Improved mass spectrometric identification of gel-separated hydrophobic membrane proteins after sodium dodecyl sulfate removal by ion-pair extraction |
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Authors: | Zischka Hans Gloeckner Christian Johannes Klein Christian Willmann Stefanie Swiatek-de Lange Magdalena Ueffing Marius |
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Affiliation: | GSF National Research Centre for Environment and Health, Institute for Human Genetics, Oberschleissheim, Germany. |
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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. |
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Keywords: | Bacteriorhodopsin Matrix-assisted laser desorption/ionization-time of flight Membrane proteomics |
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