应用iTRAQ-HPLC-MS技术筛选奶牛脂肪肝病尿液蛋白标志物

doi:10.11843/j.issn.0366-6964.2014.05.025

畜牧兽医学报 ›› 2014, Vol. 45 ›› Issue (5): 844-852.doi: 10.11843/j.issn.0366-6964.2014.05.025

应用iTRAQ-HPLC-MS技术筛选奶牛脂肪肝病尿液蛋白标志物

孙雨航，夏成^*，舒适，孙玲伟，徐闯

(黑龙江八一农垦大学动物科技学院，大庆 163319)

收稿日期:2013-10-30 出版日期:2014-05-23 发布日期:2014-05-23
通讯作者: 夏成，教授，博士生导师，主要从事动物营养代谢病研究，Tel：0459-6819207， E-mail：xcwlxyf@sohu.com
作者简介:孙雨航（1989-），女，满族，辽宁锦州人，硕士生，主要从事临床兽医学专业，动物营养代谢病研究， E-mail：758375006@qq.com
基金资助:
国家自然基金青年基金项目（31001094）；“十二五”农村领域国家科技课题（2012BAD12B05-2；2012BAD12B03-2）；科技支撑计划（2013BAD21B01）；国家自然基金面上项目（31672625）；科技部星火计划（2012GA670001）；黑龙江省新世纪优秀人才项目（1252-NCET-003）

Screening Protein Markers in Urine of Dairy Cows with Fatty Liver Disease Based on ITRAQ-HPLC-MS Technology

SUN Yu-hang， XIA Cheng^*， SHU Shi， SUN Ling-wei， XU Chuang

(College of Animal Science and Veterinary Medicine，Heilongjiang Bayi Agricultural University，Daqing 163319， China)

Received:2013-10-30 Online:2014-05-23 Published:2014-05-23

摘要/Abstract

摘要：

应用同位素标记相对和绝对定量(iTRAQ)技术筛选并鉴定脂肪肝病奶牛尿液中的差异表达蛋白，为诊断奶牛脂肪肝病提供新的生物标志物。收集40头奶牛的尿液，分为脂肪肝病组A1、A2和健康对照组B1、B2，每组各10头奶牛。组内每10个样品等量混合后进行试验，将符合要求的样品进行iTRAQ标记后利用高效液相色谱法层析，经串联质谱鉴定。共筛选出110个差异表达蛋白，其中50个表达下调，60个表达上调；通过生物信息学分析得到了4个关键性蛋白：玻连蛋白 (Vitronectin，VTN)、脂质运载蛋白（ Lipocalin，LCN2)、凝血酶原(Prothrombin，F2)和丛集素 (Clusterin，CLU)。本试验成功筛选出脂肪肝病奶牛尿液中的差异表达蛋白，并鉴定出其中4种与奶牛脂肪肝病存在密切关系的关键性调节因子，如能进一步证实为奶牛脂肪肝病的重要生物标志物，将会为奶牛脂肪肝病的早期检测和诊断提供新的方法。

Abstract:

To obtain new protein markers of fatty liver disease in dairy cows，the relative and absolute quantification isotope labeling (iTRAQ) technology was used to screen and identify differentially expressed proteins in urine of dairy cows with fatty liver disease.40 cows urines were collected and divided into diseased groups A1，A2 and healthy groups B1，B2，respectively including 10 cows in each group.Every 10 urine samples in the same group was equally mixed into 1 sample for the experiment.Subsequently，the samples which the requirements were marked by an iTRAQ kit.Finally，the high-performance liquid chromatography (HPLC) and tandem mass spectrometry were used.In this way，we got a series of data as a result，then the results were conducted by a series of bioinformatics analysis.Totally，110 differentially expressed proteins were screened，in which 50 were upregulated and 60 were downregulated.4 key proteins were acquired by bioinformatic analysis：Vitronectin (VTN),Lipocalin (LCN2)，Prothrombin (F2) and Clusterin (CLU).Differentially expressed proteins in the urine of dairy cows with fatty liver disease were screened in this experiment，4 key regulatory factors were closely related to fatty liver disease，and should be further confirmed as important biological markers of fatty liver disease in dairy cows，which will provide a new method in early detection and diagnosis of fatty liver disease of dairy cows.

