Protein identification from two-dimensional gel electrophoresis analysis ofKlebsiella pneumoniaeby combined use of mass spectrometry data and raw genome sequences.
Separation of proteins by two-dimensional gel electrophoresis (2-DE) coupled with identification of proteins through peptide mass fingerprinting (PMF) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is the widely used technique for proteomic analysis. This approach relies, however, on the presence of the proteins studied in public-accessible protein databases or the availability of annotated genome sequences of an organism. In this work, we investigated the reliability of using raw genome sequences for identifying proteins by PMF without the need of additional information such as amino acid sequences. The method is demonstrated for proteomic analysis ofKlebsiella pneumoniaegrown anaerobically on glycerol. For 197 spots excised from 2-DE gels and submitted for mass spectrometric analysis 164 spots were clearly identified as 122 individual proteins. 95% of the 164 spots can be successfully identified merely by using peptide mass fingerprints and a strain-specific protein database (ProtKpn) constructed from the raw genome sequences ofK. pneumoniae. Cross-species protein searching in the public databases mainly resulted in the identification of 57% of the 66 high expressed protein spots in comparison to 97% by using the ProtKpn database. 10dharegulon related proteins that are essential for the initial enzymatic steps of anaerobic glycerol metabolism were successfully identified using the ProtKpn database, whereas none of them could be identified by cross-species searching. In conclusion, the use of strain-specific protein database constructed from raw genome sequences makes it possible to reliably identify most of the proteins from 2-DE analysis simply through peptide mass fingerprinting.
Wang
Wei
W
TU-BCE, - German Research Centre for Biotechnology Mascheroder Weg 1, 38124 Braunschweig, Germany.
Sun
Jibin
J
Department of Genome Analysis, - German Research Centre for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany.
Nimtz
Manfred
M
Department of Structure Study, - German Research Centre for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany.
Deckwer
Wolf-Dieter
WD
TU-BCE, - German Research Centre for Biotechnology Mascheroder Weg 1, 38124 Braunschweig, Germany.
Zeng
An-Ping
AP
Department of Genome Analysis, - German Research Centre for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany.