水牛卵泡液差异蛋白质双向电泳方法的建立及质谱分析

doi:10.11843/j.issn.0366-6964.2013.08.010

畜牧兽医学报 ›› 2013, Vol. 44 ›› Issue (8): 1244-1250.doi: 10.11843/j.issn.0366-6964.2013.08.010

水牛卵泡液差异蛋白质双向电泳方法的建立及质谱分析

黄愉淋，黄德伦，官俊良，潘秋庄，付强，黄凤玲，张明^*，卢克焕

（广西大学，动物繁殖研究所，亚热带农业生物资源保护与利用国家重点实验室，南宁 530004）

收稿日期:2013-01-30 出版日期:2013-08-23 发布日期:2013-08-23
通讯作者: 张明，教授，E-mail：mingzhang@gxu.edu.cn
作者简介:黄愉淋（1988-），女，壮族，广西大化人，硕士，主要从事动物蛋白质组学研究，E-mail：huangyulin1016@163.com
基金资助:
广西自然科学基金（2011GXNSFB018028）；国家自然科学基金（30860192）；高等学校博士点科研基金（20104501110001）

Establishment of Two Dimensional Electrophoresis Method and Mass Spectrumetry Analysis of the Differential Proteins of Buffalo Follicular Fluid

HUANG Yu-lin, HUANG De-lun, GUAN Jun-liang, PAN Qiu-zhuang, FU Qiang,HUANG Feng-ling, ZHANG Ming^*, LU Ke-huan

（State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources，Animal Reproduction Institute，Guangxi University，Nanning 530004， China）

Received:2013-01-30 Online:2013-08-23 Published:2013-08-23

摘要/Abstract

摘要：

旨在研究沼泽型水牛成熟前后卵泡内差异表达蛋白质的变化规律。采用双向凝胶电泳技术分离成熟卵泡液和未成熟卵泡液总蛋白质，建立和优化了卵泡液的双向电泳体系，并使用质谱鉴定差异表达蛋白点。结果显示，丙酮沉淀法处理得到的总蛋白质样品后，在24 cm（pH 4~7）胶条且上样量350 μg时得到分辨率较好的双向电泳图谱。软件分析得到11个差异蛋白点，以成熟卵泡液作为对照，5个蛋白点表达上调，3个蛋白点表达下调，1个蛋白点缺失，2个蛋白点在未成熟卵泡液中特异性表达。质谱成功鉴定出4个蛋白质：过氧化物酶-2、醛糖还原酶、牛纤维蛋白原的晶体结构、转甲状腺素蛋白。该研究建立了良好的卵泡液双向电泳体系，分析并鉴定一批水牛卵泡液差异蛋白质，对于研究水牛卵母细胞的发育微环境和成熟机制提供了新的研究线索。

Abstract:

This study was undertaken to evaluate protein expression differences between mature and immature follicular fluids of swamp buffalo in Guangxi. The research used two-dimensional gel electrophoresis (2DE) coupled to Matrix Assisted Laser Desorption/Ionization Time-of-flight Tandem Mass Spectrometry (MALDI-TOF/TOF) to analyze the different proteins, and the establishment of a suitable 2DE system for separating buffalo follicular fluid proteins was described. The results indicated that acetone precipitation in a loading volume of 350 μg in 24 cm IPG strips (pH 4-7) improved resolution and the quality of the 2DE maps, and 11 different expression protein spots were found with Image Master 2D platinum software. Compared with the mature follicular fluid 2DE map, 5 of these proteins were up-regulated in immature buffalo follicular fluid, 3 were down-regulated, 1 deleted and 2 proteins specifically expressed. Finally, 4 proteins were successfully identified by mass spectrometry; these are peroxiredoxin-2, aldose reductase, fibrinogen gamma-B chain precursor, and transthyretin precursor. In conclusion, an appropriate 2DE system for separating buffalo follicular fluid proteins was established and optimized. Different expression proteins from mature and immature buffalo follicular fluids were successfully analyzed and identified to provide new research insight into the developmental microenviroment and maturation mechanism for buffalo oocytes.

中图分类号:

S823.8⁺3 .2

黄愉淋，黄德伦，官俊良，潘秋庄，付强，黄凤玲，张明，卢克焕. 水牛卵泡液差异蛋白质双向电泳方法的建立及质谱分析[J]. 畜牧兽医学报, 2013, 44(8): 1244-1250.

HUANG Yu-lin, HUANG De-lun, GUAN Jun-liang, PAN Qiu-zhuang, FU Qiang,HUANG Feng-ling, ZHANG Ming, LU Ke-huan. Establishment of Two Dimensional Electrophoresis Method and Mass Spectrumetry Analysis of the Differential Proteins of Buffalo Follicular Fluid[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(8): 1244-1250.

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水牛卵泡液差异蛋白质双向电泳方法的建立及质谱分析

Establishment of Two Dimensional Electrophoresis Method and Mass Spectrumetry Analysis of the Differential Proteins of Buffalo Follicular Fluid

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