利用TMT标记结合2D LC-MS/MS技术分析不同时期水牛睾丸曲精细管差异蛋白质组

doi:10.11843/j.issn.0366-6964.2014.08.009

畜牧兽医学报 ›› 2014, Vol. 45 ›› Issue (8): 1265-1273.doi: 10.11843/j.issn.0366-6964.2014.08.009

利用TMT标记结合2D LC-MS/MS技术分析不同时期水牛睾丸曲精细管差异蛋白质组

黄愉淋¹，付强¹，黄德伦¹，潘宏¹，黄凤玲¹，梁贤威^2*，张明^1*

（1.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；2.中国农业科学院广西水牛研究所，南宁 530001）

收稿日期:2014-01-20 出版日期:2014-08-23 发布日期:2014-08-23
通讯作者: 梁贤威，研究员，博士生导师，E-mail：Liangbri@126.com；张明，教授，博士生导师，E-mail：mingzhang@gxu.edu.cn
作者简介:黄愉淋（1988-），女，壮族，广西大化人，博士生，主要从事动物蛋白质组学的研究，E-mail：huangyulin1016@163.com
基金资助:
国家自然科学基金（30860192）；广西水牛遗传繁育重点实验室开放课题（SNKF-2012-05;SNKF-2013-02）；亚热带农业生物资源保护与利用国家重点实验室开放课题（KSL-CUSAb-2012-06）

Comparative Analysis of Proteomics at Different Developmental Stages of Buffalo Testicular Seminiferous Tubules by TMT-Coupled 2D LC-MS/MS

HUANG Yu-lin¹，FU Qiang¹，HUANG De-lun¹，PAN Hong¹,HUANG Feng-ling¹,LIANG Xian-wei^2*，ZHANG Ming^1*

（1.State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources，Animal Reproduction Institute，Guangxi University，Nanning 530004，China；2.Buffalo Research Institute，Chinese Academy of Agricultural Sciences，Nanning 530001，China）

Received:2014-01-20 Online:2014-08-23 Published:2014-08-23

摘要/Abstract

摘要：

旨在采用TMT标记结合二维高效液相色谱/串联质谱联用的技术对不同时期水牛睾丸曲精细管的差异蛋白质组进行分析。分别提取不同时期（幼年期、老年期）水牛睾丸曲精细管总蛋白，TMT试剂标记后进行强阳离子交换柱分离多肽、nano LC分离，同时在线连接电喷雾串联Orbitrap质谱进行分析，通过SEQUEST软件搜库并对鉴定的差异蛋白数据进行生物信息学分析。结果表明：共筛选出定量差异倍数≥2倍的差异蛋白89个，以幼年期水牛睾丸曲精细管作为对照，上调蛋白质51个，下调蛋白质38个。生物信息学分析表明，TMT标记技术结合二维液相色谱串联质谱可以对不同发育时期水牛睾丸曲精细管蛋白进行有效地分离和鉴定。本研究结果为探索精子发生过程中蛋白质表达的变化规律提供了试验依据，找出可能与精子发生相关的标志性蛋白：谷胱甘肽S转移酶P（GSTP1）、过氧化氢酶（CAT）、热休克蛋白(HSP90AA1和HSPA)。

Abstract:

To investigate expression of proteins from buffalo testicular seminiferous tubules at different developmental stages (nepionic and senescence).Tandem mass tag (TMT) coupled to two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS).Identification was based on tandem mass tag (TMT)-coupled to 2D liquid chromatography tandem mass spectrometry (2D LC-MS/MS) using LC-ESI LTQ-Orbitrap Elite MS/MS equipment.Detected proteins were processed by bioinformatics analysis using the SEQUEST search engine.Eighty-nine different proteins were obtained with a more than two-fold change between developmental stages.Compared with the nepionic stage，51 proteins were up-regulated and 38 proteins were down-regulated.TMT-coupled 2D LC-MS/MS was an appropriate method for isolating and identifying protein groups of buffalo testicular seminiferous tubules at different developmental stages.The results provided an experimental basis for exploring changes in protein expression during spermatogenesis，and found biological markers of spermatogenesis，possibly Glutathione S-transferase P，Catalase，Heat shock protein 90-alpha and Heat shock 70 ku protein 1-like.

中图分类号:

S823.8⁺3.2

黄愉淋，付强，黄德伦，潘宏，黄凤玲，梁贤威，张明. 利用TMT标记结合2D LC-MS/MS技术分析不同时期水牛睾丸曲精细管差异蛋白质组[J]. 畜牧兽医学报, 2014, 45(8): 1265-1273.

HUANG Yu-lin，FU Qiang，HUANG De-lun，PAN Hong,HUANG Feng-ling,LIANG Xian-wei，ZHANG Ming. Comparative Analysis of Proteomics at Different Developmental Stages of Buffalo Testicular Seminiferous Tubules by TMT-Coupled 2D LC-MS/MS[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(8): 1265-1273.

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利用TMT标记结合2D LC-MS/MS技术分析不同时期水牛睾丸曲精细管差异蛋白质组

Comparative Analysis of Proteomics at Different Developmental Stages of Buffalo Testicular Seminiferous Tubules by TMT-Coupled 2D LC-MS/MS

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