Journal
PROTEOMICS
Volume 7, Issue 14, Pages 2318-2329Publisher
WILEY
DOI: 10.1002/pmic.200700003
Keywords
de novo sequencing; LC-MS/MS; MS BLAST; organisms with unknown genomes; sequence-similarity searches
Funding
- NIGMS NIH HHS [1R01GM070986-01A1] Funding Source: Medline
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LC-MS/MS analysis on a linear ion trap LTQ mass spectrometer, combined with data processing, stringent, and sequence-similarity database searching tools, was employed in a layered manner to identify proteins in organisms with unsequenced genomes. Highly specific stringent searches (MASCOT) were applied as a first layer screen to identify either known (i.e. present in a database) proteins, or unknown proteins sharing identical peptides with related database sequences. Once the confidently matched spectra were removed, the remainder was filtered against a nonannotated library of background spectra that cleaned up the dataset from spectra of common protein and chemical contaminants. The rectified spectral dataset was further subjected to rapid batch de novo interpretation by PepNovo software, followed by the MS BLAST sequence-similarity search that used multiple redundant and partially accurate candidate peptide sequences. Importantly, a single dataset was acquired at the uncompromised sensitivity with no need of manual selection of MS/MS spectra for subsequent de novo interpretation. This approach enabled a completely automated identification of novel proteins that were, otherwise, missed by conventional database searches.
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