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Open Access Green möglich sobald Postprint bei der ZB eingereicht worden ist.
Improved mass spectrometric identification of gel-separated hydrophobic membrane proteins after sodium dodecyl sulfate removal by ion-pair extraction.
Proteomics 4, 3776-3782 (2004)
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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Publikationstyp
Artikel: Journalartikel
Dokumenttyp
Wissenschaftlicher Artikel
Schlagwörter
bacteriorhodospin; matrix-assisted laser desorption; ionization-time of flight; membrane proteomics; POLYACRYLAMIDE-GEL; SEQUENCE DATABASES; PEPTIDE; BACTERIORHODOPSIN; RHODOPSIN; FRAGMENTS; SAMPLES; LEVEL
ISSN (print) / ISBN
1615-9853
e-ISSN
1615-9861
Zeitschrift
Proteomics
Quellenangaben
Band: 4,
Heft: 12,
Seiten: 3776-3782
Verlag
Wiley
Begutachtungsstatus
Peer reviewed
Institut(e)
Institute of Human Genetics (IHG)