BACKGROUND： The use of fluorescent two-dimensional difference gel electrophoresis （2D-DIGE） has been shown to compensate for the shortcomings of conventional two-dimensional gel electrophoresis, such as poor repeat...BACKGROUND： The use of fluorescent two-dimensional difference gel electrophoresis （2D-DIGE） has been shown to compensate for the shortcomings of conventional two-dimensional gel electrophoresis, such as poor repeatability and large systematic errors. However, little information is presently available regarding the use of 2D-DIGE to investigate mechanisms of ischemic cerebrovascular diseases. Plasma and body fluids have been utilized in proteomic technology to study ischemic cerebrovascular diseases. OBJECTIVE： To perform proteomic analysis of fresh rat brain tissue in peripheral ischemic regions using 2D-DIGE 6 hours after middle cerebral artery occlusion （MCAO）, and to identify specific proteins closely associated with early ischemic cerebrovascular diseases. DESIGN, TIME AND SETTING： Proteomics-based, randomized, controlled, animal experiment was performed at the Laboratories of Neurology and Proteomics, Jilin University between January and April 2006. MATERIALS： 2, 3, 5-triphenyl tetrazolium chloride was purchased from Sigma, USA. Ettan DALTSix system, DeCyder DIA V5.0 differential analysis software, and Ettan matrix-assisted laser desorption/ionization time-of-flight mass spectrometer （MALDI-TOF-MS） were purchased from Amersham Bioscience, Sweden. METHODS： Eight healthy, male, Wistar rats were randomized to experimental and control groups, with four rats in each group. In the experimental group, rat models of focal cerebral ischemia were established by MCAO. In the control group, the internal and external carotid arteries were exposed and then immediately sutured, and the remaining procedures were identical to the experimental group. MAIN OUTCOME MEASURES： At 6 hours after cerebral ischemia, protein expression in the peripheral ischemia region of the experimental group was compared with the control group using 2D-DIGE. Protein spots that exhibited statistical differences between experimental and control groups with 〉 1.4 attributable risk were screened using DeCyder DIA V5.0 differential analysis software. Differential proteins were identified using MALDI-TOF-MS. RESULTS： Triphenyl tetrazolium chloride staining results revealed pink, normal brain tissue and white, ischemic brain tissue, suggesting successful MCAO establishment. The average matching rate of four 2D-DIGE gels was 92.4%. There were （1 758 ± 43） protein spots on each gel, with similar distribution modes. At 6 hours after focal cerebral ischemia, 13 protein spots exhibited marked expression changes, including significantly increased （n = 7） and decreased （n = 6） expression （P 〈 0.05）. MALDI-TOF-MS results revealed two differential protein spots： a-tubulin and heat shock protein 27, which were significantly decreased in the experimental group compared with the control group （P 〈 0.05）. CONCLUSION： Thirteen protein spots with expression changes were revealed by 2D-DIGE proteomics technology. Of them, a-tubulin and heat shock protein 27 expressions were markedly decreased during the early stage of cerebral ischemia. These two proteins were presumed to be proteins associated with early ischemic cerebrovascular diseases.展开更多
