The whereabouts of transmembrane proteins from rat brain synaptosomes during two-dimensional gel electrophoresis.
Little is known about what happens to transmembrane proteins (TMP) in 2-DE. In order to obtain more insight into the whereabouts of these proteins we prepared membrane-enriched synaptosomes from rat frontal cortex and washed them with 7 M urea or Na(2)CO(3). From each preparation, 200 microg protein was loaded on 2-DE gels covering the 4-7 and 6-11 pH ranges, respectively. MALDI-MS/MS analysis detected only 3 TMP among 421 identified spots. However, when the samples had been washed with Na(2)CO(3), only few well-focused spots remained detectable on the gel covering the pH 6-11 range. Instead, a heavily ruthenium-stained smear became visible at the upper edge of the gel at the location where the samples had been applied by cup loading. LC-MS/MS analysis revealed that this smear contained 38 unfocused TMP with up to 12 transmembrane helices. After transfer to the second dimension, no major areas of protein staining were left on the IPG strips. This indicates that after extraction and denaturation the TMP may form high-molecular aggregates, due to their "hydrophobic interactions". These aggregates enter the IPG strips, but do not focus regularly. They are then transferred onto the 2-DE-gels, where they remain caught at the upper edge.
Eravci
Murat
M
Department of Radiology and Nuclear Medicine (Radiochemistry), Charité Universitätsmedizin, Campus Benjamin Franklin, Berlin, Germany.
Fuxius
Sandra
S
Broedel
Oliver
O
Weist
Stephanie
S
Krause
Eberhard
E
Stephanowitz
Heike
H
Schluter
Hartmut
H
Eravci
Selda
S
Baumgartner
Andreas
A
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002254:Carbonates / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry; D010449:Peptide Mapping / Q000379:methods; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013574:Synaptosomes / Q000378:metabolism*
D002254:Carbonates; D008565:Membrane Proteins; D020543:Proteome