Improved mass spectrometric identification of gel-separated hydrophobic membrane proteins after sodium dodecyl sulfate removal by ion-pair extraction.
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.
Zischka
Hans
H
GSF National Research Centre for Environment and Health, Institute for Human Genetics, Oberschleissheim, Germany.
Gloeckner
Christian Johannes
CJ
Klein
Christian
C
Willmann
Stefanie
S
Swiatek-de Lange
Magdalena
M
Ueffing
Marius
M
D000595:Amino Acid Sequence; D000818:Animals; D001436:Bacteriorhodopsins / Q000737:chemistry*; D002462:Cell Membrane / Q000737:chemistry*; D006801:Humans; D007477:Ions; D013058:Mass Spectrometry / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D017433:Protein Structure, Secondary; D040901:Proteomics / Q000379:methods*; D011522:Protons; D017386:Sequence Homology, Amino Acid; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D007477:Ions; D008565:Membrane Proteins; D010455:Peptides; D011522:Protons; D012967:Sodium Dodecyl Sulfate; D001436:Bacteriorhodopsins