Combined proteomic analysis of liver tissue and serum in chronically alcohol-fed rats.
Combined proteomic analysis of liver tissue and serum in chronically alcohol-fed rats.
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DOI：
10.1111/j.1530-0277.2012.01883.x
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发表时间：
2013
期刊：
影响因子：
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通讯作者：
M. Yamada;M. Satoh;Masanori Seimiya;K. Sogawa;S. Itoga;T. Tomonaga;F. Nomura
中科院分区：
文献类型：
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作者：
M. Yamada;M. Satoh;Masanori Seimiya;K. Sogawa;S. Itoga;T. Tomonaga;F. Nomura
蛋白质组学方法可能为与酒精中毒相关的病理状况提供新的见解。本研究采用琼脂糖二维凝胶电泳（2-DE）和三步血清蛋白质组学分析方法对慢性酒精喂养大鼠肝组织和血清进行蛋白质组学分析。方法将12只大鼠配对饲喂含乙醇占总能量36%的营养充足的液体饲料或等热量对照饲料2个月。琼脂糖2-DE作用于大鼠肝脏匀浆和胞浆。对血清样品进行3步血清蛋白质组分析，包括免疫耗尽丰富的蛋白质，然后使用反相高效液相色谱和一维十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）进行分离。候选蛋白用胰蛋白酶消化，用质谱法鉴定。观察到的蛋白表达水平差异用Western blotting证实。结果酒精喂养的大鼠肝脏匀浆和细胞质中有46个蛋白点的表达与配对喂养的对照组有差异。最显著的变化是29 kda蛋白的下调，该蛋白随后被鉴定为碳酸酐酶III （CA III）。Western blotting证实该蛋白在酒精喂养大鼠中下调。CA III的信使RNA水平也降低。在大鼠血清中，共有41种蛋白出现差异表达。在这些蛋白中，只有甜菜碱-同型半胱氨酸甲基转移酶（BHMT）在肝脏中也被发现有差异表达。结论对慢性酒精喂养大鼠肝脏组织和血清进行的蛋白质组学分析显示，慢性酒精喂养大鼠肝脏中CA III的表达明显下调。我们的研究结果还表明，BHMT在酒精喂养大鼠的肝脏和血清中表达上调。
BACKGROUND Proteomic approaches may provide new insights into pathological conditions associated with alcoholism. The aim of this study was to conduct a proteomic analysis of liver tissue and serum in chronically alcohol-fed rats using agarose 2-dimensional gel electrophoresis (2-DE) and 3-step serum proteome analysis. METHODS A total of 12 rats were pair-fed nutritionally adequate liquid diet containing ethanol as 36% of the total energy or an isocaloric control diet for 2 months. Rat liver homogenates and cytosol fractions were subjected to agarose 2-DE. Serum samples were subjected to 3-step serum proteome analysis involving immunodepletion of abundant proteins followed by fractionation using reverse-phase high-performance liquid chromatography and 1-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Candidate proteins were digested with trypsin and identified using mass spectrometry. Observed differences in protein expression levels were confirmed using Western blotting. RESULTS A total of 46 protein spots were found to be differentially expressed in the liver homogenates and cytosol fractions of alcohol-fed rats relative to pair-fed controls. The most notable change was down-regulation of a 29-kDa protein, which was subsequently identified as carbonic anhydrase III (CA III). Down-regulation of this protein in alcohol-fed rats was confirmed by Western blotting. The messenger RNA level of CA III was decreased as well. In rat serum, a total of 41 proteins were differentially expressed. Of these proteins, only betaine-homocysteine methyltransferase (BHMT) was also found to be differentially expressed in the liver. CONCLUSIONS A combined proteomic analysis of liver tissue and serum in chronically alcohol-fed rats revealed that the expression of CA III is significantly down-regulated in the liver of alcohol-fed rats. Our results also showed that BHMT expression is up-regulated in both the liver and serum of alcohol-fed rats.