Globozoospermia is a severe form of teratozoospermia characterized by round-headed spermatozoa with an absent acrosome, an aberrant nuclear membrane and midpiece defects. Globozoospermia is diagnosed by the presence o...Globozoospermia is a severe form of teratozoospermia characterized by round-headed spermatozoa with an absent acrosome, an aberrant nuclear membrane and midpiece defects. Globozoospermia is diagnosed by the presence of 100% round-headed spermatozoa on semen analysis, and patients with this condition are absolutely infertile. The objective of this study was to investigate the differences in protein expression between human round- headed and normal spermatozoa. Two-dimensional （2-D） fluorescence difference gel electrophoresis （DIGE） coupled with mass spectrometry （MS） was used in this study. Over 61 protein spots were analysed in each paired normal/round-headed comparison, using DIGE technology along with an internal standard. In total, 35 protein spots identified by tandem mass spectrometry （MS/MS） exhibited significant changes （paired t-test, P 〈 0.05） in the expression level between normal and round-headed spermatozoa. A total of nine proteins were found to be upregulated and 26 proteins were found to be downregulated in round-headed spermatozoa compared with normal spermatozoa. The differentially expressed proteins that we identified may have important roles in a variety of cellular processes and structures, including spermatogenesis, cell skeleton, metabolism and spermatozoa motility.展开更多
Aim： To extend the analysis of the proteome of human spermatozoa and establish a 2-D gel electrophoresis （2-DE） reference map of human spermatozoal proteins in a pH range of 3.5-9.0. Methods： In order to reveal mo...Aim： To extend the analysis of the proteome of human spermatozoa and establish a 2-D gel electrophoresis （2-DE） reference map of human spermatozoal proteins in a pH range of 3.5-9.0. Methods： In order to reveal more protein spots, immobilized pH gradient strips （24 cm） of broad range of pH 3-10 and the narrower range of pH 6-9, as well as different overlapping narrow range pH immobilized pH gradient （IPG） strips, including 3.5-4.5, 4.0-5.0, 4.5-5.5, 5.0-6.0 and 5.5-6.7, were used. After 2-DE, several visually identical spots between the different pH range 2-D gel pairs were cut from the gels and confirmed by mass spectrometry and used as landmarks for computer analysis. Results： The 2-D reference map with pH value from 3.5 to 9.0 was synthesized by using the ImageMaster analysis software. The overlapping spots were excluded, so that every spot was counted only once. A total of 3 872 different protein spots were identified from the reference map, an approximately 3-fold increase compared to the broad range pH 3-10 IPG strip （1 306 spots）. Conclusion： The present 2-D pattern is a high resolution 2-D reference map for human fertile spermatozoal protein spots. A comprehensive knowledge of the protein composition of human spermatozoa is very meaningful in studying dysregulation of male fertility.展开更多
Proteomics is one of the most active research fields in the post-genomic era. Here we briefly introduce the scientific background of the origination of proteomics and its content, research method. The new developments...Proteomics is one of the most active research fields in the post-genomic era. Here we briefly introduce the scientific background of the origination of proteomics and its content, research method. The new developments of proteomics at the levels of individual plants, tissues, organs and organells, as well as its applications in the area of plant genetic diversity, mutant characterization, and plant physiology, etc are reviewed. At last, the challenge and prospect of proteomics are discussed.展开更多
