Proteome analysis of round-headed and normal spermatozoa by 2-D fluorescence difference gel electrophoresis and mass spectrometry

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.

Preparation of protein samples for gel electrophoresis by sequential extraction

Since preparation and solubilization of protein samples are crucial factors in proteome research,the authors established a sequential extraction technique to prepare protein samples from the body wall of the 5th instar larvae of silkworm.Bombyx mori.Two kinds of protein samples were obtained from the body wall using the method.Between the two types of samples only about 15% proteins were identical;the majority were different,indicating that more species of proteins could be obtained with the sequential extraction method;which will be useful for preparation of protein samples for proteome study.

Establishment of a high-resolution 2-D reference map of human spermatozoal proteins from 12 fertile sperm-bank donors

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.

An improved Coomassie Brilliant Blue (CBB R-250) staining to proteins in gels

An improved CBB staining with higher sensitivity than that of the typical CBB staining was reported.The main improvement was using a fixing step of 25% trichloroacetic acid(TCA) before CBB staining.For most proteins studied,the sensitivity of the improved CBB staining was about twice as high as that of the typical method.For basic and low molecular weight proteins such as ribosomal proteins,the sensitivity of this improved staining method was about 3.5-28 times that of the typical method.It was speculated that the improved procedure would be suitable for exact quantitative analysis of proteins fractionated by SDS-PAGE,especially for basic and low molecular weight proteins.On the other hand,this new modified method might be also applied to multidisciplinary studies,such as biological researches and nuclear sciences.

熊果酸对人胃癌细胞BGC-823的作用研究

目的:检测熊果酸作用于人胃癌细胞BGC-823引起的细胞内蛋白质表达水平改变,探讨熊果酸可能的抑癌作用机制。方法:双向电泳和飞行质谱分析熊果酸作用细胞后的蛋白质表达变化,MTT法和流式细胞术检测细胞增殖和凋亡。结果:熊果酸作用BGC-823细胞引起胞浆内丝切蛋白cofilin-1表达下调,钙网蛋白CRT表达上调,抑制细胞增殖,诱导细胞凋亡,阻滞细胞于G2/M期。结论:熊果酸可能通过调节胞内蛋白cofilin-1和CRT表达水平来诱导细胞凋亡,促进凋亡细胞被吞噬。

白粉菌侵染Pm21近等基因系的早期蛋白组差异研究

分析接种小麦白粉菌E09后Pm21近等基因系及其亲本叶片的总蛋白质差异,从蛋白质组调控角度揭示Pm21基因抗白粉病的抗病机理。以Pm21近等基因系及其亲本百农3217为试材,显微观察接种后5 d内的叶片表面细胞与白粉菌孢子表型差异;采用非离子去污剂提取总蛋白质,运用双向电泳、质谱分析与数据库检索技术,比较Pm21近等基因系及其亲本接菌第4天后的叶片总蛋白质差异。超显微和显微观察结果表明,12 h后百农3217叶细胞表面与Pm21近等基因系相比褶皱明显;在接菌第2天后,在Pm21近等基因系白粉菌孢子生长明显慢于其亲本表面孢子,且孢子侵入个数较少,不萌发现象较多;在第4天后Pm21近等基因系被侵染叶片细胞出现过敏性反应;通过MALDI-TOF-MS/MS质谱测序和MASCOT软件序列分析,注释了15个表达上调蛋白点;功能分类表明,差异蛋白质分别参与叶绿体光合呼吸作用(40%)、放氧作用(13%)、代谢物合成(20%)、抗病、动力蛋白、运输等生理功能。蛋白质组水平发现,光合呼吸蛋白和叶谷氨酰胺合成酶在Pm21近等基因系感病3～4 h后就大量表达,可能是导致叶片明显变绿原因;叶谷氨酰胺合成酶的增加与光合呼吸蛋白的增加密不可分;光合呼吸蛋白和抗坏血酸过氧化物酶的上调表达为植物体在高H2O2浓度下正常生长提供了保障。另外,β-1,3-葡聚糖苷酶的过量表达也是Pm21基因前期抵御白粉菌侵染有效物质。钾离子通道蛋白可能在抗病防御过程中起到了信号转导的作用。

Preprocessing of 2-Dimensional Gel Electrophoresis Images Applied to Proteomic Analysis： A Review

