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

Abstract

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.