慢性间歇性缺氧大鼠肾脏组织蛋白质组的变化

目的:比较慢性间歇性缺氧大鼠与正常大鼠肾脏组织蛋白质双向电泳图谱,寻找和鉴定慢性间歇性缺氧大鼠肾脏组织中的差异蛋白表达谱。方法:以固相pH梯度等电聚焦为第一向和垂直SDS-PAGE为第二向,分别对正常对照大鼠和慢性间歇性缺氧大鼠的肾脏组织蛋白质样品进行二维分离,2-DE图谱经ImageMaster 2D Platinum V5.0软件分析,选取4个差异蛋白点用基质辅助激光解吸附离子化飞行时间质谱(MALDI-TOF-MS)进行鉴定。结果:通过对2-DE图谱蛋白斑点的匹配及对比分析,与慢性间歇性缺氧相关的差异表达蛋白斑点为112个;经质谱鉴定的4个差异表达的蛋白斑点,慢性间歇性缺氧组量下调的差异点1个,即线粒体ATP合成酶δ亚基;上调的差异点3个,分别为己糖激酶、儿茶酚-O-甲基转移酶、脱嘌呤/脱嘧啶核酸内切酶/氧化还原因子-1。结论:大鼠肾脏组织慢性间歇性缺氧损伤相关的差异蛋白表达变化可能通过多种途经影响肾脏功能。     

甲状腺乳头状癌的蛋白质组学研究

目的 对甲状腺乳头状癌(PTC)的蛋白质组进行研究,并与正常组织对比,寻找其中差异显著的蛋白.方法 应用蛋白质组学技术,提取10例PTC与周围正常甲状腺组织(NT)的蛋白质样品进行双向电泳分离及对比,应用Image Master软件对电泳图谱进行分析;将选出的差异性蛋白质点进行质谱鉴定和数据库查询.结果 得到了PTC及NT的双向电泳图谱;通过软件分析、对比,从中初步选取4个差异蛋白质点进行质谱鉴定,确认为4种相应的蛋白质:其中脂皮质蛋白(annexinⅠ)、peroxiredoxin Ⅰ、线粒体顺乌头酸酶(mitochondrial aconitase)在PTC中表达上升,碳酸酐酶Ⅰ(carbonic anhydrase Ⅰ)在PTC中表达下降.结论 蛋白质组学为PTC的研究提供了新的发展方向,在寻找肿瘤的特异性蛋白标记物、进一步探究肿瘤的发病机制以及寻找治疗肿瘤的分子靶点等方面发挥作用.     

蛋白质组学研究中双向电泳的样品制备

蛋白质组学是目前生命科学研究中的一个热点,而双向电泳技术是现阶段大多数蛋白质组学研究中分离蛋白质混合物时所采用的主要技术.在双向电泳技术中,样品制备是整个分离过程中的关键一步,对实验结果将起到决定性作用.文章介绍了双向电泳技术的样品来源,并重点对样品制备过程一般包括的步骤,如样品匀质化、蛋白质溶解、干扰物去除和蛋白质富集等进行了详细说明.随着样品制备技术的发展,双向电泳技术将在蛋白质组学的研究中发挥更为显著的作用.     

先天性马蹄内翻足胎鼠脊髓组织的蛋白质组学分析

目的 在构建动物模型的基础上,运用蛋白质组学实验方法,寻找马蹄内翻足畸形相关蛋白。方法 提取单纯性马蹄内翻足畸形胎鼠及正常对照胎鼠脊髓总蛋白,进行双向电泳;经PDQuest软件分析,选择差异点进行质谱鉴定,采用生物信息学方法分析结果。结果 经双向电泳分离后,畸形胎鼠与正常对照相比,脊髓组织的烯醇化酶没有表达,微管蛋白表达下调,载脂蛋白表达上调,ATP合酶F1复合体组装因子2(ATP12P)额外表达。结论 畸形胎鼠脊髓组织与正常对照比较,存在蛋白质组差异,畸形胎鼠微管蛋白、ATP12P表达改变,可能与马蹄内翻足畸形相关。     