Various methods and specialized software programs are available for processing two- dimensional gel electrophoresis （2-DGE） images. However, due to the anomalies present in these images, a reliable, automated, and highly reproducible system for 2-DGE image analysis has still not been achieved. The most common anomalies found in 2-DGE images include vertical and hor- izontal streaking, fuzzy spots, and background noise, which greatly complicate computational anal- ysis. In this paper, we review the preprocessing techniques applied to 2-DGE images for noise reduction, intensity normalization, and background correction. We also present a quantitative comparison of non-linear filtering techniques applied to synthetic gel images, through analyzing the performance of the filters under specific conditions. Synthetic proteins were modeled into a two-dimensional Gaussian distribution with adjustable parameters for changing the size, intensity, and degradation. Three types of noise were added to the images： Gaussian, Rayleigh, and exponen- tial, with signal-to-noise ratios （SNRs） ranging 8-20 decibels （dB）. We compared the performanceof wavelet, contourlet, total variation （TV）, and wavelet-total variation （WTTV） techniques using parameters SNR and spot efficiency. In terms of spot efficiency, contourlet and TV were more sen- sitive to noise than wavelet and WTTV. Wavelet worked the best for images with SNR ranging 10- 20 dB, whereas WTTV performed better with high noise levels. Wavelet also presented the best per- formance with any level of Gaussian noise and low levels （20-14 dB） of Rayleigh and exponential noise in terms of SNR. Finally, the performance of the non-linear filtering techniques was evaluated using a real 2-DGE image with previously identified proteins marked. Wavelet achieved the best detection rate for the real image.

Proteomic analysis for developing new biomarkers of hepatocellular carcinoma

AIM: To identify new markers of hepatocellular carcinoma (HCC) using a proteomic analysis. METHODS: Patients with liver cirrhosis of the three most frequent etiologies: hepatitis C virus, hepatitis B virus and alcoholic liver disease, were included in the study. The samples were analysed by 2D-electrophoresis in order to determine the differential protein expression. The proteins were separated according to the charge in immobilized pH 3-10 gradient strips and then by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins of interest were excised, digested with trypsin and the resulting peptides were separated and identified. RESULTS: Three differentially expressed apolipoproteins (Apo) were identified based on the protein profile using proteomic techniques: Apo-A1, Apo-A4 and Apo-E. Apo-A4 levels were significantly lower in HCC than in non-HCC patients regardless of etiology (P < 0.01). Multivariate logistic regression showed that Apo-A4 and Apo-A1 were the only independent factors related to HCC diagnosis (P < 0.05). The receiver operating characteristic (ROC) curve including both Apo-A4 and Apo-A1 showed an area under the ROC of 0.944 (P < 0.001), a sensitivity of 0.89 and a specificity of 0.81 for diagnosis of HCC. CONCLUSION: Apo-A4 and Apo-A1 may be used clinically as biomarkers of HCC with a high sensibility and specificity. These findings may provide additional insights into the mechanism of HCC development and progression.

Parkinson disease drug screening based on the interaction between D2 dopamine receptor and beta-arrestin 2 detected by capillary zone electrophoresis

Parkinson’s disease is the second most common neurodegenerative disease in the world.Beta-arrestin-2 has been reported to be an important protein involved in D2 dopamine receptor desensitization,which is essential to Parkinson’s disease.Moreover,the potential value of pharmacological inactivation of G protein-coupled receptor kinase or arrestin in the treatment of patients with Parkinson’s disease has recently been shown.We studied the interaction between D2 dopamine receptor and beta-arrestin-2 and the pharmacological regulation of chemical compounds on such interaction using capillary zone electrophoresis.The results from screening more than 40 compounds revealed three compounds that remarkably inhibit the beta-arrestin-2/D2 dopamine receptor interaction among them.These compounds are promising therapies for Parkinson’s disease,and the method used in this study has great potential for application in large-scale drug screening and evaluation.

Comparative Analysis of the Endosperm Proteins Separated by 2-D Electrophoresis for Two Cultivars of Hybrid Rice （Oryza sativa L,）

Liangyoupeijiu is a two-parental-line, and Shanyou63 is a three-parental-line hybrid rice （Oryza sativa L.）. Although both belong to the indica subspecies, they have obvious differences with respect to morphology, physiology and grain quality. Variations in endosperm protein compositions were studied by comparing the 2-D electrophoresis （2-DE） maps for these two cultivars of hybrid rice. After matrix-assisted laser desorption/ionization time of flight mass spectrometry （MALDI-TOF/MS） analysis, a 21-kDa precursor of 19- kDa globulin was identified as the major storage protein for both cultivars. Some isoforms of peroxiredoxin and seed maturation protein were found to only exist in Shanyou63, whereas aldose reductase and starch granule-bound starch synthase were only detected in Liangyoupeijiu. These data might provide a foundation for further comparative studies of these two cultivars of hybrid rice.

