title: Targeted Proteomics of Human Metapneumovirus in Clinical Samples and Viral Cultures.
abstract: The rapid, sensitive, and specific identification of infectious pathogens from clinical isolates is a critical need in the hospital setting. Mass spectrometry (MS) has been widely adopted for identification of bacterial pathogens, although polymerase chain reaction remains the mainstay for the identification of viral pathogens. Here, we explored the capability of MS for the detection of human metapneumovirus (HMPV), a common cause of respiratory tract infections in children. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) sequencing of a single HMPV reference strain (CAN97-83) was used to develop a multiple reaction monitoring (MRM) assay that employed stable isotope-labeled peptide internal standards for quantitation of HMPV. Using this assay, we confirmed the presence of HMPV in viral cultures from 10 infected patients and further assigned genetic lineage based on the presence/absence of variant peptides belonging to the viral matrix and nucleoproteins. Similar results were achieved for primary clinical samples (nasopharyngeal aspirates) from the same individuals. As validation, virus lineages, and variant coding sequences, were confirmed by next-generation sequencing of viral RNA obtained from the culture samples. Finally, separate dilution series of HMPV A and B lineages were used to further refine and assess the robustness of the assay and to determine limits of detection in nasopharyngeal aspirates. Our results demonstrate the applicability of MRM for identification of HMPV, and assignment of genetic lineage, from both viral cultures and clinical samples. More generally, this approach should prove tractable as an alternative to nucleic-acid based sequencing for the multiplexed identification of respiratory virus infections.
authors.forename: Matthew W
authors.initials: MW
authors.lastname: Foster
authors.affiliation: Waters Corporation , Milford, Massachusetts 01757, United States.
authors.forename: Geoff
authors.initials: G
authors.lastname: Gerhardt
authors.affiliation: Department of Molecular Medicine, Department of Microbiology, Immunology and Infectious Diseases, Université Laval , Québec City, Québec, Canada G1V 0A6.
authors.forename: Lynda
authors.initials: L
authors.lastname: Robitaille
authors.affiliation: Department of Molecular Medicine, Department of Microbiology, Immunology and Infectious Diseases, Université Laval , Québec City, Québec, Canada G1V 0A6.
authors.forename: Pier-Luc
authors.initials: PL
authors.lastname: Plante
authors.affiliation: Department of Molecular Medicine, Department of Microbiology, Immunology and Infectious Diseases, Université Laval , Québec City, Québec, Canada G1V 0A6.
authors.forename: Guy
authors.initials: G
authors.lastname: Boivin
authors.affiliation: Department of Molecular Medicine, Department of Microbiology, Immunology and Infectious Diseases, Université Laval , Québec City, Québec, Canada G1V 0A6.
authors.forename: Jacques
authors.initials: J
authors.lastname: Corbeil
authors.forename: M Arthur
authors.initials: MA
authors.lastname: Moseley
mesh_terms: D002478:Cells, Cultured; D002853:Chromatography, Liquid; D006801:Humans; D029121:Metapneumovirus / Q000737:chemistry* / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification; D018184:Paramyxoviridae Infections / Q000821:virology*; D020543:Proteome / Q000032:analysis*; D040901:Proteomics; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D053719:Tandem Mass Spectrometry; D014764:Viral Proteins / Q000032:analysis*
chemical_list: D020543:Proteome; D012367:RNA, Viral; D014764:Viral Proteins