":"math-cs"}],"cited_in_dossiers":[]}