Extensive De Novo Sequencing of New Parvalbumin Isoforms Using a Novel Combination of Bottom-Up Proteomics, Accurate Molecular Mass Measurement by FTICR−MS, and Selected MS/MS Ion Monitoring

Parvalbumins (PRVB) (11.20−11.55 kDa) are considered the major fish allergens. In this work, we propose a novel strategy for extensive characterization of this group of proteins based on the integration of a classical Bottom-Up proteomics approach with accurate M_r determination by FTICR−MS of intact proteins and selected MS/MS ion monitoring (SMIM) of peptide mass gaps. For each PRVB, mass spectra obtained by LC−ESI−IT−MS/MS from two digests (trypsin, Glu-C) were de novo sequenced manually with help of two programs (PEAKS, DeNovoX). The deduced peptide sequences were arranged and the theoretical M_r for the resulting sequences was calculated. Experimental M_r for each PRVB was measured with high mass accuracy by FTICR−MS (0.05−4.47 ppm). The masses of several missing peptide gaps were estimated by comparing the theoretical and experimental M_r, and the MS/MS spectra corresponding to these ions were obtained by LC−ESI−IT−MS/MS in the SMIM scanning mode. Finally, all peptide sequences were combined to generate the final protein sequences. This approach allowed the complete de novo MS-sequencing of 25 new PRVB isoforms. These new sequences belong to 11 different species from the Merlucciidae family, organisms for which genomes remain unsequenced. This study constitutes the report accounting for the higher number of new proteins completely sequenced making use of MS-based techniques only.