为建立一套适合海滨木槿叶片蛋白质组学分析的双向电泳体系,以海滨木槿无性系叶片为材料,分别采用裂解液法、三氯乙酸-丙酮沉淀法、改良酚抽法分离纯化蛋白质,选用24 cm pH 3～10的IPG胶条,并对不同上样量、等电聚焦程序、平衡时间等电...为建立一套适合海滨木槿叶片蛋白质组学分析的双向电泳体系,以海滨木槿无性系叶片为材料,分别采用裂解液法、三氯乙酸-丙酮沉淀法、改良酚抽法分离纯化蛋白质,选用24 cm pH 3～10的IPG胶条,并对不同上样量、等电聚焦程序、平衡时间等电泳条件进行优化。结果表明:采用改良酚抽法提取蛋白质,上样量为每IPG胶条1 000μg,总聚焦功率90 kV.h,平衡时间15 min,胶体考马斯亮蓝染色,可得到蛋白质点数目多、分辨率高的双向电泳图谱。重复性试验结果显示,该体系具有很好的稳定性及可重复性,可满足海滨木槿蛋白质组学研究的要求。展开更多
采用双向电泳技术,对重金属镉暴露后海洋鱼类大弹涂鱼肝脏组织的差异蛋白质进行了研究。pH=5～8、7 cm胶条电泳图谱经PDquest软件分析,结果表明,对照组、慢性胁迫组和急性胁迫组平均检测到的蛋白点数分别为512±35、509±29和53...采用双向电泳技术,对重金属镉暴露后海洋鱼类大弹涂鱼肝脏组织的差异蛋白质进行了研究。pH=5～8、7 cm胶条电泳图谱经PDquest软件分析,结果表明,对照组、慢性胁迫组和急性胁迫组平均检测到的蛋白点数分别为512±35、509±29和532±22,匹配率约为90%,急性镉胁迫后差异蛋白点数为14个,其中7个蛋白点在胁迫后表达量显著下调,4个蛋白点显著上调,消失1个蛋白点,并新增1个蛋白点。对其中7个差异蛋白点进行肽指纹图谱分析(MALDI-TOFMS),差异蛋白分别为K18,prohibitin,GTP-binding protein Ypt1,two-componentsystem response regulator,similar to T-complex protein 1 subunit theta(TCP-1-theta)(CCT-theta),transcriptional regulator,CopG family和mitogenactivatedprotein kinase homologue。而慢性镉胁迫后差异表达点数为15个,其中8个蛋白点在胁迫后表达量显著上升,5个蛋白点显著下调,消失1个蛋白点,并新增1个蛋白点。对其中6个差异蛋白点进行肽指纹图谱分析(MALDI-TOF-MS),差异蛋白分别为GTP-binding protein Ypt1,two-component system response regulator,hypothetical protein BT-1634,keratin9,anscriptional regulator,CopG family和mitogen-activated protein kinase homologue。通过分析各蛋白的变化,探讨了慢性(0.05 mg/L,30 d)和急性(5mg/L,3 d)重金属镉胁迫的致毒机制。在急慢性镉胁迫胁迫下,不仅大弹涂鱼体内的蛋白表达发生相应的变化,鱼体内环境的动态平衡也被打破。这种变化超过大弹涂鱼的自我解毒和保护能力,就会使得肝脏细胞的正常代谢受到破坏,引起细胞凋亡。展开更多
目的建立血清蛋白质组学研究中高丰度蛋白的去除方法并评价其去除效果。方法应用Sigma公司2006年最新推出的血清白蛋白和IgG去除试剂盒(ProteoPrep Immunoaffinity Albumin and IgG Depletion Kit)去除血清中高丰度的白蛋白和IgG等,再...目的建立血清蛋白质组学研究中高丰度蛋白的去除方法并评价其去除效果。方法应用Sigma公司2006年最新推出的血清白蛋白和IgG去除试剂盒(ProteoPrep Immunoaffinity Albumin and IgG Depletion Kit)去除血清中高丰度的白蛋白和IgG等,再用双向凝胶电泳(2-DE)分析去除的效果。结果本实验建立的血清中高丰度蛋白的去除方法,约95%的白蛋白和IgG能够被去除,2-DE图谱中蛋白质点更加清楚,部分低丰度蛋白得以显现。结论成功建立血清中高丰度蛋白的去除方法,可为今后的血清蛋白质组学研究奠定基础。展开更多
基金Acknowledgment We thank Beijing Proteome Research Center, （Beijing, China） for its enthusiastic technological support and for the theory of 2-D DIGE. We also thank（Changsha, China） College of Life Sciences at Hunan Normal University for supporting the MS technology. Finally, we are very grateful to our collaborators for their help, as well as their valuable discussions and suggestions during the course of this work. This work was supported by two grants from the National Natural Science Foundation of China （NO. 30170480 and NO. 30470884）.
文摘Globozoospermia is a severe form of teratozoospermia characterized by round-headed spermatozoa with an absent acrosome, an aberrant nuclear membrane and midpiece defects. Globozoospermia is diagnosed by the presence of 100% round-headed spermatozoa on semen analysis, and patients with this condition are absolutely infertile. The objective of this study was to investigate the differences in protein expression between human round- headed and normal spermatozoa. Two-dimensional （2-D） fluorescence difference gel electrophoresis （DIGE） coupled with mass spectrometry （MS） was used in this study. Over 61 protein spots were analysed in each paired normal/round-headed comparison, using DIGE technology along with an internal standard. In total, 35 protein spots identified by tandem mass spectrometry （MS/MS） exhibited significant changes （paired t-test, P 〈 0.05） in the expression level between normal and round-headed spermatozoa. A total of nine proteins were found to be upregulated and 26 proteins were found to be downregulated in round-headed spermatozoa compared with normal spermatozoa. The differentially expressed proteins that we identified may have important roles in a variety of cellular processes and structures, including spermatogenesis, cell skeleton, metabolism and spermatozoa motility.
