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.     

中国对虾肝胰腺蛋白质组学双向电泳技术体系的建立

建立中国对虾肝胰腺蛋白质组学双向电泳技术体系。利用双向电泳技术,以中国对虾肝胰腺为研究对象,通过离心等手段提取蛋白,双向电泳分离,硝酸银染色,进行图谱分析。结果表明:从双向电泳图谱上获得蛋白质斑点。验证了用双向电泳技术研究对虾免疫及其他生理现象的可行性。通过建立中国对虾蛋白质组学双向电泳技术体系,可极大拓宽对虾免疫生理研究的途径,从蛋白质水平探讨对虾发病机制,寻找有效药物靶点,为生物信息学分析奠定了基础,提供了理论依据。     

蛋白质组学在医学研究中的应用与进展

BACKGROUND: Studies on the proteomics contribute not only to exploring the laws governing life activities, but also to elucidating the pathogenesis of a variety of diseases to find the treatment strategies. OBJECTIVE: To summarize the application of proteomics in the plateau medical research. METHODS: A computer-based online search was conducted in PubMed and Wanfang databases from 1995 to 2015 to screen the relevant literatures using the key words “proteomics, medical research, plateau medicine” in English and Chinese, respectively. A total of approximately 200 English and 60 Chinese relevant literatures were selected and the 59 eligible literatures were included after screening finally. RESULTS AND CONCLUSION: Currently, the human genome has been decoded through the studies on exploring the proteomics changes under the pathological conditions and the underlying mechanisms. Construction of physiological and pathological mapping based on the human proteome contributes to revealing the novel treatment targets, diagnostic markers, and drugs for the prevention and treatment of diseases. Proteomics has become the frontier and hot research field both at home and abroad, including all the proteins expressed in the tissue, cell or organism, and becomes a bridge between the genome and clinical application. 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

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。     

鹿茸再生及其蛋白质组学研究进展

背景：鹿茸是目前已知的惟一可以周期性再生的哺乳动物器官。这是一种来源于角柄骨膜并基于干细胞的再生过程。鹿茸又以角柄骨膜致敏区的干细胞作为再生基础,鹿茸及角柄骨膜中的蛋白质组,对于揭开鹿茸所具有的独特生物学活性以及再生机制具有重要的意义。 目的：综述鹿茸再生和目前鹿茸蛋白质组学研究的两种主要途径与研究现状等。 方法：应用计算机在PubMed数据库(http://www.ncbi.nlm.nih.gov/pubmed/)与CNKI数据库(http://www.cnki.net/)中进行检索。在PubMed数据库中的检索词为“deer antler,antler regeneration,antler proteome”;在CNKI数据库中的检索词为“鹿茸再生,鹿茸蛋白质组”。将涉及到鹿茸再生组织学与形态学,鹿茸干细胞以及鹿茸蛋白质组学研究相关的文章找出来,内容无关与重复的文章排除掉,最后纳入43条文献进行综述。 结果与结论：在PubMed与CNKI数据库中通过初检与筛选共找到了43篇文献。鹿茸是能够周期性再生的,并且通过组织学等相关实验表明这种再生是来源于角柄骨膜干细胞的,而角柄骨膜分为致敏区与休眠区,两者是通过与皮肤接触的紧密程度来划分的。正是致敏区骨膜与其所覆盖皮肤的相互作用才最终促使角柄骨膜干细胞发育成完整的鹿茸组织。同时,鹿茸及角柄骨膜中的蛋白质组在这些过程中起着重要作用。通过还原蛋白质谱的完整情况并进一步通过基因工程等后续手段来研究未知蛋白的功能对于了解鹿茸具有的独特生物学活性与再生作用奠定重要基础,同时对于相关蛋白质在哺乳动物器官再生中所具有的调节作用提供参考。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

