Improved mass spectrometric identification of gel-separated hydrophobic membrane proteins after sodium dodecyl sulfate removal by ion-pair extraction

Proteomics. 2004 Dec;4(12):3776-82. doi: 10.1002/pmic.200400851.


Separation and identification of hydrophobic membrane proteins is a major challenge in proteomics. Identification of such sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-separated proteins by peptide mass fingerprinting (PMF) via matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) is frequently hampered by the insufficient amount of peptides being generated and their low signal intensity. Using the seven helical transmembrane-spanning proton pump bacteriorhodopsin as model protein, we demonstrate here that SDS removal from hydrophobic proteins by ion-pair extraction prior to in-gel tryptic proteolysis leads to a tenfold higher sensitivity in mass spectrometric identification via PMF, with respect to initial protein load on SDS-PAGE. Furthermore, parallel sequencing of the generated peptides by electrospray ionization-mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) was possible without further sample cleanup. We also show identification of other membrane proteins by this protocol, as proof of general applicability.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Bacteriorhodopsins / chemistry*
  • Cell Membrane / chemistry*
  • Humans
  • Ions
  • Mass Spectrometry / methods*
  • Membrane Proteins / chemistry*
  • Molecular Sequence Data
  • Peptides / chemistry
  • Protein Structure, Secondary
  • Proteomics / methods*
  • Protons
  • Sequence Homology, Amino Acid
  • Sodium Dodecyl Sulfate / chemistry*
  • Spectrometry, Mass, Electrospray Ionization
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization


  • Ions
  • Membrane Proteins
  • Peptides
  • Protons
  • Sodium Dodecyl Sulfate
  • Bacteriorhodopsins