应用SELDI-TOF-MS技术对患脂肪肝奶牛血浆差异蛋白的分离鉴定及生物学分析

doi:10.3864/j.issn.0578-1752.2016.08.016

摘要/Abstract

摘要： 【目的】脂肪肝是围产期奶牛主要代谢性疾病之一，50%产后奶牛受其影响。产后奶牛生产力降低、繁殖性能低下、免疫功能减弱、肝功能衰竭和过早死亡都与脂肪肝有关。表面增强激光解析/电离飞行时间质谱（SELDI-TOF-MS）是一种对生物样本蛋白质研究的新型高敏感性蛋白质组学方法，文章旨在探讨脂肪肝奶牛血浆蛋白质组学特征。【方法】于黑龙江某集约化养牛场选择产后7—28d，1—2胎次荷斯坦奶牛40头。清晨空腹尾静脉采血10mL，应用肝素抗凝（150U）迅速离心（3 000 r/min，5 min）分离血浆,置于-80℃冰箱冷冻保存。依据血浆甘油三酯（TG＞0.20 mmol·L^-1）, 血浆β-羟丁酸（BHBA＞1.2mmol·L^-1）浓度和临床症状，将其分为试验组（T）20头和健康对照组（C）20头。应用SELDI-TOF-MS技术测得血浆蛋白质谱，利用Ciphergen Protein Chip Software (Version 3.1.1)对在试验组和对照组所有样品中得到的峰图进行分析，得到原始数据。将两组之间的数据峰值做wilcoxon sum rank test统计检验，用计算出的P值判断峰在两组中是否有显著差异。以0.01作为P值的阈值，筛选出P值小于0.01的差异峰。在得到的差异峰中，将差异峰的m/z值与Swissport蛋白数据库中多肽的理论荷质比 (mass to charge ratio，m/z)进行比较，找到最相似的蛋白作为差异峰可能检测蛋白的预测结果。【结果】与对照组相比，试验组奶牛血浆蛋白质谱存在39个差异峰，在得到的39个差异峰中，将差异峰的m/z值与Swissport蛋白数据库中多肽的理论荷质比值进行比较，最终获得26个可预测的差异峰，预测蛋白11种。结果显示，相对于C组，11种预测蛋白在T组中均表达下调。通过生物信息学（Network，GO，Pathway）分析，应用Cytoscape软件对获得的11种蛋白进行牛类基因网络搜索，得到Networks分析结果图。在数据库中搜索到了试验结果中的淀粉样肽前体蛋白(amyloid precursor protein, APP)和纤维蛋白原α链(fibrinogen alpha chain, FGA)两种差异蛋白，并得到其相关的Networks分析结果。应用Cytoscape软件中BinGO插件对11种蛋白进行GO分析，并得到GO分析结果。对这些蛋白质经软件搜库得到已经注释的差异蛋白有9种，分别为FGA、血清淀粉样蛋白A (serum amyloid A protein, SAA)、血浆蛋白酶c1抑制剂（plasma protease c1 inhibitor, SERPING1 or C1INH）、血清载脂蛋白-CⅢ（apolipoprotein c-Ⅲ，ApoCⅢ）、海帕西啶（hepcidin, HAMP）、骨桥蛋白（osteopontin, OPN or SPP1）、甲状腺素运转蛋白（transthyretin, TTR）、胱蛋白酶抑制剂C （cystatin-c, CysC or CST）、神经分泌血管生长因子蛋白（neurosecretory protein, VGF）。应用KEGG（Kyoto Encyclopedia of Genes and Genomes）pathway数据库搜索。搜索结果显示只有6种蛋白质可在数据库中搜到，分别是FGA、SERPING1、APO-CⅢ、APP、CysC、SPP1。并得到其相关的Pathway分析结果。这些蛋白可能是与奶牛脂肪肝发病机制相关的物质。【结论】应用SELDI-TOF-MS技术有效的分离了健康牛与患病牛之间的血浆差异表达蛋白，均在肝脏代谢或脂肪肝疾病发生发展过程中起重要的调节作用，对探究奶牛脂肪肝发病机理及其对机体生物学功能的影响具有重要的理论价值。差异蛋白对奶牛脂肪肝的发病机理的影响尚待进一步研究。

