Automated nanoflow liquid chromatography/tandem mass spectrometric identification of proteins from Shewanella putrefaciens separated by two-dimensional polyacrylamide gel electrophoresis.
The implementation of nanoflow liquid chromatography offers unique opportunities for automation of proteomics research. We demonstrate that automated nanoflow LC/MS/MS allowed the unambiguous identification of proteins from the omnipotent bacterium Shewanella putrefaciens, based on similarity searches against the completely determined genome of related microorganisms and against non-redundant databases. Total protein extracts were separated by 2-dimensional polyacrylamide electrophoresis. Only 1/20th of a tryptic digest mixture obtained from a single Coomassie Blue stained spot was loaded on the nanoflow LC column using a preconcentration/desalting step, and analyzed on-line on a hybrid quadrupole time-of-flight mass spectrometer with an automated MS-to-MS/MS switching protocol. This method allowed the de novo peptide sequence determination of several tryptic fragments and the identification of different proteins. CopyrightCopyright 2000 John Wiley & Sons, Ltd.
Devreese
B
B
Laboratory of Protein Biochemistry and Protein Engineering, KL Ledeganckstraat 35, B-9000 Gent, Belgium.
Vanrobaeys
F
F
Van Beeumen
J
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification; D020614:Shewanella putrefaciens / Q000737:chemistry* / Q000235:genetics
D001426:Bacterial Proteins; D010446:Peptide Fragments