肿瘤细胞诱导T细胞下调表达腺苷脱氨酶

目的 蛋白质组学分析鉴定与肿瘤细胞共培养T细胞的表达差异蛋白.方法 双向电泳分离有/无肿瘤细胞共培养的T细胞蛋白,PDQuest软件分析筛选肿瘤细胞诱导表达异常的蛋白点,用基质辅助激光解析电离飞行时间质谱(MAL-DI-TOF MS)对酶解消化样品进行肽质量指纹图谱(PMF)分析,结合数据库搜索鉴定蛋白质.结果 PDQuest分析有/无肿瘤细胞共培养的T细胞蛋白双向电泳图谱,70余个蛋白点表达水平差异显著,其中与肿瘤细胞共培养的T细胞有一明显表达下调的蛋白点,质谱分析结合数据库检索表明该蛋白点为腺苷脱氨酶.结论 肿瘤细胞诱导T细胞下调表达腺苷脱氨酶.     

正常组与人工模拟寒环境组大鼠肺组织蛋白组学差异分析

目的:探讨正常组与人工模拟寒环境组大鼠肺组织蛋白组学差异表达及质谱分析。方法:选取20只SD大鼠随机分为正常对照组与寒环境组,每组10只。寒环境组模型复制使用人工气候箱:(T)=(6±2)℃,每天刺激4 h(其余时间为正常组温湿度);正常组(T)=(21±2)℃,每天持续24 h。造模7 d后,两组大鼠于麻醉前12 h禁食、禁水,称量体重。麻醉后开胸取出左肺组织10 mg/只,每组取3个样本进行样品蛋白质制备、双向电泳实验、图像分析、质谱检测。结果:对两组差异蛋白图谱进行蛋白质组学分析,鉴定出9个蛋白在两组之间差异表达,寒环境组与正常对照组比较,其中4个表达上调、5个表达下调。结论:通过蛋白质组学分析,鉴定出的蛋白与外寒环境有相关性。     

霍乱弧菌菌体蛋白双向电泳技术的建立

目的建立霍乱弧菌全菌体蛋白的双向电泳技术，获得分辨度高、重复性好的双向电泳图谱。方法利用适当的裂解液处理霍乱弧菌，提取全菌蛋白；采用pH梯度等电聚焦对全菌蛋白进行双向电泳；考马斯亮蓝染色后获得的双向电泳图谱，并利用ImageMaster 2D Elite5．0图象分析软件进行分析，所得的数据用SPSS15．0进行统计分析。结果得到了（1081±16）个蛋白斑点，蛋白主要集中在pI4．24～7．20之间，重复胶的匹配点数为（1057±28），匹配率为97．85％。结论建立了霍乱弧菌全菌双向电泳分析方法，2-DE图谱中蛋白位点的分辨率和重复性非常高，获得了较为理想、清晰的双向电泳图谱，为进一步研究其蛋白质组学奠定了基础。     

大鼠附睾液蛋白质组二维液相色谱分离的实验研究

目的：建立一种利用二维液相色谱分离和质谱鉴定大鼠附睾液蛋白质组的实验方法,为以后研究其它哺乳动物附睾蛋白质组提供实验基础。方法：分离提取大鼠附睾头体尾部官腔蛋白,样品经脱盐、浓缩,利用起始缓冲液置换,进行一维色谱聚焦分离,收集pH8.5-4.0之间的组份进行二维反相色谱分离,并通过自动馏分收集器收集分离组份;最后将获得的二维UV图通过ProteoVue软件转换成二维PI/UV图谱;选择含高丰度蛋白的馏份经冷冻干燥浓缩后进行质谱鉴定。结果：通过二维液相色谱成功分离了大鼠头体尾部附睾液蛋白并建立了二维PI/UV图谱;对二维获得的含高丰度蛋白的馏分进行了质谱鉴定,获得大鼠附睾头体尾部主要蛋白。结论：为进一步利用二维液相色谱全面分离附睾蛋白和研究附睾功能奠定了基础。     

正常及肺气虚大鼠肺组织蛋白组学差异分析

目的:探讨正常及肺气虚大鼠肺组织蛋白组学差异表达及质谱分析。方法:选取20只SD大鼠随机分为正常对照组与肺气虚组每组10只。肺气虚组模型复制采用气管注射脂多糖与烟熏的复合方法。造模28 d后,两组大鼠于麻醉前12 h禁食、禁水,称量体重。麻醉后开胸取出左肺组织10 mg/只,每组取3个样本进行样品蛋白质制备、双向电泳实验、图像分析、质谱检测。结果:对两组差异蛋白图谱进行蛋白质组学分析,鉴定出14个蛋白在两组之间差异表达,肺气虚组与正常对照组比较,其中5个表达上调、9个表达下调。结论:通过蛋白质组学分析,鉴定出的蛋白与肺气虚证有相关性。     

