iTRAQ技术筛选急性心肌梗死动物血清早期潜在标记物的试验研究
|
摘要
目的利用同位素标记相对和绝对定量(isobaric tags for relative and absolute quantitation, iTRAQ)技术筛选急性心肌梗死(AMI)后表达的差异蛋白,寻找可早期诊断AMI的潜在血清标记物。方法 32条成年比格犬分为假手术组和AMI组(采用冠状动脉注射聚乙烯微球悬浮制剂和凝血酶粉剂制作动物模型),分别收集造模前和造模后1、2、3、6h血清。利用8标iTRAQ技术分析两组血清中蛋白质的表达情况,将表达差异倍数>1.2倍(上下调)且P<0.05的蛋白视为差异蛋白,并对差异蛋白进行基因本体(Geneontology,GO)功能注释和富集分析。结果 AMI后1h出现的差异蛋白有51种,上调28种,下调23种。AMI后2h出现的差异蛋白有54种,上调28种,下调26种。AMI后3h出现的差异蛋白有43种,上调24种,下调19种。AMI后6h出现的差异蛋白有54种,上调29种,下调25种。综合分析前6h差异蛋白,查阅文献资料排除未命名蛋白和犬特有蛋白后,共发现15种差异蛋白在AMI后6h内稳定表达,上调7种,下调8种。结论利用iTRAQ技术筛选出15种与早期AMI相关蛋白,对筛选出的差异蛋白进行功能分析并查阅相关文献,其中肌球蛋白作为早期诊断AMI的新型血清标记物可能有较大潜力。
Objective To screen potential serum biomarkers for early diagnosis of acute myocardial infarction(AMI) by using isobaric tags for relative and absolute quantitation(iTRAQ) technique. Methods Thirty two adult Beagle dogs were divided into sham operation group and AMI group. Serum samples were collected before AMI and at 1 h, 2 h, 3 h, 6 h after AMI respectively. The expression of proteins in serum was analyzed by 8-labeled iTRAQ technique. Proteins expression with a fold change >1.2(P <0.05) were considered differentially expressed, Gene Ontology(GO) analysis and enrichment analysis of differential proteins were performed. Results There were 51 differentially expressed proteins at 1 h after AMI, 28 were up-regulated and 23 were down-regulated; 54 differentially expressed proteins at 2 h after AMI, 28 were up-regulated and 26 were down-regulated; 43 differentially expressed proteins at 3 h after AMI, 24 were up-regulated and 19 were down-regulated; 54 differentially expressed proteins at 6 h after AMI, 29 were up-regulated and 25 were down-regulated. The unnamed protein and dog specific proteins were excluded, and 15 differential proteins were stably expressed within 6 h after AMI, 7 of them were up-regulated and 8 were down-regulated. Conclusion In this study 15 differential proteins related to early AMI were screened out by iTRAQ techniqu. Through functional analysis and literature review, we find that among 15 proteins, myosin may be used as a new serum marker for early diagnosis of AMI.
Objective To screen potential serum biomarkers for early diagnosis of acute myocardial infarction(AMI) by using isobaric tags for relative and absolute quantitation(iTRAQ) technique. Methods Thirty two adult Beagle dogs were divided into sham operation group and AMI group. Serum samples were collected before AMI and at 1 h, 2 h, 3 h, 6 h after AMI respectively. The expression of proteins in serum was analyzed by 8-labeled iTRAQ technique. Proteins expression with a fold change >1.2(P <0.05) were considered differentially expressed, Gene Ontology(GO) analysis and enrichment analysis of differential proteins were performed. Results There were 51 differentially expressed proteins at 1 h after AMI, 28 were up-regulated and 23 were down-regulated; 54 differentially expressed proteins at 2 h after AMI, 28 were up-regulated and 26 were down-regulated; 43 differentially expressed proteins at 3 h after AMI, 24 were up-regulated and 19 were down-regulated; 54 differentially expressed proteins at 6 h after AMI, 29 were up-regulated and 25 were down-regulated. The unnamed protein and dog specific proteins were excluded, and 15 differential proteins were stably expressed within 6 h after AMI, 7 of them were up-regulated and 8 were down-regulated. Conclusion In this study 15 differential proteins related to early AMI were screened out by iTRAQ techniqu. Through functional analysis and literature review, we find that among 15 proteins, myosin may be used as a new serum marker for early diagnosis of AMI.
引文
[1]张宗彬,鲍杰.心肌标记物的实验室检测及临床应用研究进展[J].中国医药指南,2008,6(15):76-78.DOI:10.15912/j.cnki.gocm.2008.15.154.
[2]王林纤,戴勇,涂植光.iTRAQ标记技术与差异蛋白质组学的生物标记物研究[J].生命的化学,2010,1:135-140.DOI:10.13488/j.smhx.2010.01.016
[3]Wi niewski JR,Zougman A,Nagaraj N,et al.Universal sample preparation method for proteome analysis[J].Nature Methods,2009,6(5):359-362.DOI:10.1038/nmeth.1322.
[4]朱永辉,粟勇.急性心肌梗死心肌损伤生化标记物检测进展[J].国际检验医学杂志,2010,31(9):995-997.DOI:1673-4130(2010)09-0995-03.
[5]潘柏申.心肌损伤标记物的研究进展与心肌梗死诊断标准的修订[J].临床检验杂志,2002,20(3):129-132.DOI:10.13602/j.cnki.jcls.2002.03.004.
[6]谢秀枝,王欣,刘丽华,等.iTRAQ技术及其在蛋白质组学中的应用[J].中国生物化学与分子生物学报,2011,27(7):616-621.DOI:10.13865/j.cnki.cjbmb.2011.07.013.
