牦牛卵泡液差异蛋白质组双向电泳图谱的构建与质谱分析

doi:10.16431/j.cnki.1671-7236.2015.11.031

›› 2015, Vol. 42 ›› Issue (11): 3037-3043.doi: 10.16431/j.cnki.1671-7236.2015.11.031

牦牛卵泡液差异蛋白质组双向电泳图谱的构建与质谱分析

郭宪, 裴杰, 褚敏, 王宏博, 丁学智, 阎萍

中国农业科学院兰州畜牧与兽药研究所, 甘肃省牦牛繁育工程重点实验室, 兰州 730050

收稿日期:2015-04-14 出版日期:2015-11-20 发布日期:2015-11-26
通讯作者: 阎萍 E-mail:yanping@caas.cn
作者简介:郭宪(1978-),男,甘肃环县人,博士,副研究员,研究方向:动物遗传育种与繁殖,Tel:0931-2115257;E-mail:guoxian@caas.cn
基金资助:
国家自然科学基金(31301976);现代农业(肉牛牦牛)产业技术体系建设专项(CARS-38);国家科技支撑计划项目子课题(2013BAD16B09);中国农业科学院创新工程项目(CAAS-ASTIP-2014-LIHPS-01)

Establishment of Two-dimensional Electrophoresis Mapping and Mass Spectrometry Analysis of the Differential Proteome of Yak Follicular Fluid

GUO Xian, PEI Jie, CHU Min, WANG Hong-bo, DING Xue-zhi, YAN Ping

Key Laboratory of Yak Breeding Engineering of Gansu Province, Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agricultural Sciences, Lanzhou 730050, China

Received:2015-04-14 Online:2015-11-20 Published:2015-11-26

摘要/Abstract

摘要： 从蛋白质水平了解牦牛季节性繁殖规律,利用双向电泳与质谱鉴定技术分析牦牛卵泡液与血浆蛋白质组分变化。以青海高原牦牛卵泡液与血浆为研究对象,采用双向电泳技术构建牦牛卵泡液与血浆蛋白质双向电泳图谱,银染后利用Image Master 2D Platinum软件分析并采用MALDI-TOF-MS进行质谱鉴定。用试剂盒ProteoExtract Albumin/IgG Removal Kit去除高丰度蛋白质后,利用2-DE技术获得了分辨率较高的卵泡液与血浆蛋白质电泳图谱,卵泡液与血浆蛋白质图谱对比分析共发现了24个差异表达蛋白质点,其中2个蛋白质点表达上调,22个蛋白质点表达下调。经质谱分析,最终成功鉴定出8个蛋白质点、5个未知蛋白质点。本研究成功构建了蛋白质图谱及分离鉴定的差异蛋白质,为从蛋白质水平揭示牦牛卵泡发育规律及了解卵母细胞发育的微环境提供了试验依据。

关键词: 牦牛; 卵泡液; 血浆; 差异蛋白质组; 双向电泳; 质谱鉴定

Abstract: In order to know the law of seasonal reproduction of yak (Bos grunniens) at protein level,the composition changes of follicular fluid and plasma protein were analyzed by two-dimensional electrophoresis (2-DE) and mass spectrometry (MS).The study employed Qinghai plateau yak follicular fluid and plasma as the research objects.The maps were obtained by 2-DE.After silver staining,the differential proteome was analyzed using the Image Master 2D Platinum software and MALDI-TOF-MS (matrix-assisted laser desorption ionization time of flight mass spectrometry).First we described the purification of follicular fluid and plasma samples using ProteoExtract Albumin/IgG Removal Kit,and then described the maps of protein from these purified follicular fluid and plasma using the 2-DE system.24 different expression protein spots were found with Image master 2D platinum soteware,2 of these proteins were up-regulated,and 22 of these proteins were down-regulated in yak follicular fluid.Through MALDI-TOF-MS identification,8 protein spots were successfully identified and 5 protein spots were unknown in NCBI_Bos tarus.Establishment of 2-DE maps,isolation and identification of differential protein were successfully to provide experimental basis for revealing the law of follicular development and developmental microenviroment of oocytes at protein level in yak.

Key words: yak; follicular fluid; plasma; differential proteome; two-dimensional electrophoresis; mass spectrometry identification

中图分类号:

Q591.2

郭宪, 裴杰, 褚敏, 王宏博, 丁学智, 阎萍. 牦牛卵泡液差异蛋白质组双向电泳图谱的构建与质谱分析[J]. , 2015, 42(11): 3037-3043.

