title: MALDI-TOF/TOF de novo sequence analysis of 2-D PAGE-separated proteins from Halorhodospira halophila, a bacterium with unsequenced genome.
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.
authors.affiliation: Department of Biochemistry, Physiology and Microbiology, Laboratory of Protein Biochemistry and Protein Engineering, Ghent University, Gent, Belgium. bart.samyn@UGent.be
authors.forename: Bart
authors.initials: B
authors.lastname: Samyn
authors.forename: Kjell
authors.initials: K
authors.lastname: Sergeant
authors.forename: Samy
authors.initials: S
authors.lastname: Memmi
authors.forename: Griet
authors.initials: G
authors.lastname: Debyser
authors.forename: Bart
authors.initials: B
authors.lastname: Devreese
authors.forename: Jozef
authors.initials: J
authors.lastname: Van Beeumen
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000032:analysis*; D015180:Electrophoresis, Gel, Two-Dimensional; D016680:Genome, Bacterial / Q000235:genetics; D020607:Halorhodospira halophila / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D001426:Bacterial Proteins