基于纳升高效液相色谱-四极杆-线性离子阱-静电场轨道阱高分辨质谱技术研究广地龙中的蛋白质
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摘要
该文以新鲜广地龙(来源钜蚓科动物参环毛蚓Pheretima aspergillum)为研究对象,采用SDS-PAGE法对广地龙总蛋白进行分离,并进行胶内酶切,利用纳升高效液相色谱-四极杆-线性离子阱-静电场轨道阱高分辨质谱技术研究广地龙中的蛋白质。采用Proteome Discoverer软件,检索环节动物门蛋白数据库(Annelida. fasta)鉴定蛋白。结果共鉴定到386个蛋白质,包括珠蛋白、甘油醛-3-磷酸脱氢酶、纤溶活性蛋白等,大多数蛋白质与细胞结构、能量供给有关,纤溶活性蛋白、胍乙基磷酸丝氨酸蛋白酶可能与抗凝血或溶栓活性有关。利用PANTHER对蛋白进行基因本体功能分类,KEGG进行代谢通路富集分析,发现涉及多种信号转导通路,包括代谢途径、碳代谢通路,氨基酸的生物合成通路,糖酵解/糖异生通路,柠檬酸循环等54种。该研究为阐明广地龙的蛋白物质基础以及其功能研究提供了实验依据。
In the present study,fresh Guangdilong( GD),originating from Pheretima aspergillum,was taken as the object. The total proteins from GD were firstly separated by SDS-PAGE according to their molecular weights and in-gel digestion was then performed.After that,the peptides were analyzed by nano LC/orbitrap fusion lumos high resolution mass spectrometry( nano LC/orbitrap fusion lumos HR-MS). Protein identification was implemented by comparison with Annelida. fasta database using Proteome Discoverer software.As a result,386 proteins were tentatively identified,including chain F,globin B chain,glyceraldehyde-3-phosphate dehydrogenase,fibrinolytic protein,and so on. Most of the proteins took part in cell structure and energy metabolism,and fibrinolytic protein and lombricine kinase might be related to fibrinolytic activity. Protein classification based on gene ontology was carried out using PANTHER and KEGG for metabolic pathway enrichment. The results indicated that these proteins were related to diverse signal transduction pathways,including metabolic pathways,central carbon metabolism,biosynthesis of amino acids,ribosome,glycolysis,citrate cycle( TCA cycle),and so on. This study would lay the foundation for the further research on the proteins in GD and also their functions.
In the present study,fresh Guangdilong( GD),originating from Pheretima aspergillum,was taken as the object. The total proteins from GD were firstly separated by SDS-PAGE according to their molecular weights and in-gel digestion was then performed.After that,the peptides were analyzed by nano LC/orbitrap fusion lumos high resolution mass spectrometry( nano LC/orbitrap fusion lumos HR-MS). Protein identification was implemented by comparison with Annelida. fasta database using Proteome Discoverer software.As a result,386 proteins were tentatively identified,including chain F,globin B chain,glyceraldehyde-3-phosphate dehydrogenase,fibrinolytic protein,and so on. Most of the proteins took part in cell structure and energy metabolism,and fibrinolytic protein and lombricine kinase might be related to fibrinolytic activity. Protein classification based on gene ontology was carried out using PANTHER and KEGG for metabolic pathway enrichment. The results indicated that these proteins were related to diverse signal transduction pathways,including metabolic pathways,central carbon metabolism,biosynthesis of amino acids,ribosome,glycolysis,citrate cycle( TCA cycle),and so on. This study would lay the foundation for the further research on the proteins in GD and also their functions.
引文
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[13]赵霞,岳庆喜,谢正兰.蛋白质组学技术在中药复杂体系研究中的应用[J].生命科学,2013,25(3):334.
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[15] Liu R,Wang F,Huang Q,et al. Available sustainable alternatives replace endangered animal horn based on their proteomic analysis and bio-effect evaluation[J]. Sci Rep,2016,6:36027.
[16]陈银芳,傅应军,刘超,等.基于NanoLC-LTQ-Orbitrap技术的蛋白质组学法探索人参干预“气虚”大鼠的生物学基础[J].中国中药杂志,2016,41(19):3649.
[17]刘培,尚尔鑫,钱大玮,等.基于nano LC-LTQ-Orbitrap-MS/MS的香附四物汤干预原发性痛经模型小鼠卵巢组织差异表达蛋白筛选[J].中国中药杂志,2016,41(16):3060.
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[19] Patron N J,Rogers M B,Keeling P J. Gene replacement of fructose-1,6-bisphosphate aldolase supports the hypothesis of a single photosynthetic ancestor of chromalveolates[J]. Eukaryot Cell,2004,3(5):1169.