霍乱弧菌O1群有毒株和无毒株的蛋白质谱特征分析

目的 利用双向电泳和质谱鉴定技术探讨环境和人体中霍乱弧菌蛋白表达的差异.方法 利用适当的裂解液处理霍乱弧菌,提取全菌蛋白;采用pH梯度等电聚焦对全菌蛋白进行双向电泳;考马斯亮蓝染色后获得双向电泳图谱,并利用ImageMaster 2D Elite 5.0图像分析软件进行分析,所得的数据采用SPSS15.0进行统计分析,找出差异蛋白;在此基础上,胰蛋白酶消化这些特殊差异蛋白,并进行质谱(MALDI-TOF-MS)分析.结果 获得1032±22个蛋白斑点,蛋白主要集中在等电点(PI)4.00～7.20之间,重复胶的匹配点数为1025±24,匹配率为96.30%;发现了有明显差异的21个蛋白点,并用质谱鉴定了其中4种蛋白.结论 获得了环境和人体中霍乱弧菌蛋白的差异表达蛋白.     

Analysis of mitochondrial antigens reveals inner membrane succinate dehydrogenase flavoprotein subunit as autoantigen to antibodies in anti-M7 sera

The role of mitochondrial proteins as antigens to antibodies of anti-M7 sera was analysed by flavin fluorescence, one- and two-dimensional Western blots and blue native gel electrophoresis. Flavin fluorescence of succinate dehydrogenase (SucDH, complex II of the respiratory chain) of rat liver inner mitochondrial membranes correlated with the immunoreactivity of a representative anti-M7 myocarditis serum. Antigens of isolated bovine heart mitochondria reacting with antibodies of myocarditis serum on two-dimensional Western blots were identified by MALDI-TOF and NanoESI mass spectrometry as myosin heavy chain beta and as dihydrolipoamide dehydrogenase of the mitochondrial 2-oxoacid dehydrogenase complexes. The SucDH-flavoprotein was not resolved as a discrete protein spot on two-dimensional polyacrylamide gels. However, separation of the rat liver inner mitochondrial membrane complexes by blue native gel electrophoresis followed by Western blotting, and Western blots of purified Escherichia coli SucDH complex revealed that anti-M7 sera contained antibodies directed against the SucDH-flavoprotein subunit.     

Fungal proteomics: mapping the mitochondrial proteins of a<Emphasis Type="Italic"> Trichoderma harzianum</Emphasis> strain applied for biological control

Mitochondria are essential for cellular functions across organisms. A proteomic approach was taken to separate and identify mitochondrial proteins from a strain of Trichoderma harzianum with well established biocontrol properties. We optimized a method for the preparation of a sample enriched with mitochondria by ensuring efficient cell lysis and including several washing steps to minimize cytoplasmic contamination. We separated hundreds of proteins, using two-dimensional gel electrophoresis and identified 31 protein spots from T. harzianum, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and liquid chromatrography with mass spectrometry. Over 50% of the identified proteins were known to localize in the mitochondria. Three protein spots were identified from T. harzianum and a further five protein spots from the genera Trichoderma and Hypocrea. The remaining protein spots were identified by cross-species identification from other filamentous fungi, including Neurospora crassa and Aspergillus spp, and yeasts, including Saccharomyces cerevisiae and Schizosaccharomyces pombe. In total, we identified 25 protein spots from T. harzianum, representing proteins that have not been characterized in existing Trichoderma protein databases. To our knowledge, this is the first two-dimensional mitochondrial protein map of a filamentous fungus.Communicated by U. Kück     

Comparison of hippocampal synaptosome proteins in young-adult and aged rats

The hippocampus is important in learning and memory functions but its ability to aid in these functions declines during aging. In this study, we examined hippocampal proteins whose expressions changed in the aging process. A comparison of synaptosome proteins of hippocampus prepared from young-adult (9-week-old) rats with those from aged (30-month-old) rats by two-dimensional fluorescence difference gel electrophoresis revealed 24 spots that were expressed differently among about 1000 spots detected in both young-adult and aged rat samples. Nineteen of these 24 spots were identified by peptide mass fingerprinting. These proteins included chaperone proteins and proteins related to the cytoskeleton, neurotransmission, signal transduction and energy supply. The cytoskeleton-related proteins included actin and T-complex 1, which is thought to play a role in actin folding. Actin was up-regulated but T-complex 1 was down-regulated in aged rat synapses. These results suggest that age-dependent changes of actin filament formation are related to neuronal dysfunction associated with aging.     