中图分类号:

孙雨航，夏成，舒适，孙玲伟，徐闯. 应用iTRAQ-HPLC-MS技术筛选奶牛脂肪肝病尿液蛋白标志物[J]. 畜牧兽医学报, 2014, 45(5): 844-852.

SUN Yu-hang， XIA Cheng， SHU Shi， SUN Ling-wei， XU Chuang. Screening Protein Markers in Urine of Dairy Cows with Fatty Liver Disease Based on ITRAQ-HPLC-MS Technology[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(5): 844-852.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.xmsyxb.com/CN/10.11843/j.issn.0366-6964.2014.05.025

https://www.xmsyxb.com/CN/Y2014/V45/I5/844

参考文献

［1］JORRITSMA R，JORRITSMA H，SCHUKKEN Y H，et al.Prevalence and indicators of postpartum fatty infiltration of the liver in nine commercial dairy herds in the Netherlands［J］.Livest Prod Sci，2001，68(1):53-60.
［2］GERLOFF B J，HERDT T H，EMERY R S.Relationship of hepatic lipidosis to health and performance in dairy cattle［J］.J Am Vet Med Assoc，1986，188(8):845-850.
［3］LEBLANC S.Metabolic health of dairy cattle in the transition perriod［J］.J Reprod Dev，2010，(Suppl):S29-35.
［4］WOLFF S，OTTO A，ALBRECHT D，et al.Gel-free and gel-based proteomics in Bacillus subtilis：A comparative study［J］.Mol Cell Proteomics，2006，5(7):1183-1192.
［5］XU C，WANG Z，LIU G W，et al.Metabolic charateristic of the liver of dairy cows during ketosis based on comparative proteomics［J］.Asian-Aust J Anim Sci，2008，21(7):1003-1010.
［6］XU C，WANG Z.Comparative proteomics analyze of ketotic cows liver［J］.Vet Res Commun，2008，32(3):263-273.
［7］ROSS P L，HUANG Y N，MARCHESE J N，et al.Multiplexed protein quantitation in saccharomyces cerevisiate using amine-reactive isobaric tagging reagents［J］.Mol Cell Proteomics，2004，3(12):1154-1169.
［8］HARDT M，WITKOWSKA H E，WEBB S，et al.Assessing the effects of diurnal variation on the composition of human parotid saliva-quantitative analysis of native peptides using iTRAQ reagents［J］.J Anal Methods Chem，2005，77(15):4947-4954.
［9］HAURA E B，MÜLLER A，BREITWIESER F P，et al.Using iTRAQ combined with tandem affinity purification to enhance low-abundance proteins associated with somatically-mutateed EGFR core complexes in lung cancer［J］.J Proteome Res，2011，10(1):182-190.
［10］HERGENROEDER G，REDELL J B，MOORE A N，et al.Identification of serum biomarkers in brain-injured adults-potential for predicting elevated intracranial pressure［J］.J Neurotrauma，2008，25(2):79-93.
［11］CULNAN D M，COONEY R N，STANLEY B，et al.Apolipoprotein A-IV，a putative satiety antiatherogenic facter，rises after gastric bypass［J］.Obesity (Silver Spring)，2009，17(1):46-52.
［12］SRINIVASAN S，THANGAVELU M，ZHANG L，et al.iTRAQ quantitative proteomics in the analysis of tears in dry eye patients［J］.Invest Ophthalmol Vis Sci，2012，53(8):5052-5059.
［13］DESOUZA L，DIEHL G，RODRIGUES M J，et al.Search for cancer markers from endometrial ts using differentially labeled tags i-TRAQ and c-ICAT with multid imensional liquild chromatography and tandem mass spectrometry［J］.J Proteome Res，2005，4(2):377-386.
［14］MA D，LI L.Searching for reliable premortem protein biomarkers for prion diseases［J］.Exp Rev Proteomics，2012，9(3):267-280.
［15］XU C，LIU G W，LI X B，et al.Decreased complete oxidation capacity of fatty acid in the liver of ketotic cows［J］.Asian-Aust J Anim Sci，2010，3:312-317.
［16］HERDT T H，GOEDERS L，LIESMAN J S，et al.Test for estimation of bovine hepatic lipid content［J］.J Am Vet Med Assoc，1983，182(9):953-955.
［17］RUNGROJ N，SUDTACHAT N，NETTUWAKUL C，et al.Association between human prothrombin variant (T165M) and kidney stone disease［J］.PLoS ONE，2012，7(9):e45533.