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
To gain insight into the molecular mechanisms of resveratrol-mediated neuroprotection, two-dimensional difference gel electrophoresis in combination with matrix-assisted laser desorption ionization time-of-flight mass...To gain insight into the molecular mechanisms of resveratrol-mediated neuroprotection, two-dimensional difference gel electrophoresis in combination with matrix-assisted laser desorption ionization time-of-flight mass spectrometry was used to identify proteins differentially-expressed in SH-SY5Y cells treated with resveratrol. Compared with the control group, resveratrol treatment significantly affected the expression of four proteins： endoplasmic reticulum oxidoreductin 1-like protein alpha, p21-activated kinase 1, Archain 1, and T cell receptor beta chain. The former three were downregulated and the latter was upregulated. These proteins are primarily associated with endoplasmic reticulum stress, intracellular trafficking, and immune function.展开更多
This study sought to identify differentially expressed proteins in SH-SY5Y cells treated with valproic acid, using two-dimensional difference gel electrophoresis analysis. Three proteins were unambi-guously identified...This study sought to identify differentially expressed proteins in SH-SY5Y cells treated with valproic acid, using two-dimensional difference gel electrophoresis analysis. Three proteins were unambi-guously identified： the eukaryotic translation initiation factor 4A isoform 1 and ATP6V1B2 protein were downregulated, while the heterogeneous nuclear ribonucleoprotein K was upregulated. Moreover, all three proteins are associated with altered expression due to oxidative stress. Ma-trix-assisted laser desorption/ionization-time of flight mass spectrometry and protein immunoblotting assay confirmed the differential expression of eukaryotic translation initiation factor 4A isoform 1. The results indicate that valproic acid exerts an antioxidation effect by regulating the expression of eukaryotic translation initiation factor 4A isoform 1.展开更多
目的寻找冠心病血瘀证患者冠脉事件关键血小板功能蛋白。方法纳入冠心病血瘀证组患者,设立正常对照组,每组分4个小组,即两组各有4次重复。抽取外周血,分离血小板并提取蛋白,将各个提取血小板蛋白样本按小组等体积混合、定量。用荧光差...目的寻找冠心病血瘀证患者冠脉事件关键血小板功能蛋白。方法纳入冠心病血瘀证组患者,设立正常对照组,每组分4个小组,即两组各有4次重复。抽取外周血,分离血小板并提取蛋白,将各个提取血小板蛋白样本按小组等体积混合、定量。用荧光差异显示二维凝胶电泳技术筛选、基质辅助激光解析/电离-飞行时间质谱技术鉴定血小板差异功能蛋白。对鉴定成功差异蛋白,用Western-blotting进一步验证。结果筛选出13个差异蛋白点,质谱成功鉴定7个:isoform 1 of integrinalpha-Ⅱb、isoform 2 of integrinalpha-Ⅱb、actin-cytoplasmic1、actin-cytoplasmic 2、cDNAFLJ52842、cDNAFLJ55253、cDNAFLJ43573fis。其中isoform2ofintegrinalpha-Ⅱb(CD41)和actin-cytoplasmic2(Actinγ)成功验证。结论 CD41和Actinγ是冠心病血瘀证的标志蛋白。其他血小板功能蛋白的异常表达可能在冠心病血瘀证事件发生发展中起关键作用。展开更多
