少弱精子症患者精子蛋白质组学的生物信息学研究

目的:采用TMT技术筛选少弱精子症患者精子差异蛋白,并通过生物信息学分析,旨在为少弱精子症的研究提供研究方向和理论依据。方法:收集并随机取30例少弱精子症患者和30例正常男性的精液标本,运用TMT技术蛋白质组学分析筛选出少弱精子症患者精子差异表达的蛋白质,并进行基因本体(GO)分析和KEGG信号通路生物信息学分析。结果:通过蛋白质组学技术获得差异蛋白1 199种,其中上调蛋白663种,下调蛋白536种。GO分析结果初步提示差异蛋白主要聚集在核糖体成分、核糖体功能方面。KEGG信号通路分析发现差异蛋白涉及244条信号通路。结论:少弱精子症患者精子差异蛋白涉及复杂的生物过程、分子功能和信号通路,并且蛋白质组学筛查与其生物信息学分析有助于少弱精子症发病机制的研究。     

蛋白质组学在胃肠肿瘤研究中的应用进展

蛋白质组学主要研究基因编码的所有蛋白质,近年来已成为全面分析蛋白质表达变化的有力手段.蛋白质组学在胃肠肿瘤研究中的应用开始越来越多,通过双向凝胶电泳等各种技术,已经筛选出多种与胃肠肿瘤预后相关的差异蛋白,发现了多种潜在的胃肠肿瘤标志物;在胃肠肿瘤的发生、发展机制方面,通过蛋白质组学发现了多种与肿瘤细胞营养摄取、细胞代谢等影响肿瘤发生的蛋白,发现了多种影响细胞黏附、迁移和细胞骨架重塑的转移相关蛋白;利用蛋白质组学技术还发现了化疗药物和分子靶向治疗耐药的相关机制.蛋白质组学在胃肠肿瘤的基础和临床研究中发挥了越来越重要的作用.     

蛋白质组学在肾脏病研究中的应用

简要介绍目前蛋白质组研究的技术及其进展。蛋白质组研究是对基因组研究的重要补充 ,它是在蛋白质水平定量、动态、整体性研究生物体。二维凝胶电泳是目前分离蛋白质组成分的技术 ,是其研究的核心 ,图像分析依靠计算机为基础的数据处理 ,定量分析可确定蛋白质的基本属性如等电点和分子。随着蛋白质组学技术的不断发展 ,已有越来越多的研究者将蛋白质组学运用到肾脏疾病研究中 ,即运用二维凝胶电泳、质谱等手段来观察肾脏在生理和各种病理状态下的蛋白质组学变化 ,并对这些疾病的发病机制给予独特的解释。与此同时 ,肾脏正常和一些疾病模型的相关蛋白质组数据库也在建设之中 ,这将大大方便更多的蛋白质组研究。本文综述了近年来使用蛋白质组学在肾脏研究中取得的进展。     

蛋白质组学在肾脏病研究中的应用

简要介绍目前蛋白质组研究的技术及其进展。蛋白质组研究是对基因组研究的重要补充，它是在蛋白质水平定量、动态、整体性研究生物体。二维凝胶电泳是目前分离蛋白质组成分的技术．是其研究的核心，图像分析依靠计算机为基础的数据处理，定量分析可确定蛋白质的基本属性如等电点和分子。随着蛋白质组学技术的不断发展，已有越来越多的研究者将蛋白质组学运用到肾脏疾病研究中，即运用二维凝胶电泳、质谱等手段来观察肾脏在生理和各种病理状态下的蛋白质组学变化，并对这些疾病的发病机制给予独特的解释。与此同时，肾脏正常和一些疾病模型的相关蛋白质组数据库也在建设之中，这将大大方便更多的蛋白质组研究。本文综述了近年来使用蛋白质组学在肾脏研究中取得的进展。     

肾移植排斥反应的蛋白质组学研究

蛋白质作为基因转录和翻译的产物,其所具备的特点已经引起了研究者的广泛关注,与基因组学的静态表现不同,蛋白质组学有其独有的动态变化特点.从基因转录到蛋白表达,许多因素可以影响蛋白质水平的变化.本文就肾移植领域的蛋白质组学研究作如下综述.     