胎脑与成年脑皮质组织的蛋白质组学分析

目的 应用蛋白质组技术研究成年和胚胎脑皮质蛋白质组表达差异,初步探索在胎脑与成年人中脑蛋白的变化情况.方法 收集成人无病变区正常脑皮质1份和水囊引产胎脑组织脑皮质2份(3月胎脑组织和5月胎脑组织),提取总蛋白,通过双相电泳分离蛋白,每组样品均重复电泳,使用PDQuest7.0图像分析软件分析电泳图谱,得到具有表达差异的蛋白质,分别选取在大脑发育过程中逐步降低和逐步升高的差异点及在胎脑成脑蛋白表达中差异两倍以上的蛋白点,应用胶内酶切和基质辅助激光解析电离飞行时间质谱(matrix-assisted laser desorption/ionization-time of flight-time of flight,MALDI-TOF-TOF)进行鉴定,得到肽指纹图谱,并查阅蛋白质数据库得到蛋白质的相关信息.结果 (1)成人脑组织、3月胎脑组织和5月胎脑组织的脑组织蛋白图谱分别测得642、511和527个点,图象匹配率达87%.成人脑蛋白图谱中碱性蛋白点明显多于胎脑的;(2)成人脑组织、3月胎脑组织和5月胎脑组织的脑组织单独特有蛋白分别为:172、171和152个;(3)与成人脑组织比较,3月胎脑组织和5月胎脑组织的差异蛋白质分别为131和115个;差异在2倍以上的蛋白点为60和40个,其中低表达的蛋白点分别有24和17个,高表达蛋白点为36和23个;(4)白蛋白、磷酸丙糖异构酶等8种蛋白表达均有不同的变化.脂肪酸结合蛋白7和未命名蛋白只在3月胎脑高表达,而5月胎脑和成人组均无表达;核酮糖1,5-二磷酸羧化酶/加氧酶和转导素只在成人脑中高表达,胎脑无表达.血清白蛋白却随着大脑发育逐渐降低.ATP合成酶、线粒体F0复合体亚基、磷酸丙糖异构酶随着大脑发育而逐渐升高.结论 人类从胚胎期到成人发育过程中脑部蛋白质可能会发生明显改变,这些差异蛋白的发现为研究脑发育机理提供了极其有益的线索.

Abstract：

Objective To study the differences of protein expression levels in the brain cortex of human fetus and adult with proteomics technique, and provide preliminary data on the change of proteins during brain development. Methods Proteins extracted from human temporal lobes in fetal (3 month and 5 month respectively) and adult (30 years old) brain were separated by two-dimensional gel electrophoresis (2DE).The proteins were then stained with colloidal Coomassie blue to produce a high-resolution map of the proteiome.The differential protein spots were analyzed by PDQuest 7.0 software and 8 spots,which were gradually reduced or gradually increased in brain development process and the protein spots of difference over two-fold in the brain,were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/TOF).Results (1) On average, 642, 511 and 527 protein spots could be obtained in the temporal lobes of adult, 3 month and 5 month fetus. The matching rate of images was 87%. The basic proteins in adult brain were obviously much more than that in the fetus; (2) There were 172, 171 and 152 singular protein spots in temporal lobes of adult, 3 month and 5 month fetus respectively. (3) Compared with adult, there were 131 and 115 different protein spots in the 3 month and 5 month fetus respectively. There were 60 and 40 protein spots with more than 2 fold difference, among which 24 and 17 were down-regulated, and 36 and 23 were up-regulated respectively. (4) There was different expression in proteins such as serum albumin, triosephosphate isomerase, etc. in the 3 groups. Fatty acid binding protein 7 and unnamed proteins were only highly expressed in the 3 month brain;ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit and transducin beta-1 subunit were up-regulated in adult brain. Serum albumin decreases gradually with brain development. However, ATP synthase, mitochondrial F0 complex, and triosephosphate isomerase increase gradually with brain development. Conclusion The proteins of human brain cortex were obviously changed from embryonic stage to adult. The differentially displayed proteins may provide further insight into the understanding of development of human brain.     