Differential protein expression in low-grade astrocytomas and peritumoral human brain tissues

Differential protein expression between various pathological grades of glioma has been shown in studies of glioma proteomics. However, very little data is available regarding normal brain tissues and glioma differential protein expression, because normal human brain tissues are difficult to harvest. The present study selected samples from low-grade astrocytomas and peritumoral brain tissues to analyze differential protein expression by two-dimensional （2D） electrophoresis and mass spectrometry techniques. Results revealing 36 protein spots by 2D electrophoresis, including 23 spots revealing increased expression and 13 spots revealing decreased expression. However, 25 differential proteins were identified by mass spectrometry, including 16 proteins with increased expression and 9 with decreased expression. Western blot analysis confirmed the mass spectrometry results, i.e., heat shock protein 70 （HSP70） and human transthyretin （TTR） expressions were increased, but glial fibrillary acidic protein （GFAP） was decreased, in astrocytomas. The present study constructed a 2D electrophoresis pattern between low-grade astrocytomas in the human brain and peritumoral tissues. Results demonstrated that a majority of differential proteins, such as HSP70, TTR, and GFAP, participate in malignant progression of gliomas.

肝片吸虫18.6kD蛋白抗原双向电泳及免疫印迹分析

从分子水平上研究了肝片吸虫的抗原组分 ,用制备性 SDS- PAGE纯化 ES18.6 k D蛋白抗原 ,并用它成功制备了单因子兔抗血清 ,用这种抗血清来分析肝片吸虫各部分抗原 ,证实各部分抗原的 18.6 k D多肽具有相同的抗原决定簇 ,而且这种抗原决定簇不存在于其它分子量的多肽中。体抗原 (BA)和分泌排泄抗原 (ES)的双向电泳(Two- dimention Electrophoresis,2 - D)分析研究表明 ,肝片吸虫同一种分子量的蛋白质大多由多种等电点不同的多肽组成 ;同时也显示了体抗原和分泌 -排泄抗原分子量相同的蛋白质所包含的多肽数量和性质是有差异的。

Comparative analysis of bed density, total phenol content and protein expression pattern in <i>Posidonia oceanica</i>(L.) Delile

Posidonia oceanicameadows are experiencing a progressive decline, and monitoring their status is crucial for the maintenance of theseecosystems. We performed a comparativeanalysis of bed density, total phenol content and protein expression pattern to assess the conservation status ofPosidoniaplants from the S. Marinella (Rome, Italy) meadow. The total phenol content was inversely related to maximum beddensity, confirming the relationship betweenhigh phenol content and stressful conditions. In addition, protein expression pattern profilesshowed that the number of differentially expressed proteins was dramatically reduced in the latest years compared to previous analyses. Our results support the usefulness of integrating solid descriptors, such as phenol content, with novel biochemical/molecular approaches in the monitoring of meadows.

Effect of high salinity on cell growth and protein production of Antarctic ice microalgae Chlamydomonas sp. ICE-L

Antarctic ice microalgae Chlamydomonas sp.ICE-L can survive and thrive in Antarctic sea ice.In this study,Chlamydomonas sp.ICE-L could survive at the salinity of 132‰ NaCl.SDS-PAGE showed that the density of 2 bands（26 and 36 kD） decreased obviously at the salinity of 99‰ NaCl compared to at the salinity of 33‰ NaCl.The soluble proteins in Chlamydomonas sp.ICE-L grown under salinity of 33‰ and 99% NaCl were compared by 2-D gel electrophoresis.After shocking with high salinity,8 protein spots were found to disappear,and the density of 28 protein spots decreased.In addition,19 protein spots were enhanced or induced,including one new peptide（51 kD）.The changes of proteins might be correlated with the resistance for Chlamydomonas sp.ICE-L to high salinity.

鳜和鲢肌肉蛋白质氨基酸组成及其蛋白质组成的比较分析(英文)

采用高压液相色谱和二维电泳方法,对鳜和鲢肌肉组织蛋白质组成成分以及肌肉蛋白质双向电泳分布图谱进行了比较研究。研究结果证明,鳜肌肉组织蛋白质干重为34.7%,而鲢肌肉干重为33.6%;鳜肌肉组织中必需氨基酸含量和鲜味氨基酸(天冬氨酸和甘氨酸)均高于鲢。同时,将两种鱼肌肉蛋白进行2D-gel双向电泳分离,发现鳜和鲢肌肉蛋白质组成和分布也存在明显的差异。本研究将肌肉蛋白质组成成分和蛋白的双向电泳技术结合起来,进行蛋白质组分归类分析,可为进一步研究两种鱼肌肉肉质研究打下基础,为水产业提供有价值的参考,为鱼类的养殖和肉质的改良、利用提供一个指标。

