Discovery of Species-unique Peptide Biomarkers of Bacterial Pathogens by Tandem Mass Spectrometry-based Proteotyping

Peptide biomarker candidates for the respiratory tract pathogens Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and Staphylococcus aureus are presented. First, bacterial cultures representing the genetic variability in each of the four species, were analyzed. The peptide biomarker candidates were then experimentally verified to be present in a clinical situation by analyzing true positive clinical samples. The most promising peptide biomarkers were used in a targeted MS mode, demonstrating their use for future clinical implementation. [Display omitted] Highlights •Discovery of peptide biomarker candidates of respiratory tract pathogens S. pneumoniae, H. influenzae, M. catarrhalis and S. aureus as target pathogens.•Peptide biomarker candidates were experimentally verified in clinical samples.•Targeted MS using promising peptide biomarker candidates shown as proof-of-concept. 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 condition