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Improved mass spectrometric identification of gel‐separated hydrophobic membrane proteins after sodium dodecyl sulfate removal by ion‐pair extraction
Author(s) -
Zischka Hans,
Gloeckner Christian Johannes,
Klein Christian,
Willmann Stefanie,
Swiatekde Lange Magdalena,
Ueffing Marius
Publication year - 2004
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200400851
Subject(s) - chemistry , chromatography , mass spectrometry , bottom up proteomics , protein mass spectrometry , sodium dodecyl sulfate , electrospray ionization , matrix assisted laser desorption/ionization , sample preparation in mass spectrometry , top down proteomics , peptide mass fingerprinting , tandem mass spectrometry , gel electrophoresis , bacteriorhodopsin , membrane , proteomics , biochemistry , desorption , organic chemistry , adsorption , gene
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.