Automated nanoflow liquid chromatography/tandem mass spectrometric identification of proteins from Shewanella putrefaciens separated by two-dimensional polyacrylamide gel electrophoresis
✍ Scribed by Bart Devreese; Frank Vanrobaeys; Jozef Van Beeumen
- Publisher
- John Wiley and Sons
- Year
- 2000
- Tongue
- English
- Weight
- 260 KB
- Volume
- 15
- Category
- Article
- ISSN
- 0951-4198
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✦ Synopsis
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.