特发性弱精子症精子与正常精子蛋白质双向电泳图谱分析

目的:探讨双向电泳技术在人类精子差异表达蛋白研究中的应用。方法:运用固相pH梯度双向凝胶电泳分离4例正常人和4例弱精子症患者精液标本的总蛋白质,凝胶银染后,用PDQuest软件进行分析,分辨出差异表达蛋白。结果:按照5倍的表达量计算,发现有差异表达的蛋白质点7个,2个点在弱精子症精子中高表达,而在正常精子为低表达;相反,在正常精子中高表达的5个点,而在弱精子症为低表达。结论:初步建立了正常精子和弱精子症患者精子的双向电泳图谱,并发现两者之间存在一些差异表达蛋白,为进一步研究特发性弱精子症精子差异表达蛋白的分离及鉴定奠定了良好基础。     

一例供精人工授精反复失败的精子蛋白质组学分析

目的分析1例供精人工授精(AID)反复失败捐献者精子的蛋白质组成差异,探讨精子蛋白质组学变化与妊娠结局的相关性。方法 1例连续9个AID周期失败的捐献者作为实验组;AID成功率在50%以上的3例捐献者作为对照组。收集两组精液样品,每位志愿者取3份精液,每次取精前禁欲3～7d,提取精子全蛋白,应用双向凝胶电泳分离精子蛋白,并行MALDI-TOF/TOF-MS质谱鉴定。结果实验组在分析区域内检测到432个蛋白点,对照组有460个蛋白点,两组中共有347个匹配蛋白点,匹配率达80.32%。检测到22个差异蛋白点,其中实验组有11个蛋白点高表达,8个蛋白点低表达,在对照组中3个蛋白点是缺失的。结论本研究利用蛋白质组学技术研究了1例AID反复失败供精者的精子蛋白质表达差异,发现22个差异蛋白点,其生物学意义有待进一步的研究。     

蛋白质组学与骨科疾病

蛋白质组学技术在骨科疾病中的应用研究已取得一些可喜的进步.蛋白质组学技术可鉴定人骨关节软骨分泌释放的新蛋白,为大样本人骨关节软骨的系统研究提供了一条可行的技术路线;可用于研究雌激素在骨蛋白质表达中的作用,为阐明雌激素丢失相关骨质疏松症的分子机制提供了实验依据;可鉴定人骨肉瘤细胞和成熟成骨细胞之间的蛋白质组差别,为研究骨肉瘤增生分化机制提供了技术手段.蛋白质组学技术可从整体水平上探究脊髓损伤这类多基因、多蛋白参与的损伤修复过程,还可用于研究椎间盘软骨细胞外基质小分子蛋白功能及在椎间盘病变中的改变等.该文就相关研究进展作一综述.     

人精浆中与精子前向运动相关蛋白的研究—蛋白的分离及活性检测

我们运用ConA包被珠的蛋白质亲和层析分离技术、蛋白质超滤技术对热处理后的人精浆进行蛋白分离、浓缩，结果发现：蛋白亲和层析洗脱浓缩液加茶碱的实验组与公牛附睾头部的精子共同孵育后，经计算机辅助的精子分析技术显示精子的前向运动速度显著高于仅用茶碱的对照组（P＜0．01）。蛋白新和层析洗脱浓缩液经聚丙酰胺凝胶电泳和SDS－聚丙酰胺凝胶电泳及Coomassie blue染色后显示：蛋白条带由聚丙酰胺凝胶中的高分子量解率成SDS－聚丙酰胺凝胶中的低分子量，经SDS－聚丙酰西安凝胶电泳分离的主要蛋白其分子量分别为46．8和57．5kDa。精浆中与精子运动相关蛋白的分离及深入的研究对进一步了解其作用机制具有十分重要的基础理论和临床意义。     

葡萄糖调节蛋白前体GRP78和肿瘤排斥抗原GP96为仓鼠附睾头部精子特有

哺乳动物睾丸中的精子经过“附睾成熟”期,由静止状态转变成为运动状态。该过程中精子从附睾头部向附睾尾部移动,同时精子发生了一系列的形态、生理和生化改变,如蛋白组成和蛋白修饰的改变可能会影响精子获能的潜能。本实验使用基质辅助激光解吸／电离串联质谱（MALDI-MS／MS）法分析仓鼠睾丸头部和尾部精子的蛋白组学,成功发现了113个蛋白质点。对113个蛋白质点进一步对照比较发现30个蛋白质点（对应20个蛋白）的密度发生了显著改变,其中附睾尾部精子5个蛋白的密度增加,11个蛋白密度减少;此外,葡萄糖调节蛋白前体GRP78和肿瘤排斥抗原GP96为仓鼠附睾头部精子特有,而纤维蛋白原样蛋白1为附睾尾部精子所特有。几个蛋白密度增加可能与附睾成熟过程中精子代谢和ATP产生相关。一些蛋白如ERp57,GRP78,GP96,Hsp60,Hsp70和二氢硫辛酰胺S-乙酰转移酶的密度改变通过免疫印迹法得到验证。本研究首次报道了仓鼠精子的蛋白质组学研究,全面展示了仓鼠精子附睾成熟过程中的蛋白结构改变。     

