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Abstract:
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 BLASTsequence-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.