Proteomic analysis of human ovaries from normal and polycystic ovarian syndrome

Polycystic ovary syndrome (PCOS) is the most common cause of anovulatory infertility, affecting 5-10% of females of reproductive age. Currently, little is known about the changes in whole proteins between PCOS and normal ovaries. In the present study, a proteomic approach comprised two-dimensional gel electrophoresis (2DE) analysis and mass spectroscopy was used to identify proteins and examine expression patterns in three PCOS and normal ovaries. One hundred and ten protein spots were separated and showed different intensities between PCOS and normal ovaries. Sixty-nine proteins associated with cellular metabolism and physiological process were identified from 72 spots. Fifty-four proteins were up-regulated in PCOS ovaries and 15 other proteins were up-regulated in normal ovaries. These data demonstrate, for the first time, the complexity in the regulation of ovarian protein expression in human PCOS, and will provide important insight for a better understanding of the pathogenetic mechanisms underlying this clinical disorder.     

Proteomic analysis of mitochondrial proteins in cardiomyocytes from rats subjected to intermittent hypoxia

Intermittent hypoxia (IH) markedly enhances cardiac tolerance against ischemia/reperfusion injury, but its mechanism and molecular basis remain unclear. For exploring the expression of mitochondrial proteins induced by IH, two-dimensional electrophoresis and Thermo Finnigan LTQ mass spectrometer (MS) were applied. After comparing the protein profiles of myocardial mitochondria between IH and normoxic hearts, 14 protein spots were found to be altered more than threefold between the two groups, 11 of which were identified by Finnigan LTQ MS. Among these 11 proteins, 9 were involved in energy metabolism, including 7 that were increased after IH. The latter were identified as aldehyde dehydrogenase, methylmalonate-semialdehyde dehydrogenase, ATP synthase β chain, mitochondrial aconitase, malate dehydrogenase, electron transfer flavoprotein α subunit and sirtuin 5. Two other proteins, ubiquinol-cytochrome C reductase iron-sulfur subunit and aspartate aminotransferase, were decreased after IH. Biochemical tests for energy metabolism in mitochondria supported the proteomic results. IH exposure also increased the expression of a molecular chaperone—heat shock protein 60 and an antioxidant protein, peroxiredoxin 5. These findings will provide clues for understanding the mechanism of IH-induced cardiac protection and may lead to the development of interventional strategies designed to utilize the advantages of IH clinically.     

Proteomic profiling and neurodegeneration in West-Nile-virus-infected neurons

West Nile virus, a mosquito-borne flavivirus, is a human, equine, and avian pathogen. High-resolution two-dimensional differential-gel electrophoresis (2D-DIGE) was used to characterize protein expression in primary rat neurons and to examine the proteomic profiling to understand the pathogenesis of West-Nile-associated meningoencephalitis. Three pH ranges, 3-10, 4-7, and 5-6, were used to analyze the protein spots. The proteins are labeled with fluorescent dyes Cy3 and Cy5 before being separated on the basis of charge and size respectively on a two-dimensional platform. About 55 proteins showed altered expression levels. These were then subsequently digested and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis using peptide mass fingerprinting and database searching. These cellular proteins could represent distinct roles during infection related to apoptosis. Our findings show that two-dimensional differential gel electrophoresis combined with mass spectrometry is a powerful approach that permits the identification of proteins whose expression was altered due to West Nile virus infection.     