[7]Foth BJ,Goededecke MC,Soldati D.New insights into myosin evolution and classification[J].Proceedings of the National Academy of Sciences of the United States of America,2006,103(10):3681-3686.DOI:10.1073/pnas.0506307103.
[8]Nambiar R,Mcconnell RE,Tyska MJ.Control of cell membrane tension by myosin-I[J].Proceedings of the National Academy of Sciences of the United States of America,2009,106(29):11972-11977.DOI:10.1073/pnas.0901641106.
[9]Tyska MJ,Mackey AT,Huang JD,et al.Myosin-1a is critical for normal brush border structure and composition[J].Molecular biology of the cell,2005,16(5):2443-2457.DOI:10.1091/mbc.e04-12-1116.
[10]Guziklendrum S,Heissler SM,Billington N,et al.Mammalian myosin-18A,a highly divergent myosin[J].Journal of Biological Chemistry,2013,288(13):9532-9548.DOI:10.1074/jbc.M112.441238.
[11]Ishigami M,Swertfeger DK,Granholm NA,et al.Apolipoprotein E inhibits platelet-derived growth factor-induced vascular smooth muscle cell migration and proliferation by suppressing signal transduction and preventing cell entry to G1 phase[J].Journal of Biological Chemistry,1998,273(32):20156.DOI:10.1074/jbc.273.32.20156.
[12]张雪梅,刘秉文.载脂蛋白E研究进展[J].中国动脉硬化杂志,2001,9(2):165-168.DOI:10.3969/j.issn.1007-3949.2001.02.025.
[13]Luo Y,Mok TS,Lin X,et al.SWATH-based proteomics identified carbonic anhydrase 2 as a potential diagnosis biomarker for nasopharyngeal carcinoma[J].Scientific reports,2017,7:41191.DOI:10.1038/srep41191.
[14]张鑫,杨永健,朱文玲,等.血管紧张素Ⅱ受体拮抗剂对梗死心脏纤连蛋白的调节[J].中华心血管病杂志,2006,34(11):1029-1034.DOI:10.3760/j:issn:0253-3758.2006.11.020.
[15]Korkmaz B,Horwitz MS,Jenne DE,et al.Neutrophil elastase,proteinase3,and cathepsin G as therapeutic targets in human diseases[J].Pharmacological Reviews,2010,62(62):726-759.DOI:10.1124/pr.110.002733.
[2]王林纤,戴勇,涂植光.iTRAQ标记技术与差异蛋白质组学的生物标记物研究[J].生命的化学,2010,1:135-140.DOI:10.13488/j.smhx.2010.01.016
[3]Wi niewski JR,Zougman A,Nagaraj N,et al.Universal sample preparation method for proteome analysis[J].Nature Methods,2009,6(5):359-362.DOI:10.1038/nmeth.1322.
[4]朱永辉,粟勇.急性心肌梗死心肌损伤生化标记物检测进展[J].国际检验医学杂志,2010,31(9):995-997.DOI:1673-4130(2010)09-0995-03.
[5]潘柏申.心肌损伤标记物的研究进展与心肌梗死诊断标准的修订[J].临床检验杂志,2002,20(3):129-132.DOI:10.13602/j.cnki.jcls.2002.03.004.
[6]谢秀枝,王欣,刘丽华,等.iTRAQ技术及其在蛋白质组学中的应用[J].中国生物化学与分子生物学报,2011,27(7):616-621.DOI:10.13865/j.cnki.cjbmb.2011.07.013.
[7]Foth BJ,Goededecke MC,Soldati D.New insights into myosin evolution and classification[J].Proceedings of the National Academy of Sciences of the United States of America,2006,103(10):3681-3686.DOI:10.1073/pnas.0506307103.
[8]Nambiar R,Mcconnell RE,Tyska MJ.Control of cell membrane tension by myosin-I[J].Proceedings of the National Academy of Sciences of the United States of America,2009,106(29):11972-11977.DOI:10.1073/pnas.0901641106.
[9]Tyska MJ,Mackey AT,Huang JD,et al.Myosin-1a is critical for normal brush border structure and composition[J].Molecular biology of the cell,2005,16(5):2443-2457.DOI:10.1091/mbc.e04-12-1116.
[10]Guziklendrum S,Heissler SM,Billington N,et al.Mammalian myosin-18A,a highly divergent myosin[J].Journal of Biological Chemistry,2013,288(13):9532-9548.DOI:10.1074/jbc.M112.441238.
[11]Ishigami M,Swertfeger DK,Granholm NA,et al.Apolipoprotein E inhibits platelet-derived growth factor-induced vascular smooth muscle cell migration and proliferation by suppressing signal transduction and preventing cell entry to G1 phase[J].Journal of Biological Chemistry,1998,273(32):20156.DOI:10.1074/jbc.273.32.20156.
[12]张雪梅,刘秉文.载脂蛋白E研究进展[J].中国动脉硬化杂志,2001,9(2):165-168.DOI:10.3969/j.issn.1007-3949.2001.02.025.
[13]Luo Y,Mok TS,Lin X,et al.SWATH-based proteomics identified carbonic anhydrase 2 as a potential diagnosis biomarker for nasopharyngeal carcinoma[J].Scientific reports,2017,7:41191.DOI:10.1038/srep41191.
[14]张鑫,杨永健,朱文玲,等.血管紧张素Ⅱ受体拮抗剂对梗死心脏纤连蛋白的调节[J].中华心血管病杂志,2006,34(11):1029-1034.DOI:10.3760/j:issn:0253-3758.2006.11.020.
[15]Korkmaz B,Horwitz MS,Jenne DE,et al.Neutrophil elastase,proteinase3,and cathepsin G as therapeutic targets in human diseases[J].Pharmacological Reviews,2010,62(62):726-759.DOI:10.1124/pr.110.002733.