骨科疾病中蛋白质组学的应用进展

背景：蛋白质组学是后基因组时代的新研究领域,它以组织或细胞的全部蛋白质结构与功能为研究对象,通过检测蛋白质来分析各种疾病的重要病理生理变化。 目的：就目前蛋白质组学相关技术在骨科各种疾病中的应用与展望作一综述。 方法：以“组织构建/蛋白质组学/骨科学/椎间盘退变/骨质疏松/骨性关节炎/成骨细胞/破骨细胞/综述”为中文检索词,以“proteomics/orthopedics/review/osteoblasts metabolism/osteoclasts metabolism/ intervertebraldisc degeneration/osteoporosis/osteoarthritis/serum related orthopedic diseases”为英文检索词,检索珠江三角洲数字图书馆联盟期刊全文数据库和Pubmed外文数据库2001年1月至2013年9月有关蛋白质组学在骨科各类疾病当中的基础和临床研究的相关文献。纳入有关报告蛋白质组学在椎间盘病变、骨质疏松症及骨性关节炎等方面的应用的文献。排除重复性研究和不典型报道。 结果与结论：蛋白质组学已成功应用于成骨细胞、破骨细胞代谢,椎间盘退变、骨质疏松症、骨性关节炎及骨科相关疾病的血清等方面,以上所提及的疾病均为各类人群的常见多发病,这些相关的基础临床研究无疑为人类健康带来了巨大的福音,相信蛋白质组学相关技术在基础医学和临床医学中会取得更大的突破,将展示广阔的发展前景。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

布鲁氏菌外膜蛋白免疫蛋白质组学方法的建立

目的 建立布鲁氏菌外膜蛋白的免疫蛋白质组学研究方法,筛选布鲁氏菌保护性抗原.方法 利用双向电泳技术对实验条件下培养的布鲁氏菌疫苗株M5外膜蛋白进行分离,结合WB(Western blotting)技术寻找发生免疫反应的蛋白质.结果 21个免疫蛋白点经胶内酶切、肽提取后用基质辅助激光解析/电子飞行时间质谱(MALDI-TOF-MS)进行鉴定.每个蛋白点的肽质量指纹图谱用Mascot进行检索后,代表了12个开放阅读框.这些蛋白不全是外膜蛋白,还存在胞浆蛋白,其功能涉及生物合成和物质代谢等领域,还有一个功能未知的蛋白.结论 成功建立了布鲁氏菌外膜蛋白的免疫蛋白质组学研究方法,为寻找保护性抗原及为新型疫苗抗原候选提供新思路.     

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。     

人胃黏膜组织蛋白质组二维电泳图像的获得

目的初步建立并优化人类胃黏膜组织蛋白质组分析所需的双向凝胶电泳技术，提高其分辨率及重复性。方法刮取手术胃黏膜组织，对以固相pH梯度为第一向的双向凝胶电泳的关键因素与环节，如样品处理、上样量、电泳参数、凝胶浓度和SDS凝胶电泳染色方法等进行一系列的优化。以固相pH梯度——IPG胶条(pH=3—10)进行第一向等电聚焦，以SDS均一胶(13％)的垂直电泳为第二向。结果成功地得到了胃黏膜组织的双向凝胶电泳图谱。     

The apoE polymorphism studied by two-dimensional, high-resolution gel electrophoresis of serum

The apoE polymorphism of human very low density lipoprotein (VLDL) was studied by the two-dimensional high-resolution gel electrophoresis technique of O'Farrell, which combines isoelectric focusing and SDS-polyacrylamide gel electrophoresis. With whole serum, six major patterns and two "sub-variants" of apoE were observed. A series of twin pairs (21 monozygotic (MZ) and 13 dizygotic (DZ) pairs) as well as unrelated people were analyzed. Both members of MZ pairs always exhibited the same pattern, whereas DZ twins were often discordant. The patterns observed would be consistent with the hypothesis that three apoE isopeptides are coded for by three different alleles at one single locus. In this small series, all three postulated homozygous patterns, namely apoE-II, apoE-III and apoE-IV as well as the three heterozygous patterns apoE-11, 111; apoE-III, IV and apoE-ll, IV were seen.     

