Identification of gel-separated proteins by liquid chromatography-electrospray tandem mass spectrometry: comparison of methods and their limitations

Electrophoresis
P A HaynesR Aebersold

Abstract

We have compared several different experimental systems currently in use in our laboratory for protein identification by high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The efficiency of peptide recovery from trypsin-digested gel bands or electroblotted membrane slices was examined using 35S-labeled yeast proteins, and was found to be in excess of 80%. A dilution series of two standard proteins, bovine serum albumin (BSA) and carbonic anhydrase (CA), was analyzed by HPLC-ESI-MS/MS to determine what amount of protein could be loaded onto a gel and successfully identified, a measure we refer to as the practical detection limit. We were able to identify both standards at the 500 ng level in samples prepared from gel slices, using either a regular spray or a flow-split microspray HPLC-MS interface system. In samples prepared from membrane pieces, carbonic anhydrase was also identified at the 500 ng level, while bovine serum albumin could only be identified in samples of more than 1000 ng. In general, protein identification was slightly better in samples prepared from gels rather than membranes. A dilu...Continue Reading

References

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Citations

Sep 18, 1998·Electrophoresis·P A HaynesR Aebersold
Feb 19, 2002·Current Opinion in Biotechnology·T J GriffinR Aebersold
Apr 16, 2002·Journal of the American Society for Mass Spectrometry·Roger E MooreTerry D Lee
Jul 3, 2003·Journal of the American Society for Mass Spectrometry·Bret CooperPaul A Haynes
Jan 12, 2007·Molecular BioSystems·Joohyun Lee, Bret Cooper
Aug 7, 2002·Proceedings of the National Academy of Sciences of the United States of America·Antonius KollerJohn R Yates
Sep 10, 2010·The Journal of Biological Chemistry·Haijuan WangRachel A Altura
Jun 9, 2001·Biochemical and Biophysical Research Communications·Z ZhuJ T Dalton
Jan 19, 2013·Journal of Chromatographic Science·Qian LiuJeffrey N Agar
Sep 1, 2000·FEBS Letters·J S Andersen, M Mann
Jul 22, 2005·Proteomics·Martha L MedinaWilson A Francisco
Apr 30, 2003·Rapid Communications in Mass Spectrometry : RCM·Hiroyuki KatayamaTakeshi Nagasu
Jul 27, 2007·Journal of Separation Science·Joohyun LeeBret Cooper
May 30, 2012·Proteomics. Clinical Applications·Zuzana ReicheltováJan E Dyr
Jul 17, 2010·Biotechnology and Bioengineering·Roberto Olivares-HernándezJens Nielsen
Jun 4, 2013·International Journal of Hyperthermia : the Official Journal of European Society for Hyperthermic Oncology, North American Hyperthermia Group·Michael W GranerEmmanuel Katsanis
Oct 5, 2001·The Journal of Biological Chemistry·T J Griffin, R Aebersold
Feb 8, 2006·The Journal of Biological Chemistry·Yutaka MaedaJeffrey A Whitsett
Jul 4, 2002·Molecular & Cellular Proteomics : MCP·Timothy J GriffinRuedi Aebersold

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