[20]张玲,刘峰,崔书健,等.吡喹酮处理日本血吸虫成虫蛋白质组学分析[J].中国寄生虫学与寄生虫病杂志,2008,26(4):258.
[21] De C J,Karbstein K,Woolford Jr J L. Functions of ribosomal proteins in assembly of eukaryotic ribosomes in vivo[J]. Annu Rev Biochem,2015,84:93.
[22] Hedstrom L. Serine protease mechanism and specificity[J].Chem Rev,2003,34(6):4501.
[2]张晓丽,杨洪武,吴品昌.蚯蚓用于抗血栓的加工方法[J].中国实验方剂学杂志,2011,17(22):24.
[3] Mihara H,Sumi H,Yoneta T,et al. A novel fibrinolytic enzyme extracted from the earthworm,Lumbricus rubellus[J]. Jpn J Physil,1991,41:461.
[4] Nakajima N,Mihara H,Sumi H. Characterization of potent fibrinolytic enzymes in earthworm[J]. Biosci Biotech Biochem,1993,57:1726.
[5] Wu J X,Zhao X Y,Pan R,et al. Glycosylated trypsin-like proteases from earthworm Eisenia fetida[J]. Int J Biol Macromol,2007,40:399.
[6] Cho I H,Choi E S,Lim H G,et al. Purification and characterization of six fibrinolytic serine-proteases from earthworm Lumbricus rubellus[J]. J Biochem Mol Biol,2004,37:199.
[7] Cho I H,Choi E S,Lee H H. Molecular cloning,sequencing,and expression of a fibrinolytic serine-protease gene from the earthworm Lumbricus rubellus[J]. J Biochem Mol Biol,2004,37:574.
[8]张东方,周美环,单玉,等.参环毛蚓中纤溶活性蛋白酶研究[J].药物生物技术,2006,13(1):49.
[9]唐鼎,凃乾,李娟,等.药用地龙的药理作用和临床研究进展[J].中国药师,2015,18(6):1016.
[10]张玉,董文婷,霍金海,等.基于UPLC-Q-TOF-MS技术的广地龙化学成分分析[J].中草药,2017,48(2):252.
[11] Zhang J X,Yang W Z,Li S R,et al. An intelligentized strategy for endogenous small molecules characterization and quality evaluation of earthworm from two geographic origins by ultra-high performance HILIC/QTOFMSEand progenesis QI[J]. Anal Bioanal Chem 2016,408(14):3881.
[12]朱宇伟,于庆云,刘培,等.蛋白质组学在中医药科学研究中的应用进展[J].中国现代中药,2016,18(5):661.
[13]赵霞,岳庆喜,谢正兰.蛋白质组学技术在中药复杂体系研究中的应用[J].生命科学,2013,25(3):334.
[14] Xue L,Feng S,Gong L,et al. Species-specific identification of collagen components in Colla corii asini using a nano-liquid chromatography tandem mass spectrometry proteomics approach[J].Int J Nanomed,2017,12:4443.
[15] Liu R,Wang F,Huang Q,et al. Available sustainable alternatives replace endangered animal horn based on their proteomic analysis and bio-effect evaluation[J]. Sci Rep,2016,6:36027.
[16]陈银芳,傅应军,刘超,等.基于NanoLC-LTQ-Orbitrap技术的蛋白质组学法探索人参干预“气虚”大鼠的生物学基础[J].中国中药杂志,2016,41(19):3649.
[17]刘培,尚尔鑫,钱大玮,等.基于nano LC-LTQ-Orbitrap-MS/MS的香附四物汤干预原发性痛经模型小鼠卵巢组织差异表达蛋白筛选[J].中国中药杂志,2016,41(16):3060.
[18]尚海旭,井然,贾弘禔,等. GAPDH功能多样性[J].生理科学进展,2011,42(5):371.
[19] Patron N J,Rogers M B,Keeling P J. Gene replacement of fructose-1,6-bisphosphate aldolase supports the hypothesis of a single photosynthetic ancestor of chromalveolates[J]. Eukaryot Cell,2004,3(5):1169.
[20]张玲,刘峰,崔书健,等.吡喹酮处理日本血吸虫成虫蛋白质组学分析[J].中国寄生虫学与寄生虫病杂志,2008,26(4):258.
[21] De C J,Karbstein K,Woolford Jr J L. Functions of ribosomal proteins in assembly of eukaryotic ribosomes in vivo[J]. Annu Rev Biochem,2015,84:93.
[22] Hedstrom L. Serine protease mechanism and specificity[J].Chem Rev,2003,34(6):4501.