Automated nanoflow liquid chromatography/tandem mass spectrometric identification of proteins from Shewanella putrefaciens separated by two-dimensional polyacrylamide gel electrophoresis

• Published in: Rapid communications in mass spectrometry 2001-01, Vol.15 (1), p.50-56
• Main Authors: Devreese, Bart, Vanrobaeys, Frank, Van Beeumen, Jozef
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Chromatography, High Pressure Liquid - instrumentation; Chromatography, High Pressure Liquid - methods; Electrophoresis, Gel, Two-Dimensional; Mass Spectrometry - methods; Molecular Sequence Data; Peptide Fragments - genetics; Peptide Fragments - isolation & purification; Shewanella putrefaciens - chemistry; Shewanella putrefaciens - genetics
• ISSN: 0951-4198; 1097-0231
• DOI: 10.1002/1097-0231(20010115)15:1<50::AID-RCM191>3.0.CO;2-V

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. Copyright© 2000 John Wiley & Sons, Ltd.