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Background  Robust biomarkers are needed to improve microbial identification and diagnostics. Proteomics methods based on mass spectrometry can be used for the discovery of novel biomarkers through their high sensitivity and specificity. However, there has been a lack of a coherent pipeline connecting biomarker discovery with established approaches for evaluation and validation. We propose such a pipeline that uses  in silico  methods for refined biomarker discovery and confirmation.    Results  The pipeline has four main stages: Sample preparation, mass spectrometry analysis, database searching and biomarker validation. Using the pathogen  Clostridium botulinum  as a model, we show that the robustness of candidate biomarkers increases with each stage of the pipeline. This is enhanced by the concordance shown between various database search algorithms for peptide identification. Further validation was done by focusing on the peptides that are unique to  C. botulinum  strains and absent in phylogenetically related  Clostridium  species. From a list of 143 peptides, 8 candidate biomarkers were reliably identified as conserved across  C. botulinum  strains. To avoid discarding other unique peptides, a confidence scale has been implemented in the pipeline giving priority to unique peptides that are identified by a union of algorithms.    Conclusions  This study demonstrates that implementing a coherent pipeline which includes intensive bioinformatics validation steps is vital for discovery of robust biomarkers. It also emphasises the importance of proteomics based methods in biomarker discovery.

Coherent pipeline for biomarker discovery using mass spectrometry and bioinformatics