水稻种子胚乳蛋白的双向电泳分析技术

为探讨水稻灌浆期间与籽粒充实相关蛋白表达的变化,建立了一套适于水稻种子胚乳蛋白双向电泳分析技术,探讨了胚乳蛋白的提取、纯化以及双向电泳的一些关键技术.结果表明,通过乙醇铵沉淀、高压等电聚焦和两步平衡等方法可以获得较好的分析结果,获得胚乳蛋白斑点数约350个,等电点范围在3.0～9.0,其中大部分集中在4.0～7.0.

大鼠骨组织蛋白质组样品提取方法的建立

蛋白质组学是目前研究的重要热点.该文拟建立大鼠骨组织蛋白质样品提取的最佳方法,用于双向电泳分离,为进一步进行蛋白组学分析奠定基础.实验对3种不同方法制备的样本进行同一条件的二维电泳(2- DE)图谱,比较2- DE的结果.结果发现,丙酮沉淀法最佳.这些结果提示,只有结合骨组织成分特征与双向电泳的具体要求,选择恰当的样品提取液,采用合理的提取步骤,获得蛋白质样品,才能得到清晰高分辨率的电泳图谱,满足蛋白质组学分析需要.

镉胁迫下嗜温鞘氨醇杆菌降解丁草胺的蛋白质组学研究

为了从蛋白表达水平阐释丁草胺降解菌应答镉胁迫及胁迫情况下降解丁草胺的分子机制,对丁草胺高效降解菌嗜温鞘氨醇杆菌(Sphingobacterium thalpophilum)在重金属镉胁迫下蛋白质组变化进行研究,设置10 mg·L-1镉胁迫处理和不加镉的对照,7 d后分别取样提取菌株总蛋白,利用双向(2-D)电泳和质谱鉴定技术分析两者之间的差异表达蛋白,并进行差异蛋白功能分析。2-D电泳结果分析共获得93个差异蛋白点,其中上调蛋白49个,下调蛋白44个。选取重复性好且差异最为明显的23个蛋白质点酶解脱盐后进行MALDI-TOF-MS分析,成功鉴定出15个差异蛋白,其中8个为上调蛋白,7个为下调蛋白;差异蛋白分别按GO和COG进行了蛋白功能的生物信息学分类。研究表明:差异显著蛋白主要为参与丁草胺代谢的蛋白、响应镉胁迫敏感蛋白及在镉胁迫下参与丁草胺代谢的蛋白;获得2个镉高度敏感蛋白(蛋白编号分别为A0A0M7HIV1和A0A0D6IIE6)以及1个抗镉胁迫同时参与丁草胺代谢的蛋白(蛋白编号为D4FMF4),推测它们在镉胁迫下丁草胺的降解中起重要作用。

植物蛋白质组学技术研究进展

蛋白质组学被认为是后基因组研究中的最主要部分。与基因组学相比较,蛋白质组学的研究内容更为复杂,同时也更为直观地揭示生命过程。近年来新的蛋白质组学研究技术层出不穷,极大地推动了生命科学的研究进展。简要介绍了蛋白质组学的概念、蛋白质组学的研究内容以及一些新的研究技术在植物中的应用等。

通过生物素化标记分析细胞膜亚蛋白质组

细胞膜在许多基本生物学作用过程中扮演着重要角色,比如细胞-细胞相互作用、信号转导和物质运输.分析不同生理或病理状态细胞的膜蛋白的表达变化,有助于了解这些生物学过程以及发现新的诊断、治疗靶位.成功地进行膜蛋白分析最重要的是获得足够浓度与纯度的膜蛋白.这里报告一种分离高纯度膜蛋白的方法,主要包括三个步骤:(1)生物素化活细胞表面的膜蛋白,(2)链亲和素琼脂糖亲和吸附,(3)强烈的清洗条件去除非特异吸附的胞质蛋白.最后用化学方法洗脱生物素标记的膜蛋白进行双向电泳分离分析.用该方法制备了HeLa细胞的膜蛋白.免疫印迹结果表明,生物素标记的膜蛋白基本上都是低丰度蛋白.这样制备得到的膜蛋白双向电泳分离出2400多个蛋白质点,而且大多数点的亮度相近,分布得相对分散没有聚集成群.这种图谱非常便于比较分析找到差异蛋白.多次重复制备膜蛋白、电泳表明该方法的重复性和稳定性很好.