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

Paul A. Haynes, Natelaine Fripp, Ruedi Aebersold*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

61 Citations (Scopus)

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 dilution series of lesser amounts of the same standard proteins was also analyzed using a gradient capillary LC system and we were able to successfully identify 50 ng of carbonic anhydrase and 100 ng of BSA.

Original languageEnglish
Pages (from-to)939-945
Number of pages7
JournalElectrophoresis
Volume19
Issue number6
DOIs
Publication statusPublished - May 1998
Externally publishedYes

Keywords

  • Electrospray mass spectrometry
  • Microbore liquid chromatography
  • Microelectrospray
  • Protein electrophoresis
  • Protein identification

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