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Automated nanoflow liquid chromatography/tandem mass spectrometric identification of proteins from Shewanella putrefaciens separated by two‐dimensional polyacrylamide gel electrophoresis
Author(s) -
Devreese Bart,
Vanrobaeys Frank,
Van Beeumen Jozef
Publication year - 2000
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/1097-0231(20010115)15:1<50::aid-rcm191>3.0.co;2-v
Subject(s) - chemistry , chromatography , mass spectrometry , shewanella putrefaciens , capillary electrophoresis , polyacrylamide gel electrophoresis , proteomics , gel electrophoresis , isotachophoresis , tandem mass spectrometry , coomassie brilliant blue , bacteria , biochemistry , genetics , electrolyte , enzyme , medicine , staining , pathology , gene , biology , electrode
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.