影响人类精子耐冻性的蛋白质分析

目的:利用比较蛋白质组学技术研究影响人类精子耐冻性的蛋白质。方法:将10例供精志愿者的31份精液标本依据冷冻复苏率的不同分为高冷冻复苏率组和低冷冻复苏率组,应用二维电泳技术和质谱分析鉴定技术寻找两组标本的精子蛋白和精浆蛋白在表达量上的差异。结果:在精子蛋白和精浆蛋白中共发现了22个差异表达蛋白。其中精浆蛋白12个,分别为细胞角蛋白1、神经元导向因子3、Rho2样蛋白、Krueppel样因子10、FERM域蛋白8、GMP合成酶、鸟嘌呤结合蛋白G、溶质载体家族12、ATP结合盒亚家族D1、6-磷酸果糖-2-激酶、肌钙蛋白C、肌球蛋白轻链;精子蛋白9个,分别为卷曲螺旋结构域蛋白113、RAF原癌基因丝氨酸/苏氨酸蛋白激酶、Ras相关蛋白Rab-31、α微管蛋白3C/D、锌指蛋白879、胆固醇26-羟化酶、DNA修复和重组蛋白、细胞角蛋白73、钙磷蛋白;精浆和精子共有蛋白1个,为细胞周期依赖性蛋白激酶1。文献调研发现这些差异蛋白主要与精子的能量代谢、成熟、运动、DNA修复等有关。结论:鉴定了多种可能影响人类精子耐冻性的蛋白质,为以后从分子水平上揭示精子冷冻复苏原理提供了依据。     

The 'omics revolution and our understanding of sperm cell biology

The foundations of proteomics are to study gene products and their regulatory roles within cells. Paradoxically, the only evidence that sperm cells make new proteins is through mitochondrial protein synthesis. Yet despite this, spermatozoa are the perfect candidates for mass spectrometry and hence, proteomic analysis. These enterprising cells use a plethora of post-translational modifications in order to gain functionality following their production within the testis. By using a combination of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and more recently liquid chromatography-mass spectrometry (LC-MS)/MS, recent advances in sperm cell biology, through the use of proteomics, is making unparalleled progress. The protein inventory lists being generated have shed light on transmembrane proteins, kinases and chaperones never previously recognized. In addition, the ability to isolate either phosphopeptides or glycopeptides and quantify the differences between cells of two different populations make proteomic analysis of spermatozoa a real chance to finally answer some age old questions.     

双向凝胶电泳和质谱技术在睾丸蛋白表达研究中的应用

目的 :探讨双向凝胶电泳和质谱技术在睾丸蛋白表达研究中的应用。　方法 :用双向凝胶电泳分离成年雄性ICR小鼠睾丸总蛋白 ;从胶上切下 2个蛋白点 ,经胶内原位酶解后 ,用质谱仪测得其肽质量指纹谱 ;再通过数据库检索鉴定蛋白质。　结果 :从胶上切下的 2个蛋白点数据库检索结果是小鼠血清白蛋白和蛋白质二硫化物异构酶。　结论 :用双向凝胶电泳分离睾丸蛋白样品取得了很高的分辨率 ,质谱技术鉴定蛋白快速方便 ,可用于从蛋白质组水平上研究睾丸表达的蛋白     

Proteomics and the genetics of sperm chromatin condensation

Spermatogenesis involves extremely marked cellular, genetic and chromatin changes resulting in the generation of the highly specialized sperm cell. Proteomics allows the identification of the proteins that compose the spermatogenic cells and the study of their function. The recent developments in mass spectrometry (MS) have markedly increased the throughput to identify and to study the sperm proteins. Catalogs of thousands of testis and spermatozoan proteins in human and different model species are becoming available, setting up the basis for subsequent research, diagnostic applications and possibly the future development of specific treatments. The present review intends to summarize the key genetic and chromatin changes at the different stages of spermatogenesis and in the mature sperm cell and to comment on the presently available proteomic studies.     

Comparative proteomic study between human normal motility sperm and idiopathic asthenozoospermia

Purpose

Idiopathic asthenozoospermia is considered as one of the causes of male infertility and characterized by reduced sperm motility. For a better determination of pathogenic mechanism of asthenozoospermia, the exploration of differentially expressed proteins in normal sperm motility and idiopathic asthenozoospermia was conducted in our study.

