Coherent pipeline for biomarker discovery using mass spectrometry and bioinformatics

Robust biomarkers are needed to improve microbial identification and diagnostics. Proteomics

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.

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.