目的:筛查甲胎蛋白(α-fetoprotein,AFP)阴性的肝癌患者和肝硬化患者血清中的差异表达蛋白。方法:选择6例AFP阴性的肝癌患者和6例AFP阴性的肝硬化患者。应用MARS系统分离获得各组血清标本中的低丰度蛋白,采用双向荧光差异凝胶电泳(two-d...目的:筛查甲胎蛋白(α-fetoprotein,AFP)阴性的肝癌患者和肝硬化患者血清中的差异表达蛋白。方法:选择6例AFP阴性的肝癌患者和6例AFP阴性的肝硬化患者。应用MARS系统分离获得各组血清标本中的低丰度蛋白,采用双向荧光差异凝胶电泳(two-dimensional fluorescence difference gel electrophoresis,2D-DIGE)技术筛选两组间有表达差异的蛋白,并用蛋白质印迹法加以验证。结果:2D-DIGE技术共鉴定出29种差异蛋白,其中AFP阴性肝癌患者血清中高表达的蛋白有22种,低表达的蛋白有7种。差异蛋白包括激肽原1、四连接素、基膜聚糖蛋白、补体1r和锌α2糖蛋白等。蛋白质印迹法证实,AFP阴性的肝癌患者血清中锌α2糖蛋白表达水平明显低于AFP阴性的肝硬化患者(P<0.05),同DIGE结果一致。结论:采用蛋白质组学技术筛查出的一系列在AFP阴性肝硬化和AFP阴性肝癌患者血清中有表达差异的蛋白有望作为AFP阴性肝癌患者早期诊断和辅助诊断的分子标志物。展开更多
目的:筛选人急性髓系白血病细胞株HL-60甲基化沉默基因,为揭示白血病的发病机制及防治提供科学依据。方法:应用荧光差异双向凝胶电泳(two-dimensional fluorescence difference gel electro-phoresis,F-2D-DIGE)分离甲基转移酶抑制剂5-...目的:筛选人急性髓系白血病细胞株HL-60甲基化沉默基因,为揭示白血病的发病机制及防治提供科学依据。方法:应用荧光差异双向凝胶电泳(two-dimensional fluorescence difference gel electro-phoresis,F-2D-DIGE)分离甲基转移酶抑制剂5-杂氮-2-脱氧胞苷(5-aza-2-dC)处理与未处理的HL-60细胞的总蛋白质,Decyder和PDquest图像分析软件识别差异表达蛋白质点,基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)技术鉴定差异表达蛋白质。结果:建立了5-aza-2-dC处理与未处理的HL-60细胞蛋白质的F-2D-DIGE图谱,图像分析识别了53个差异表达的蛋白质点,质谱分析鉴定了35个差异表达的蛋白质,其中32个蛋白质在5-aza-2-dC处理后的HL-60细胞中表达上调,3个蛋白质表达下调。结论:35个差异表达蛋白可能与甲基化相关,为白血病的表观遗传学研究提供了有价值的信息。展开更多
以干酪乳杆菌Lactobacillus casei Zhang为出发菌株,通过适应性进化获得了干酪乳杆菌酸胁迫抗性驯化菌株。对细胞内微环境的检测发现,驯化菌株在酸胁迫过程中能够维持较高的磷酸烯醇式丙酮酸-糖转移酶系统活力,并具有较高的H^+-ATPase...以干酪乳杆菌Lactobacillus casei Zhang为出发菌株,通过适应性进化获得了干酪乳杆菌酸胁迫抗性驯化菌株。对细胞内微环境的检测发现,驯化菌株在酸胁迫过程中能够维持较高的磷酸烯醇式丙酮酸-糖转移酶系统活力,并具有较高的H^+-ATPase活性以及胞内ATP浓度。蛋白质组学分析结果表明,酸胁迫引发了细胞蛋白表达的变化,与原始菌株相比,驯化菌株保持了更高的代谢活性;同时,驯化菌株通过大量诱导应激蛋白如分子伴侣GroEL、GrpE,冷/热应激蛋白CspC、DnaK等维持了细胞的生理活性,有效提高了细胞对酸胁迫的抵御能力。本研究为进一步揭示酸胁迫下乳酸菌细胞的生理应答机制,探寻促进乳酸菌酸胁迫性能提升的最优策略,进而改善其在生产中的应用性能提供了可借鉴的思路。展开更多
基金the National Natural Science Foundation of China, No.30470588
文摘BACKGROUND： The use of fluorescent two-dimensional difference gel electrophoresis （2D-DIGE） has been shown to compensate for the shortcomings of conventional two-dimensional gel electrophoresis, such as poor repeatability and large systematic errors. However, little information is presently available regarding the use of 2D-DIGE to investigate mechanisms of ischemic cerebrovascular diseases. Plasma and body fluids have been utilized in proteomic technology to study ischemic cerebrovascular diseases. OBJECTIVE： To perform proteomic analysis of fresh rat brain tissue in peripheral ischemic regions using 2D-DIGE 6 hours after middle cerebral artery occlusion （MCAO）, and to identify specific proteins closely associated with early ischemic cerebrovascular diseases. DESIGN, TIME AND SETTING： Proteomics-based, randomized, controlled, animal experiment was performed at the Laboratories of Neurology and Proteomics, Jilin University between January and April 2006. MATERIALS： 2, 3, 5-triphenyl tetrazolium chloride was purchased from Sigma, USA. Ettan DALTSix system, DeCyder DIA V5.0 differential analysis software, and Ettan matrix-assisted laser desorption/ionization time-of-flight mass spectrometer （MALDI-TOF-MS） were purchased from Amersham Bioscience, Sweden. METHODS： Eight healthy, male, Wistar rats were randomized to experimental and control groups, with four rats in each group. In the experimental group, rat models of focal cerebral ischemia were established by MCAO. In the control group, the internal and external carotid arteries were exposed and then immediately sutured, and the remaining procedures were identical to the