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Incorporation of a proteotyping approach using mass spectrometry for surveillance of influenza virus in cell-cultured strains.

ABSTRACT: The reemergence of deadly pandemic influenza virus strains has necessitated the development of improved methods for rapid detection and subtyping of influenza viruses that will enable more strains to be characterized at the molecular level. Representative circulating strains of human influenza viruses from primary clinical specimens were grown in cell culture, purified through polyethylene glycol precipitation, proteolytically digested with an endoproteinase, and analyzed and identified by high-resolution mass spectrometry using unique signature peptides that are characteristic of type A H1N1 and H3N2 and type B influenza viruses. This proteotyping approach enabled circulating strains of type A influenza virus to be typed and subtyped, cocirculating seasonal and pandemic H1N1 viruses to be differentiated, and the lineage of type B viruses to be determined through single-ion detection by high-resolution mass spectrometry. Results were obtained using virus titers comparable to those used in reverse transcription (RT)-PCR assays with clinical specimens grown in cell cultures. The methodology represents a more rapid and direct approach than RT-PCR and can be integrated into existing procedures currently used for the surveillance of emerging pandemic and seasonal influenza viruses.

SUBMITTER: Fernandes ND

PROVIDER: S-EPMC3957765 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Incorporation of a proteotyping approach using mass spectrometry for surveillance of influenza virus in cell-cultured strains.

Fernandes Neil D ND Downard Kevin M KM

Journal of clinical microbiology 20131113 3

The reemergence of deadly pandemic influenza virus strains has necessitated the development of improved methods for rapid detection and subtyping of influenza viruses that will enable more strains to be characterized at the molecular level. Representative circulating strains of human influenza viruses from primary clinical specimens were grown in cell culture, purified through polyethylene glycol precipitation, proteolytically digested with an endoproteinase, and analyzed and identified by high- ...[more]

PMID: 24226917

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Project description:Mass spectrometry (MS) and proteomics offer comprehensive characterization and identification of microorganisms and discovery of protein biomarkers that are applicable for diagnostics of infectious diseases. The use of biomarkers for diagnostics is widely applied in the clinic and the use of peptide biomarkers is increasingly being investigated for applications in the clinical laboratory. Respiratory-tract infections are a predominant cause for medical treatment, although, clinical assessments and standard clinical laboratory protocols are time-consuming and often inadequate for reliable diagnoses. Novel methods, preferably applied directly to clinical samples, excluding cultivation steps, are needed to improve diagnostics of infectious diseases, provide adequate treatment and reduce the use of antibiotics and associated development of antibiotic resistance. This study applied nano-liquid chromatography (LC) coupled with tandem MS, with a bioinformatics pipeline and an in-house database of curated high-quality reference genome sequences to identify species-unique peptides as potential biomarkers for four bacterial pathogens commonly found in respiratory tract infections (RTIs): Staphylococcus aureus; Moraxella catarrhalis; Haemophilus influenzae and Streptococcus pneumoniae The species-unique peptides were initially identified in pure cultures of bacterial reference strains, reflecting the genomic variation in the four species and, furthermore, in clinical respiratory tract samples, without prior cultivation, elucidating proteins expressed in clinical conditions of infection. For each of the four bacterial pathogens, the peptide biomarker candidates most predominantly found in clinical samples, are presented. Data are available via ProteomeXchange with identifier PXD014522. As proof-of-principle, the most promising species-unique peptides were applied in targeted tandem MS-analyses of clinical samples and their relevance for identifications of the pathogens, i.e. proteotyping, was validated, thus demonstrating their potential as peptide biomarker candidates for diagnostics of infectious diseases.

Dataset Information

Incorporation of a proteotyping approach using mass spectrometry for surveillance of influenza virus in cell-cultured strains.

Publications

Incorporation of a proteotyping approach using mass spectrometry for surveillance of influenza virus in cell-cultured strains.

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