Protein identification from product ion spectra of peptides validated by correlation between measured and predicted elution times in liquid chromatography/mass spectrometry

Reversed‐phase liquid chromatography (LC) directly coupled with electrospray‐tandem mass spectrometry (MS/MS) is a successful choice to obtain a large number of product ion spectra from a complex peptide mixture. We describe a search validation program, ScoreRidge, developed for analysis of LC‐MS/MS data. The program validates peptide assignments to product ion spectra resulting from usual probability‐based searches against primary structure databases. The validation is based only on correlation between the measured LC elution time of each peptide and the deduced elution time from the amino acid sequence assigned to product ion spectra obtained from the MS/MS analysis of the peptide. Sufficient numbers of probable assignments gave a highly correlative curve. Any peptide assignments within a certain tolerance from the correlation curve were accepted for the following arrangement step to list identified proteins. Using this data validation program, host protein candidates responsible for interaction with human hepatitis B virus core protein were identified from a partially purified protein mixture. The present simple and practical program complements protein identification from usual product ion search algorithms and reduces manual interpretation of the search result data. It will lead to more explicit protein identification from complex peptide mixtures such as whole proteome digests from tissue samples.