experimental group. MAIN OUTCOME MEASURES： At 6 hours after cerebral ischemia, protein expression in the peripheral ischemia region of the experimental group was compared with the control group using 2D-DIGE. Protein spots that exhibited statistical differences between experimental and control groups with 〉 1.4 attributable risk were screened using DeCyder DIA V5.0 differential analysis software. Differential proteins were identified using MALDI-TOF-MS. RESULTS： Triphenyl tetrazolium chloride staining results revealed pink, normal brain tissue and white, ischemic brain tissue, suggesting successful MCAO establishment. The average matching rate of four 2D-DIGE gels was 92.4%. There were （1 758 ± 43） protein spots on each gel, with similar distribution modes. At 6 hours after focal cerebral ischemia, 13 protein spots exhibited marked expression changes, including significantly increased （n = 7） and decreased （n = 6） expression （P 〈 0.05）. MALDI-TOF-MS results revealed two differential protein spots： a-tubulin and heat shock protein 27, which were significantly decreased in the experimental group compared with the control group （P 〈 0.05）. CONCLUSION： Thirteen protein spots with expression changes were revealed by 2D-DIGE proteomics technology. Of them, a-tubulin and heat shock protein 27 expressions were markedly decreased during the early stage of cerebral ischemia. These two proteins were presumed to be proteins associated with early ischemic cerebrovascular diseases.
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.
文摘To gain insight into the molecular mechanisms of resveratrol-mediated neuroprotection, two-dimensional difference gel electrophoresis in combination with matrix-assisted laser desorption ionization time-of-flight mass spectrometry was used to identify proteins differentially-expressed in SH-SY5Y cells treated with resveratrol. Compared with the control group, resveratrol treatment significantly affected the expression of four proteins： endoplasmic reticulum oxidoreductin 1-like protein alpha, p21-activated kinase 1, Archain 1, and T cell receptor beta chain. The former three were downregulated and the latter was upregulated. These proteins are primarily associated with endoplasmic reticulum stress, intracellular trafficking, and immune function.
文摘This study sought to identify differentially expressed proteins in SH-SY5Y cells treated with valproic acid, using two-dimensional difference gel electrophoresis analysis. Three proteins were unambi-guously identified： the eukaryotic translation initiation factor 4A isoform 1 and ATP6V1B2 protein were downregulated, while the heterogeneous nuclear ribonucleoprotein K was upregulated. Moreover, all three proteins are associated with altered expression due to oxidative stress. Ma-trix-assisted laser desorption/ionization-time of flight mass spectrometry and protein immunoblotting assay confirmed the differential expression of eukaryotic translation initiation factor 4A isoform 1. The results indicate that valproic acid exerts an antioxidation effect by regulating the expression of eukaryotic translation initiation factor 4A isoform 1.