［18］RYALL R L，GROVER P K，STAPLETON A M，et al.The urinary F1 activation peptide of human prothrombin is a potent inhibitor of calcium oxalate crystallization in undiluted human urine in vitro［J］.Clin Sci，1995，89:533-541.
［19］DESOUZAL V，TAYLOR A M，LI W，et al.Multiple reaction monitoring of iTRAQ labeled peptides enables absolute quantification of endogenous levels of a potential cancer marker in cancerous and normal endometrial t8s［J］.J Proteome Res，2008，7(8):3525-3534.
［20］PREISSNER K T，REUNING U.Vitronectin in Vascular Context：Facets of a multitalented matricellular protein［J］.Semin Thromb Hemost，2011，37(4):408-424.
［21］PATE G E，WALINSKI H P，BOHUNEK L，et al.Validation of the vitronectin knockout mouse as a model for studying myocardial infarction：Vitronectin appears to influence left ventricular remodelling following myocardial infarction［J］.Exp Clin Cardiol，2013，18(1):43-47.
［22］SINGH B，JANARDHAN K S，KANTHAN R.Expression of angiostatin，integrin alphavbeta3，and vitronectin in human lungs in sepsis［J］.Exp Lung Res，2005，31:771-782.
［23］TSURUTA Y，PARK Y J，SIEGAL G P，et al.Involvement of vitronectin in lipopolysaccaride-induced acute lung injury［J］.J Immunol，2007，179(10):7079-7086.
［24］BAE H B，ZMIJEWSKI J W，DESHANE J S，et al.Vitronectin inhibits neutrophil apoptosis through activation of integrin-associated signaling pathways［J］.Am J Respir Cell Mol Biol，2012，46(6):790-796.
［25］ATKINSON S J，HOSFORD M A，MOLITORIS B A.Mechanism of actin polymerization in cellular ATP depletion［J］.J Biol Chem ，2004，279(7):5194-5199.
［26］KORN E D，CARLIER M F，PANTLONIl D.Actin polymerization and ATP hydrolysis［J］.Science，1987，238:638-644.
［27］NEUHAUS J M，WANGER M，KEISER T，et al.Treadmilling of actin［J］.J Muscle Res Cell Motil,1983，4:507-527.
［28］RUKKWAMSUK T，RUNGRUANG S，CHOOTHESA A，et al.Effect of propylene glycol on fatty liver development and hepatic fructose 1,6 bisphosphatase activity in periparturient dairy cows［J］.Livest Prod Sci，2005，95(1):95-102.
［29］KATOH N.Relevance of apolipoproteins in the development of fatty liver and fatty live-related peripartum diseases in dairy cows［J］.Vet Med Sci，2002，64:293-307.
［30］DANN H M，DRACKLEY J K.Camitine palmitoyl transferase I in liver of periparturient dairy cows effects of periparturient disorders［J］.J Dairy Sci，2005，88：3851-3859.
［31］MATERIA S，CATER M A，KLOMP L W，et al.Clusterin (Apolipoprotein J)，a molecular chaperone that facilitates degradation of the copper-ATPases ATP7A and ATP7B*［J］.J Biol Chem，2011，286:10073-10083.
［32］TROUGAKOS I P，GONOS E S.Regulation of clusterin(apolipoprotein J)，a functional homologue to the small heat shock proteins by oxidative stress in ageing and age-related diseases［J］.Free Radic Res，2006，40(12):1324-1334.
［33］ARONIS K N，VAMVINI M T，CHAMBERLAND J P，et al.Circulating clusterin (Apolipoprotein J) levels do not have any day/night variability and are positively associated with total and LDL cholesterol levels in young healthy individuals［J］.J Clin Endocrinol Metab，2011，96(11):E1871-E1875.
［34］HOOFNAGLE A N，WU M，GOSMANOVA A K，et al.Low clusterin levels in high-density lipoprotein associate with insulin resistance，Obesity，and dyslipoproteinemia.［J］.Arterioscler Thromb Vasc Biol，2010，30:2528-2534.

应用iTRAQ-HPLC-MS技术筛选奶牛脂肪肝病尿液蛋白标志物

Screening Protein Markers in Urine of Dairy Cows with Fatty Liver Disease Based on ITRAQ-HPLC-MS Technology

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 0

编辑推荐

Metrics

本文评价