1 research outputs found
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 Mr 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 Mr for the resulting sequences was calculated. Experimental Mr 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 experimentalMr, 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.Peer reviewe