文摘目的寻找冠心病血瘀证患者冠脉事件关键血小板功能蛋白。方法纳入冠心病血瘀证组患者,设立正常对照组,每组分4个小组,即两组各有4次重复。抽取外周血,分离血小板并提取蛋白,将各个提取血小板蛋白样本按小组等体积混合、定量。用荧光差异显示二维凝胶电泳技术筛选、基质辅助激光解析/电离-飞行时间质谱技术鉴定血小板差异功能蛋白。对鉴定成功差异蛋白,用Western-blotting进一步验证。结果筛选出13个差异蛋白点,质谱成功鉴定7个:isoform 1 of integrinalpha-Ⅱb、isoform 2 of integrinalpha-Ⅱb、actin-cytoplasmic1、actin-cytoplasmic 2、cDNAFLJ52842、cDNAFLJ55253、cDNAFLJ43573fis。其中isoform2ofintegrinalpha-Ⅱb(CD41)和actin-cytoplasmic2(Actinγ)成功验证。结论 CD41和Actinγ是冠心病血瘀证的标志蛋白。其他血小板功能蛋白的异常表达可能在冠心病血瘀证事件发生发展中起关键作用。
文摘目的:筛查甲胎蛋白(α-fetoprotein,AFP)阴性的肝癌患者和肝硬化患者血清中的差异表达蛋白。方法:选择6例AFP阴性的肝癌患者和6例AFP阴性的肝硬化患者。应用MARS系统分离获得各组血清标本中的低丰度蛋白,采用双向荧光差异凝胶电泳(two-dimensional fluorescence difference gel electrophoresis,2D-DIGE)技术筛选两组间有表达差异的蛋白,并用蛋白质印迹法加以验证。结果:2D-DIGE技术共鉴定出29种差异蛋白,其中AFP阴性肝癌患者血清中高表达的蛋白有22种,低表达的蛋白有7种。差异蛋白包括激肽原1、四连接素、基膜聚糖蛋白、补体1r和锌α2糖蛋白等。蛋白质印迹法证实,AFP阴性的肝癌患者血清中锌α2糖蛋白表达水平明显低于AFP阴性的肝硬化患者(P<0.05),同DIGE结果一致。结论:采用蛋白质组学技术筛查出的一系列在AFP阴性肝硬化和AFP阴性肝癌患者血清中有表达差异的蛋白有望作为AFP阴性肝癌患者早期诊断和辅助诊断的分子标志物。
基金supported by Outstanding Scholars of New Era from Ministry of Education of Peopde's Republic of China(NECT-07-0861)the Lotus Scholars Program of Hunan Province, P.R. China (2007-362)Scientific and Technological Projects of Department of Public Health of Hunan, P.R. China (B2009123)
文摘目的:筛选人急性髓系白血病细胞株HL-60甲基化沉默基因,为揭示白血病的发病机制及防治提供科学依据。方法:应用荧光差异双向凝胶电泳(two-dimensional fluorescence difference gel electro-phoresis,F-2D-DIGE)分离甲基转移酶抑制剂5-杂氮-2-脱氧胞苷(5-aza-2-dC)处理与未处理的HL-60细胞的总蛋白质,Decyder和PDquest图像分析软件识别差异表达蛋白质点,基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)技术鉴定差异表达蛋白质。结果:建立了5-aza-2-dC处理与未处理的HL-60细胞蛋白质的F-2D-DIGE图谱,图像分析识别了53个差异表达的蛋白质点,质谱分析鉴定了35个差异表达的蛋白质,其中32个蛋白质在5-aza-2-dC处理后的HL-60细胞中表达上调,3个蛋白质表达下调。结论:35个差异表达蛋白可能与甲基化相关,为白血病的表观遗传学研究提供了有价值的信息。
文摘以干酪乳杆菌Lactobacillus casei Zhang为出发菌株,通过适应性进化获得了干酪乳杆菌酸胁迫抗性驯化菌株。对细胞内微环境的检测发现,驯化菌株在酸胁迫过程中能够维持较高的磷酸烯醇式丙酮酸-糖转移酶系统活力,并具有较高的H^+-ATPase活性以及胞内ATP浓度。蛋白质组学分析结果表明,酸胁迫引发了细胞蛋白表达的变化,与原始菌株相比,驯化菌株保持了更高的代谢活性;同时,驯化菌株通过大量诱导应激蛋白如分子伴侣GroEL、GrpE,冷/热应激蛋白CspC、DnaK等维持了细胞的生理活性,有效提高了细胞对酸胁迫的抵御能力。本研究为进一步揭示酸胁迫下乳酸菌细胞的生理应答机制,探寻促进乳酸菌酸胁迫性能提升的最优策略,进而改善其在生产中的应用性能提供了可借鉴的思路。