肺腺癌SPC-A1细胞双向电泳图谱的优化

双向电泳技术＋质谱技术已经成为蛋白组学研究的基本策略之一。获得细胞或组织经过某种处理前后的双向电泳图谱，然后经专业2－D图像分析软件找到差异表达（有无的差异或是表达量的差异）的蛋白质斑点，再经过质谱分析鉴定这些蛋白质的种类，可以全面、系统地研究该处理因素对总蛋白质表达的影响。而要想准确地获得某种组织或细胞的双向电泳图谱，建立与之兼容的方法必不可少。本文通过使用和SPC－A1细胞株兼容的总蛋白质抽提方法，采用不同pH梯度的IPG（immorbilized pH gradient）干胶条，获得了较为清晰的蛋白质斑点，并重点分离了特定局部区域的蛋白质，优化了本细胞株的双向电泳图谱，为进一步分析药物处理前后的总蛋白质表达差异创造了条件。     

Application of two-dimensional electrophoresis in the research of retinal proteins of diabetic rat

Diabetes mellitus （DM） is a chronic disease which is associated with numerous serious health complications such as diabetic retinopathy, and is the leading cause of new cases of blindness in adults at the age of 20-74 years old. The aim of the study was to establish and optimize a two-dimensional polyacrylamide gel electrophoresis （2-DE） technique for retina proteomics to improve the resolution and reproducibility, and to observe the proteomic changes of retinal tissues in diabetic and normal rats. Proteins were extracted from retinal tissues of normal and 8 weeks diabetic SD rats and used in two-dimensional electrophoresis. Various conditions of retina proteomic 2-DE were adjusted, optimized and protein spots of differential expression were obtained through analysis of 2-DE images with PDQuest software. By choosing appropriate sample amount, using pre-cast IPG dry strips （pH 5-8） and casting 12% equal gel, satisfactory 2-DE images of retina were obtained and a steady 2-DE technique was established. In this way, we found 36 spots in 2-DE gel of diabetic retinas that exhibited statistically significant variations, including up-regulation of 5 proteins in diabetic rat retinas, down-regulation of 23, and disappearance of 8, in comparison with normal tissues. The differences of protein expression were observed in retinas between diabetic and normal rats. Our established 2-DE technique of retina proteins could be effectively applied in proteomics of retina diseases. Cellular ＆ Molecular Immunology. 2007;4（1）：65-70.     

双向凝胶电泳脑脊液蛋白质提取方法的比较

 目的 对 3 种常用的双向凝胶电泳脑脊液蛋白质提取方法进行比较，优选脑脊液蛋白质提取方法。 方法 收集新诊断尚未经药物治疗的 16 例帕金森病患者和 7 例头痛患者的脑脊液，分别以 3 种不同的方法提取脑脊液中的蛋白质。①透析法：用 PluseOne 微透析试剂盒处理脑脊液样品。②丙酮沉淀法：以冷丙酮溶液处理脑脊液样品，丙酮的终浓度为 80%。③三氯醋酸（TCA）/丙酮沉淀法：以 4 倍体积的 10% TCA/丙酮溶液处理脑脊液样品，TCA 的终浓度为 8%，丙酮的终浓度为 80%。取沉淀物进行双向凝胶电泳，用银染法对电泳后的凝胶进行染色，比较经 3 种不同方法提取的脑脊液蛋白质的双向凝胶电泳图谱。 结果 经透析法处理样品的电泳图谱显示的蛋白点较清晰、较圆，条纹较少，但所见几乎都是高丰度蛋白，低丰度蛋白很难显现；经丙酮沉淀法处理样品的电泳图谱显示横竖条纹均较多，大部分蛋白堆积在凝胶的上部，下面的点也显示不清晰，蛋白点出现横向漂移；经 TCA/丙酮沉淀法处理样品的电泳图谱显示横条纹相对较少，低丰度蛋白能较清晰显现，样品中所含高丰度蛋白明显少于以上两种方法处理的样品。 结论 以 TCA/丙酮沉淀法提取蛋白质进行双向凝胶电泳时聚焦效果较好，而且同时去除了大部分白蛋白，使低丰度蛋白能很好地显现出来，优于丙酮沉淀法和透析法。     