脑瘤患者脑脊液肿瘤相关蛋白的蛋白质组学初步研究

通过比较脑肿瘤患者和正常人脑脊液的二维凝胶电泳图谱（two-dimensional electrophoresis,2DE）,并对差异蛋白进行质谱鉴定,以寻找肿瘤特异脑脊液蛋白。以脑肿瘤患者和正常人的脑脊液为研究对象,采用固相pH梯度（immobilized pH gradient,IPG）2DE分离总蛋白质,凝胶经银染显后,用ImageMaster2D图像分析软件进行比较分析、识别差异表达的蛋白质。结果得到肿瘤患者脑脊液蛋白点924个,正常脑组织蛋白点507个,匹配512个,匹配率分别为55．4％和84．3％,去冗余后发现,有35个蛋白点只在脑肿瘤患者脑脊液图谱中出现。肿瘤患者脑脊液和正常对照脑脊液双向电泳图谱有明显差别,但是本实验尚未对这些差异蛋白进行质谱鉴定,所以未找出肿瘤特异蛋白。     

系统性红斑狼疮患者血清蛋白质组学研究

目的 运用蛋白质组学的方法,分析正常人及SLE患者血清蛋白质的差异表达,寻找与SLE疾病发病机制相关的蛋白质.方法 分别收集健康人及SLE患者血清各9例,同组血清等量混合,用试剂盒除去血清中的白蛋白和免疫球蛋白,再经除盐浓缩后,将血清样品采用固相pH梯度（IPG）双向凝胶电泳（2-DE）分离正常人及SLE患者血清的总蛋白质.凝胶经考马斯亮蓝染色显色后,利用Analysis2d软件对获得的蛋白图谱进行分析,寻找差异表达的蛋白质,利用基质辅助激光解析电离飞行时间质谱（MALDI-TOF-MS）进行鉴定,分析差异蛋白点.结果 对照组凝胶共检出蛋白点648个,患者组检出639个.对照组凝胶蛋白点匹配率84.5％,患者组凝胶蛋白点匹配率82.5％.通过比较分析,差异表达蛋白质点数为92个,有52个蛋白点在SLE患者组表达上调,40个表达下调,有14个点的表达水平在组间差异有统计学意义,质谱鉴定共鉴定5个蛋白质.通过文献研究显示,我们鉴定的部分蛋白在SLE的发病机制中起潜在的作用.结论 在SLE患者血清中存在着差异血清蛋白质,这些蛋白质可能是SLE发病的内在因素,并且在SLE的疾病发展过程中发挥重要作用,可能作为新的血清标志物和潜在的自身抗原.     

Alteration of the proteome profile of the pancreas in diabetic rats induced by streptozotocin

Type 1 diabetes mellitus (T1DM) is receiving increased attention. To obtain a better understanding of the mechanism underlying T1DM, we performed a proteomic study on a rat model induced by streptozotocin. Pancreatic proteins were separated by two-dimensional gel electrophoresis. Eighteen protein spots were differentially expressed (P<0.05) with 2-fold or more increased or decreased intensity in the diabetic rats as compared with controls, of which 11 protein spots were up-regulated and 7 protein spots were down-regulated. These protein spots were successfully identified by liquid chromatography-electrospray ionization tandem mass spectrometry. The 60 kDa heat shock protein, the carbonyl reductase 1 (Cbr1), the hydroxyacyl-CoA dehydrogenase, Δ(3,5),Δ(2,4)-dienoyl-CoA isomerase, the elongation factor 1-δ, the 26S protease regulatory subunit 7 and the transitional endoplasmic reticulum ATPase were up-regulated, while the 78 kDa glucose-regulated protein, peroxiredoxin 4 and plakoglobin were down-regulated. The expression change of Cbr1 which is closely related to diabetic complications was further validated by western blotting. Our results and those of the bioinformatics analysis suggest that oxidative stress, the Wnt pathway, fatty acid degradation and glucose transport may be closely related to T1DM.     

以双向凝胶电泳为基础的食管癌组织蛋白质组学分析新策略

目的：探讨采用双向电泳为基础的蛋白质组学的研究手段在食管癌组织与食管癌旁组织差异分析中的应用。方法：双向凝胶电泳分离食管癌及其癌旁组织的蛋白质，图像分析软件ImageMaster4．01分析所获得的凝胶图谱并寻找差异点，用质谱仪鉴定差异点蛋白质。结果：提出了一套可行的癌组织差异分析策略，并发现在分子量50 000-60 000，等电点5—6的区域，食管癌组织和食管癌旁组织存在2个差异表达的蛋白，质谱鉴定为丙酮酸激酶M2/M1。结论：在合理的策略指导下，以双向凝胶电泳为基础的蛋白质组学技术是研究肿瘤的一种重要手段，我们得到的丙酮酸激酶M2/M1在食管癌与其癌旁组织中存在差异表达，可能对食管癌的诊断、治疗具有重要意义。