The whereabouts of transmembrane proteins from rat brain synaptosomes during two‐dimensional gel electrophoresis

Abstract

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.

Comparison of protein precipitation methods for various rat brain structures prior to proteomic analysis

“…For the frontal cortex, the recovery was surprisingly low; only 30% of the initial protein content was recovered. This is probably due to a larger concentration of hydrophobic proteins in the frontal cortex [13]. Hydrophobic proteins are relatively difficult to dissolve in aqueous solutions.…”

Effects of 3,4‐methylenedioxymethamphetamine (MDMA) on serotonin transporter and vesicular monoamine transporter 2 protein and gene expression in rats: implications for MDMA neurotoxicity

“…Although it is preferable to use fresh tissue when preparing synaptosomes for metabolic studies, frozen samples can readily be used for synaptosomal preparations when the goal is to determine protein expression in the sample (e.g. see Eravci et al. 2008; Hallett et al.…”

“…Although it is preferable to use fresh tissue when preparing synaptosomes for metabolic studies, frozen samples can readily be used for synaptosomal preparations when the goal is to determine protein expression in the sample (e.g. see Eravci et al 2008;Hallett et al 2008). The homogenate was centrifuged at 1000 g to remove nuclei and cellular debris, and the resulting supernatant was then centrifuged at 16 000 g to yield a crude synaptosomal fraction.…”

The utility of functional interaction and cluster analysis in CNS proteomics

“…A number of articles exemplify this Eravci et al, 2008;Weist et al, 2008). The first step in the analysis of most proteomic studies, as described and illustrated above, is to simply detect candidate proteins that significantly change between control and disease tissue.…”