Clinical Proteomics Research Center, Chiba University Hospital, Japan.
Alcohol Clin Exp Res. 2011 Feb;35(2):211-7. doi: 10.1111/j.1530-0277.2010.01336.x. Epub 2010 Nov 8.
The search for biological markers of alcohol abuse is of continual interest in experimental and clinical alcohol research. We previously used gel-free proteome analysis methods such as the ProteinChip(®) system and the ClinProt™ system to search for new serum markers for alcoholism and found several novel marker candidates. As serum contains thousands of proteins and peptides that are present in a large dynamic concentration, depletion of the abundant proteins and further fractionation of the remainder is necessary to get into the deep proteome. We recently described a simple and highly reproducible three-step method for identifying potential disease-marker candidates among the low-abundance serum proteins.
Two serum samples-one on admission and one after 8 weeks of abstinence-were obtained from 8 patients with alcohol dependency. The samples were subjected to a three-step serum proteome analysis. The steps were the following: first, immunodepletion of the 6 most abundant proteins; second, fractionation using reverse-phase high-performance liquid chromatography; and third, separation using one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Differences revealed by protein staining were further confirmed by Western blotting and by enzyme-linked immunosorbent assays (ELISA).
Three-step serum proteome analysis revealed that the serum levels of 5 proteins, alpha2-HS glycoprotein, apolipoprotein A-I, glutathione peroxidase 3, heparin cofactor II, and pigment epithelial-derived factor (PEDF), were significantly greater on admission than after 8 weeks of abstinence. We focused on PEDF because alterations in its levels in alcoholic subjects are not well known. Western blotting and ELISA confirmed the upregulation of PEDF. Serum PEDF levels were significantly greater in moderate to heavy habitual drinkers (14.2 ± 7.7 μg/ml) than in healthy subjects without a drinking history (5.5 ± 3.0 μg/ml) (p < 0.001). The serum PEDF levels in subjects with nonalcoholic chronic liver diseases were comparable to the PEDF levels in healthy subjects.
Three-step serum proteome analysis reveals that excessive alcohol drinking increases the PEDF level.
在实验和临床酒精研究中，寻找酒精滥用的生物标志物一直是人们持续关注的问题。我们之前使用无胶蛋白质组分析方法，如 ProteinChip®系统和 ClinProt™系统，来寻找酒精中毒的新血清标志物，并发现了一些新的候选标志物。由于血清中含有数千种存在于大动态浓度中的蛋白质和肽，因此需要耗尽丰富的蛋白质并进一步对其余部分进行分级分离，才能深入研究蛋白质组。我们最近描述了一种简单且高度可重复的三步法，用于鉴定低丰度血清蛋白中的潜在疾病标志物候选物。
从 8 名酒精依赖患者中采集了 2 个血清样本 - 一个在入院时，一个在戒酒后 8 周。对这些样本进行了三步血清蛋白质组分析。步骤如下：首先，免疫去除 6 种最丰富的蛋白质；其次，使用反相高效液相色谱进行分级分离；最后，使用一维十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）进行分离。通过蛋白质染色揭示的差异通过 Western 印迹和酶联免疫吸附测定（ELISA）进一步证实。
三步血清蛋白质组分析显示，入院时 5 种蛋白质（α2-HS 糖蛋白、载脂蛋白 A-I、谷胱甘肽过氧化物酶 3、肝素辅因子 II 和色素上皮衍生因子（PEDF））的血清水平明显高于戒酒后 8 周。我们关注 PEDF，因为其在酒精性受试者中的水平变化尚未得到充分了解。Western 印迹和 ELISA 证实了 PEDF 的上调。中重度习惯性饮酒者（14.2 ± 7.7 μg/ml）的血清 PEDF 水平明显高于无饮酒史的健康受试者（5.5 ± 3.0 μg/ml）（p < 0.001）。非酒精性慢性肝病患者的血清 PEDF 水平与健康受试者的 PEDF 水平相当。
三步血清蛋白质组分析显示，过量饮酒会增加 PEDF 水平。