GUO Xian, PEI Jie, CHU Min, WANG Hong-bo, DING Xue-zhi, YAN Ping. Establishment of Two-dimensional Electrophoresis Mapping and Mass Spectrometry Analysis of the Differential Proteome of Yak Follicular Fluid[J]. , 2015, 42(11): 3037-3043.

参考文献

[1] Sarkar M,Sengupta D H,Bora B D,et al.Efficacy of heatsynch protocol for induction of estrus,synchronization of ovulation and timed artificial insemination in yaks (Poephagus grunniens L.)[J].Animal Reproduction Science,2008,104:299-305.
[2] Zi X D,He S M,Lu H,et al.Induction of estrus in suckled female yaks (Bos grunniens) and synchronization of ovulation in the on-sucklers for timed artificial insemination using progesterone treatments and Co-Synch regimens[J].Animal Reproduction Science,2006,92:183-192.
[3] Yu S J,Yong Y H,Cui Y.Oocyte morphology from primordial to early tertiary follicles of yak[J].Reproduction in Domestic Animals,2010,45(5):779-785.
[4] Ding X Z,Liang C N,Guo X,et al.A novel single nucleotide polymorphism in exon 7 of LPL gene and its association with carcass traits and visceral fat deposition in yak (Bos grunniens) steers[J].Molecular Biology Reports,2012,39(1):669-673.
[5] Qiu Q,Zhang G,Ma T,et al.The yak genome and adaptation to life at high altitude[J].Nature Genetics,2012,40:946-951.
[6] Eppig J J,Pendola F L,Wigglesworth K,et al.Mouse oocytes regulate metabolic cooperativity between granulosa cells and oocytes:Amino acid transport[J].Biology of Reproduction,2005,73(2):351-357.
[7] Sugiura K,Pendola F L,Eppig J J.Oocyte control of metabolic cooperativity between oocytes and companion granulosa cells:Energy metabolism[J].Developmental Biology,2005,279(1):20-30.
[8] Coleman N V,Shagiakhmetova G A,Lebedeva I Y,et al.In vitro maturation and early developmental capacity of bovine oocytes cultured in pure follicular fluid and supplementation with follicular wall[J].Theriogenology,2007,67(5):1053-1059.
[9] Kawano Y,Fukuda J,Nasu K,et al.Production of macrophage inflammatory protein-3 alpha in human follicular fluid and cultured granulose cells[J].Fertility and Sterility,2004,82:1206-1211.
[10] Nowak R.Entering the postgenome era[J].Science,1995,270:368-369.
[11] Blackstock W P,Weir M P.Proteomics:Quantitative and physical mapping of cellular proteins[J].Trends in Biotechnology,1999,17(3):121-127.
[12] 付强,杨丽,刘振方,等.水牛卵泡液液相色谱分离及差异蛋白质分析[J].中国畜牧兽医,2012,39(4):64-67.
[13] Tao J Z,Zhao G S,Zhao X X,et al.Proteomic analysis of the follicular fluid of Tianzhu White yak during diestrus[J].Int J Mol Sci,2014,15:4481-4491.
[14] Bradford M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry,1976,72:248-254.
[15] Chevallet M,Luche S,Rabilloud T.Silver staining of proteins in polyacrylamide gels[J].Nat Protoc,2006,1(4):1852-1858.
[16] 黄愉淋,付强,黄德伦,等.去除高丰度蛋白质后水牛卵泡液的双向电泳差异分析[J].畜牧兽医学报,2014,45(7):1113-1119.
[17] Angelucci S,Ciavardelli D,Di Giuseppe F,et al.Proteome analysis of human follicular fluid[J].Biochim Biophys Acta,2006,1764(11):1775-1785.
[18] Schweigert F J,Gericke B,Wolfram W,et al.Peptide and protein profiles in serum and follicular fluid of women undergoing IVF[J].Human Reproduction,2006,21(11):2960-2968.
[19] Anahory T,Dechaud H,Bennes R,et al.Identification of new proteins in follicular fluid of mature human follicles[J].Electrophoresis,2002,23:1197-1202.
[20] Lee H C,Lee S W,Lee K W,et al.Identification of new proteins in follicular fluid from mature human follicles by direct sample rehydration method of two-dimensional polyacrylamide gel electrophoresis[J].Journal of Korean Medical Science,2005,20(2):456-460.

牦牛卵泡液差异蛋白质组双向电泳图谱的构建与质谱分析

Establishment of Two-dimensional Electrophoresis Mapping and Mass Spectrometry Analysis of the Differential Proteome of Yak Follicular Fluid

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