关键词: 奶牛, 脂肪肝, SELDI-TOF-MS, 血浆蛋白质组学, 差异表达蛋白

Abstract: 【Objective】 Fatty liver is an important metabolic disorder of dairy cows in transition period, and 50% of postpartum cows are affected. Productivity redution, poor reproductive performance, weak immune function, failure of liver function and premature death are related to fatty liver in postpartum cows. Surface-enhanced laser desorption/ionization (SELDI) time-of-fight (TOF) mass spectrometry (MS) is a sensitive proteomics technology. The aim of this study was to determine the differences between the plasma proteomic profiles of health cows and those with fatty liver. 【Method】 Forty Holstein cows at 7-28 d postpartum with 1-2 parties were selected from an intensive dairy farm in Heilongjiang province. Ten milliliters of blood was taken from the jugular vein of the 40 cows, the blood was placed in an anticoagulant tube and were mixed with the anticoagulant evenly. Blood samples were centrifuged at 3000r/min for 5 min, the serum were stored at −80°C. According to the concentration of serum glycerin trilaurate (TG>0.20mmol·L^-1) and beta-hydroxybutyric acid (BHBA>0.97mmol·L^-1), 20 cows were assigned to the fatty liver group (T) and 20 to the control group (C). Plasma proteins mass spectra were tested bysurface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). The peak figures of the two groups were analyzed using the Ciphergen ProteinChip Software. The peak data of the two groups were analyzed using wilcoxon rank sum test, p values were calculated to confirm the difference of the two groups. Different peaks were selected if P<0.01. m/z values of different peaks were compared with theoretical m/z values using Swissport database. The analogous proteins were identified as a prediction result. 【Result】The 39 significantly different peptide peaks which were identified in the T group were compared with the theoretical m/z values in Swissport database. A total of 26 predictable differences peaks were obtained, 11 kinds of proteins were predicted. Results showed that the levels of these 11 proteins were reduced in T group compared with C group. The 11 kinds of protein networks figure were analyzed by bioinformatics analysis (Network，GO，Pathway) and cattle genetic network search, networks analysis results were got. The two differential protein amyloid precursor protein (APP) and fibrinogen alpha chain (FGA) were found in database, relative Network analysis results were obtained. The BinGO plug-ins in Cytoscape software were used to analyze the 11 kinds of protein and GO analysis results were got. There were 9 kinds of different proteins were found by the software search, including FGA, serum amyloid A protein (SAA), plasma protease c1 inhibitor (SERPING1 or C1INH), apolipoprotein c-Ⅲ (Apo-CⅢ), hepcidin (HAMP), osteopontin (OPN or SPP1), transthyretin (TTR), Cystatin-c (CysC, CST), neurosecretory protein (VGA). There were 6 kinds of different proteins were searched in the KEGG pathway database, including FGA, SERPING1, Apo-CⅢ, APP, CysC and SPP1. They may related to the pathogenesis of fatty liver of dairy cows. 【Conclusion】The differential expression protein in serum between health cows and fatty liver cows were separated effectively by SELDI-TOF-MS technology, which may play an important role in the process of metabolism of liver and development of fatty liver. Therefore, this study has important theoretical values to reveal new mechanism of fatty liver and its effect on bio-function of dairy cows. Further studies need to be conducted to reveal the impact of different proteins on the pathogenesis of fatty liver in dairy cows.

Key words: dairy cows, fatty liver, SELDI-TOF-MS, plasma proteomics, differential expression protein

徐闯，朱奎玲，陈媛媛，杨威，夏成，张洪友，吴凌，舒适，沈泰钰，于洪江，许秋实，张子扬. 应用SELDI-TOF-MS技术对患脂肪肝奶牛血浆差异蛋白的分离鉴定及生物学分析[J]. 中国农业科学, 2016, 49(8): 1585-1598.

XU Chuang, ZHU Kui-ling, CHEN Yuan-yuan, YANG Wei, XIA Cheng, ZHANG Hong-you, WU Ling, SHU Shi, SHEN Tai-yu, YU Hong-jiang, XU Qiu-shi, ZHANG Zi-yang . Isolation Identification and Bioinformatics of Differences Protein in Plasma of Cows Suffer from Fatty Liver with SELDI-TOF-MS Techniques[J]. Scientia Agricultura Sinica, 2016, 49(8): 1585-1598.

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