Homology‐Driven Proteomics of Dinoflagellates with Unsequenced Genomes Using MALDI‐TOF/TOF and Automated De Novo Sequencing
peer-reviewed · Evidence-Based Complementary and Alternative Medicine · 2011
| Date | 2011-01-01 |
| Type | peer-reviewed |
| Venue | Evidence-Based Complementary and Alternative Medicine |
| Publisher | Wiley |
| Contribution | downstream-application |
| DOI | 10.1155/2011/471020 |
| Citations (OpenAlex) | 27 |
Abstract
This study developed a multilayered, gel-based, and underivatized strategy for de novo protein sequence analysis of unsequenced dinoflagellates using a MALDI-TOF/TOF mass spectrometer with the assistance of DeNovo Explorer software. MASCOT was applied as the first layer screen to identify either known or unknown proteins sharing identical peptides presented in a database. Once the confident identifications were removed after searching against the NCBInr database, the remainder was searched against the dinoflagellate expressed sequence tag database. In the last layer, those borderline and nonconfident hits were further subjected to de novo interpretation using DeNovo Explorer software. The de novo sequences passing a reliability filter were subsequently submitted to nonredundant MS-BLAST search. Using this layer identification method, 216 protein spots representing 158 unique proteins out of 220 selected protein spots from Alexandrium tamarense, a dinoflagellate with unsequenced genome, were confidently or tentatively identified by database searching. These proteins were involved in various intracellular physiological activities. This study is the first effort to develop a completely automated approach to identify proteins from unsequenced dinoflagellate databases and establishes a preliminary protein database for various physiological studies of dinoflagellates in the future.
Methods and tools
- Dinoflagellate homology-driven proteomics: Layered MALDI-TOF/TOF identification of dinoflagellate proteins in which de novo sequences searched with MS-BLAST identified proteins missed by database search.
Methods it uses
- MS BLAST: Homology search of error-tolerant de novo sequences against a protein database, introduced for charting the proteomes of organisms with unsequenced genomes and later used to validate borderline identifications.