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MALDI‐TOF/TOF de novo sequence analysis of 2‐D PAGE‐separated proteins from Halorhodospira halophila , a bacterium with unsequenced genome
Author(s) -
Samyn Bart,
Sergeant Kjell,
Memmi Samy,
Debyser Griet,
Devreese Bart,
Van Beeumen Jozef
Publication year - 2006
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200500959
Subject(s) - genome , whole genome sequencing , computational biology , bacteria , biology , microbiology and biotechnology , chemistry , genetics , gene
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.