Proteomic analysis of post-nuclear supernatant fraction and percoll-purified membranes prepared from brain cortex of rats exposed to increasing doses of morphine

Background: proteomic analysis was performed in post-nuclear supernatant (pns) and percoll-purified membranes (pm) prepared from fore brain cortex of rats exposed to increasing doses of morphine (10-50 mg/kg) for 10 days.results: in pns,the 10 up (↑)- or down (↓)-regulated proteins exhibiting the largest morphine-induced change were selected,excised manually from the gel and identified by maldi-tof ms/ms: 1-(gi|148747414,guanine deaminase),↑2.5×; 2-(gi|17105370,vacuolar-type proton atp subunit b,brain isoform),↑2.6×; 3-(gi|1352384,protein disulfide-isomerase a3),↑3.4×; 4-(gi|40254595,dihydropyrimidinase-related protein 2),↑3.6×; 5-(gi|149054470,n-ethylmaleimide sensitive fusion protein,isoform craa),↑2.0×; 6-(gi|42476181,malate dehydrogenase,mitochondrial precursor),↑1.4×; 7-(gi|62653546,glyceraldehyde-3-phosphate dehydrogenase),↑1.6×; 8-(gi|202837,aldolase a),↑1.3×; 9-(gi|31542401,creatine kinase b-type),↓0.86×; 10-(gi|40538860,aconitate hydratase,mitochondrial precursor),↑1.3×. the identified proteins were of cytoplasmic (1,4,5,7,9),cell membrane (2),endoplasmic reticulum (3) and mitochondrial (6,8,10) origin and 9 of them were significantly increased,1.3-3.6×. the 4 out of 9 up-regulated proteins (4,6,7,10) were described as functionally related to oxidative stress; the 2 proteins participate in genesis of apoptotic cell death.in pm,the 18 up (↑)- or down (↓)-regulated proteins were identified by lc-ms/ms and were of plasma membrane [brain acid soluble protein,↓2.1×; trimeric gβ subunit,↓2.0x],myelin membrane [mbp,↓2.5×],cytoplasmic [internexin,↑5.2×; dpyl2,↑4.9×; ubiquitin hydrolase,↓2.0×; 60s ribosomal protein,↑2.7×; kcrb,↓2.6×; sirtuin-2,↑2.5×; peroxiredoxin-2,↑2.2×; septin-11,↑2.2×; tera,↑2.1×; syua,↑2.0×; coronin-1a,↓5.4×] and mitochondrial [glutamate dehydrogenase 1,↑2.7×; scot1,↑2.2×; prohibitin,↑2.2×; aspartate aminotransferase,↓2.2×] origin. surprisingly,the immunoblot analysis of the same pm resolved by 2d-elfo indicated that the active,morphine-induced pool of gβ subunits represented just a minor fraction of the total signal of gβ which was decreased 1.2x only. the dominant signal of gβ was unchanged.conclusion: brain cortex of rats exposed to increasing doses of morphine is far from being adapted. significant up-regulation of proteins functionally related to oxidative stress and apoptosis suggests a major change of energy metabolism resulting in the state of severe brain cell discomfort or even death. © 2014 ujcikova et al.; licensee biomed central ltd.