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
peer-reviewed · Journal of Proteome Research · 2010
| Date | 2010-09-03 |
| Type | peer-reviewed |
| Venue | Journal of Proteome Research |
| Publisher | American Chemical Society (ACS) |
| Contribution | downstream-application |
| DOI | 10.1021/pr100163e |
| Citations (OpenAlex) | 65 |
| Venue 2-year citedness | 3.83 |
Abstract
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 experimental Mr, 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.
Methods and tools
- Parvalbumin allergen isoform sequencing: Characterises new parvalbumin isoforms, the major fish allergens, by combining bottom-up proteomics, accurate intact mass by FTICR and selected MS/MS ion monitoring, with the sequences read de novo.
Methods it uses
- PEAKS: Commercial DP-based de novo
Cites (5)
- Straightforward and de Novo Peptide Sequencing by MALDI-MS/MS Using a Lys-N Metalloendopeptidase (2009) crossref
- Combining Bottom-Up and Top-Down Mass Spectrometric Strategies for De Novo Sequencing of the Crustacean Hyperglycemic Hormone from Cancer borealis (2009) crossref
- <i>De novo</i> sequencing of a 21‐kDa cytochrome <i>c</i><sub>4</sub> from <i>Thiocapsa roseopersicina</i> by nanoelectrospray ionization ion‐trap and Fourier‐transform ion‐cyclotron resonance mass spectrometry (2007) crossref
- PEAKS: powerful software for peptide de novo sequencing by tandem mass spectrometry (2003) crossref
- Peptide and protein de novo sequencing by mass spectrometry (2003) crossref