MALDI‐TOF/TOF de novo sequence analysis of 2‐D PAGE‐separated proteins from Halorhodospira halophila, a bacterium with unsequenced genome
peer-reviewed · ELECTROPHORESIS · 2006
| Date | 2006-07-01 |
| Type | peer-reviewed |
| Venue | ELECTROPHORESIS |
| Publisher | Wiley |
| Contribution | algorithm |
| DOI | 10.1002/elps.200500959 |
| Citations (OpenAlex) | 36 |
Abstract
Because protein identifications rely on matches with sequence databases, high-throughput proteomics is currently largely restricted to those species for which comprehensive sequence databases are available. The identification of proteins derived from organisms with unsequenced genomes mainly depends on homology searching. Here, we report the use of a simplified, gel-based, chemical derivatization strategy for de novo sequence analysis using a MALDI-TOF/TOF mass spectrometer. This approach allows the determination of de novo peptide sequences of up to 20 amino acid residues in length. The protocol was applied on a proteomic study of 2-D PAGE-separated proteins from Halorhodospira halophila, an extremophilic eubacterium with yet unsequenced genome. Using three different homology-based search algorithms, we were able to identify more than 30 proteins from this organism using subpicomole quantities of protein.
Methods and tools
- N-sulfonated peptide MALDI TOF/TOF de novo sequencing: N-sulfonation before MALDI TOF/TOF directs fragmentation into a single dominant series, so a peptide mass fingerprint that does not identify a protein on its own can be followed by a readable de novo spectrum.
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- Homology‐Driven Proteomics of Dinoflagellates with Unsequenced Genomes Using MALDI‐TOF/TOF and Automated De Novo Sequencing (2011) both
- De novo sequencing of peptides by MS/MS (2010) crossref
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- De novo sequence analysis and intact mass measurements for characterization of phycocyanin subunit isoforms from the blue‐green alga Aphanizomenon flos‐aquae (2009) crossref
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- De novo protein sequence analysis of Macaca mulatta (2007) semanticscholar