title: Coherent pipeline for biomarker discovery using mass spectrometry and bioinformatics.
abstract: 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.
authors.affiliation: Health Protection Agency, Centre for Infections, 61 Colindale Avenue, London NW9 5EQ, UK. ali.al-shahib@hpa.org.uk
authors.forename: Ali
authors.initials: A
authors.lastname: Al-Shahib
authors.forename: Raju
authors.initials: R
authors.lastname: Misra
authors.forename: Nadia
authors.initials: N
authors.lastname: Ahmod
authors.forename: Min
authors.initials: M
authors.lastname: Fang
authors.forename: Haroun
authors.initials: H
authors.lastname: Shah
authors.forename: Saheer
authors.initials: S
authors.lastname: Gharbia
mesh_terms: D000465:Algorithms; D001426:Bacterial Proteins / Q000032:analysis / Q000502:physiology; D015415:Biomarkers / Q000032:analysis*; D003014:Clostridium botulinum / Q000737:chemistry; D019295:Computational Biology; D017124:Conserved Sequence; D016208:Databases, Factual; D013058:Mass Spectrometry / Q000379:methods*; D040901:Proteomics / Q000379:methods*; D012680:Sensitivity and Specificity; D013045:Species Specificity
chemical_list: D001426:Bacterial Proteins; D015415:Biomarkers
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