慢性肾小球肾炎肾阴虚证血浆蛋白质双向凝胶电泳分析

目的初步观察慢性肾小球肾炎肾阴虚证患者血浆蛋白质组的变化与表达差异,为进一步探讨肾阴虚证的发生机制奠定基础。方法采用双向凝胶电泳（2-DE）技术分离慢性肾小球肾炎肾阴虚证患者、肾阳虚证患者和正常人的血浆总蛋白,银染显色;PDQest软件对凝胶图像进行定性定量差异表达分析;从中选取差异表达蛋白质斑点,通过基质辅助激光解析电离飞行时间质谱（MALDI-TOF-MS）分析,获取肽质量指纹图谱（PMF）;通过Mscot-Wizard软件进行数据库搜索,鉴定蛋白质。结果与正常人和慢性肾小球肾炎肾阳虚证患者相比,在慢性肾小球肾炎肾阴虚证患者血浆蛋白质双向凝胶电泳图谱中绝对高表达的蛋白质斑点有80个,绝对低表达的蛋白质斑点有42个;定性差异表达分析发现,在慢性肾小球肾炎肾阴虚证患者血浆蛋白质双向凝胶电泳图谱中特异出现的蛋白质斑点有2个,缺失的蛋白质斑点有13个。结论同病异证患者血浆中存在差异表达蛋白质,利用蛋白质组学技术有望对中医证候的发生机制进行阐释。     

人晶状体上皮细胞蛋白质组的双向电泳和质谱鉴定

目的： 建立人晶状体上皮细胞蛋白质组研究体系，探讨双向电泳和质谱鉴定技术在人晶状体上皮细胞蛋白质组研究中的作用。 方法: 体外培养人晶状体上皮细胞株，用两种不同方法提取总蛋白，进行固相pH梯度(IPG)等电聚焦双向凝胶电泳，凝胶通过GS-800扫描仪（Bio-Rad）获取图像并使用PDQuest专业图像分析软件分析。在此基础之上，胰酶消化蛋白质斑点并进行质谱分析。 结果: 获得了重复性较好的人晶状体上皮细胞蛋白质组电泳图谱。晶状体蛋白质斑点在等电点pH值为4-7、相对分子质量为17-72 kD之间均有分布。其中高丰度蛋白点主要分布于分子量19-50 kD、PI 5-7范围内。2个蛋白点通过质谱分析和数据库的检索得到了初步的鉴定。 结论: 建立起了一个稳定的分析人晶状体上皮细胞蛋白质组学实验体系；为进一步研究人类晶状体在生理状态及白内障等病理条件下的改变提供了蛋白质组学的研究方法和途径。     

Towards a global analysis of porcine alveolar macrophages proteins through two-dimensional electrophoresis and mass spectrometry

Alveolar macrophages (AM) are the primary phagocytes of the innate immune systems, constituting a link between innate and adaptive immunity. With the aim of studying the porcine AM biology and the dynamics of pig-pathogen cell interactions, we have obtained a reference 2-DE map of the porcine AM proteins. The proteins were separated by 2-DE using a 5–8 range pH gradient in isoelectric focusing and over 800 spots were detected. A set of proteins, covering the pI 5.2–7.4 and M_W 19 to 106 kDa ranges, was subjected to MS analysis and 106 proteins were assigned identification by PMF, this identification being confirmed by MS/MS. An important number of proteins is involved in immunological functions, signalling process, transport or apoptosis, confirming that macrophages are involved in a wide range of biological functions. This reference map provides a useful tool for identifying protein pattern changes as a result of inflammation, exposure to infectious agents or genetic diseases.