基金This work was supported by two grants from the National Natural Science Foundation of China （No. 30170480 and No. 30470884）. We would like to thank Professor Guang-Yin Lu for enthusiastic reading and amending of the manuscript. We would like to thank Mr Ji-Xian Xiong, College of Life Science at the Hunan Normal University for enthusiastic support of technology and theory of MS. We are also very grateful to collaborators in our laboratories for help and valuable discussions and suggestions during the course of this work.
文摘Aim： To extend the analysis of the proteome of human spermatozoa and establish a 2-D gel electrophoresis （2-DE） reference map of human spermatozoal proteins in a pH range of 3.5-9.0. Methods： In order to reveal more protein spots, immobilized pH gradient strips （24 cm） of broad range of pH 3-10 and the narrower range of pH 6-9, as well as different overlapping narrow range pH immobilized pH gradient （IPG） strips, including 3.5-4.5, 4.0-5.0, 4.5-5.5, 5.0-6.0 and 5.5-6.7, were used. After 2-DE, several visually identical spots between the different pH range 2-D gel pairs were cut from the gels and confirmed by mass spectrometry and used as landmarks for computer analysis. Results： The 2-D reference map with pH value from 3.5 to 9.0 was synthesized by using the ImageMaster analysis software. The overlapping spots were excluded, so that every spot was counted only once. A total of 3 872 different protein spots were identified from the reference map, an approximately 3-fold increase compared to the broad range pH 3-10 IPG strip （1 306 spots）. Conclusion： The present 2-D pattern is a high resolution 2-D reference map for human fertile spermatozoal protein spots. A comprehensive knowledge of the protein composition of human spermatozoa is very meaningful in studying dysregulation of male fertility.
文摘Proteomics is one of the most active research fields in the post-genomic era. Here we briefly introduce the scientific background of the origination of proteomics and its content, research method. The new developments of proteomics at the levels of individual plants, tissues, organs and organells, as well as its applications in the area of plant genetic diversity, mutant characterization, and plant physiology, etc are reviewed. At last, the challenge and prospect of proteomics are discussed.
文摘采用双向电泳技术,对重金属镉暴露后海洋鱼类大弹涂鱼肝脏组织的差异蛋白质进行了研究。pH=5～8、7 cm胶条电泳图谱经PDquest软件分析,结果表明,对照组、慢性胁迫组和急性胁迫组平均检测到的蛋白点数分别为512±35、509±29和532±22,匹配率约为90%,急性镉胁迫后差异蛋白点数为14个,其中7个蛋白点在胁迫后表达量显著下调,4个蛋白点显著上调,消失1个蛋白点,并新增1个蛋白点。对其中7个差异蛋白点进行肽指纹图谱分析(MALDI-TOFMS),差异蛋白分别为K18,prohibitin,GTP-binding protein Ypt1,two-componentsystem response regulator,similar to T-complex protein 1 subunit theta(TCP-1-theta)(CCT-theta),transcriptional regulator,CopG family和mitogenactivatedprotein kinase homologue。而慢性镉胁迫后差异表达点数为15个,其中8个蛋白点在胁迫后表达量显著上升,5个蛋白点显著下调,消失1个蛋白点,并新增1个蛋白点。对其中6个差异蛋白点进行肽指纹图谱分析(MALDI-TOF-MS),差异蛋白分别为GTP-binding protein Ypt1,two-component system response regulator,hypothetical protein BT-1634,keratin9,anscriptional regulator,CopG family和mitogen-activated protein kinase homologue。通过分析各蛋白的变化,探讨了慢性(0.05 mg/L,30 d)和急性(5mg/L,3 d)重金属镉胁迫的致毒机制。在急慢性镉胁迫胁迫下,不仅大弹涂鱼体内的蛋白表达发生相应的变化,鱼体内环境的动态平衡也被打破。这种变化超过大弹涂鱼的自我解毒和保护能力,就会使得肝脏细胞的正常代谢受到破坏,引起细胞凋亡。
文摘目的建立血清蛋白质组学研究中高丰度蛋白的去除方法并评价其去除效果。方法应用Sigma公司2006年最新推出的血清白蛋白和IgG去除试剂盒(ProteoPrep Immunoaffinity Albumin and IgG Depletion Kit)去除血清中高丰度的白蛋白和IgG等,再用双向凝胶电泳(2-DE)分析去除的效果。结果本实验建立的血清中高丰度蛋白的去除方法,约95%的白蛋白和IgG能够被去除,2-DE图谱中蛋白质点更加清楚,部分低丰度蛋白得以显现。结论成功建立血清中高丰度蛋白的去除方法,可为今后的血清蛋白质组学研究奠定基础。