Methods

Sperm proteins were extracted and isolated by two-dimensional electrophoresis. All significantly changed protein spots were picked up from 2D gels and identified by tandem mass spectrometry. Sixteen of the thirty-three total differentially expressed protein spots were successfully identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry.

Results

Sixteen proteins identified belonged to 15 unique protein groups. GRP78, lactoferrin, SPANXB, PGK2, flagellin, DJ-1, XPA binding protein 2, CAB2, GPX4, and GAPDH were the first to be identified as differentially expressed proteins in idiopathic asthenospermia patients. Meanwhile, the analysis of quantitative RT-PCR was carried out to compare the protein levels, and the results indicated that the expression levels of the gene and protein were not entirely consistent.

Conclusions

These experimental results expand the scope of the protein database, generating targets for further investigation of the pathogenic mechanism of idiopathic asthenozoospermia.     

小鼠成熟卵丘-卵母细胞复合体蛋白质谱图的建立

目的通过聚丙烯酰胺二维凝胶电泳技术,全面展示小鼠成熟卵丘卵母细胞复合体(COC)的蛋白质表达谱。方法采用13cmpH3～10的非线性胶条进行双向凝胶电泳,分离成熟雌鼠COC总蛋白,并作质谱鉴定。结果获得了较高分辨率的小鼠成熟COC蛋白质表达谱图,并对胶上的3个蛋白点进行了质谱鉴定。结论蛋白质组学是全面分析COC蛋白表达的有效手段。成熟COC蛋白质表达谱的建立为研究卵泡发育机理提供了依据。     

Proteomic analysis of normal human urinary proteins isolated by acetone precipitation or ultracentrifugation

BACKGROUND: Proteomic techniques have recently become available for large-scale protein analysis. The utility of these techniques in identification of urinary proteins is poorly defined. We constructed a proteome map of normal human urine as a reference protein database by using two differential fractionated techniques to isolate the proteins. METHODS: Proteins were isolated from urine obtained from normal human volunteers by acetone precipitation or ultracentrifugation, separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and identified by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry followed by peptide mass fingerprinting. RESULTS: A total of 67 protein forms of 47 unique proteins were identified, including transporters, adhesion molecules, complement, chaperones, receptors, enzymes, serpins, cell signaling proteins and matrix proteins. Acetone precipitated more acidic and hydrophilic proteins, whereas ultracentrifugation fractionated more basic, hydrophobic, and membrane proteins. Bioinformatic analysis predicted glycosylation to be the most common explanation for multiple forms of the same protein. CONCLUSIONS: Combining two differential isolation techniques magnified protein identification from human urine. Proteomic analysis of urinary proteins is a promising tool to study renal physiology and pathophysiology and to determine biomarkers of renal disease.     

Proteomic analysis of gliomas

Primary malignant brain tumours (anaplastic glioma and glioblastoma) display heterogenous histopathology and diverse genetic abnormalities. These tumours remain incurable with no significant improvement in median survival times in the last 20 years, despite significant technological advances in surgery and radiotherapy, and mechanistic insights into their aetiology. Recent clinical trials suggest molecular characterization of tumours is essential in guiding both therapy and predicting prognosis. Genetic insight into tumour biology and increasingly proteomic technology has opened new avenues for novel applied clinical research. Protein expression in human malignant glioma and matched normal brain tissues can now be reliably analysed using quantitative proteomic techniques, the most accessible of which is two-dimensional gel electrophoresis (2DGE) and matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry from which differentially expressed proteins can be identified and characterized. The potential of using differential proteomic profiling in gliomas to identify prognostic markers and to gain insight into tumour biology is currently being investigated. The current status of proteomic technology, its application to gliomas and the utility of such translational studies is reviewed.     

Differential proteomics leads to identification of domain-specific epididymal sperm proteins

The alteration in the protein signatures of the testicular sperm during its epididymal sojourn makes it functionally competent for successful fertilization. The present study was undertaken to identify the proteins acquired on its 2 domains, that is, the head and the flagellum, during the epididymal transit using a differential proteomics approach. Testicular sperm proteome was compared with cauda epididymal sperm proteome in rat. The protein spots exclusively present in the cauda epididymal sperm proteome were searched in the cauda sperm head proteome and the cauda sperm flagella proteome, and a total of 335 spots were found by alignment and auto-matching of the gels, of which 140 could be identified by mass spectrometry. Database search revealed that of these 9 proteins were novels. Gene Ontology annotation revealed that the identified proteins were distributed across different cellular components and were primarily involved in metabolic processes. The study also provides information on the localization of these proteins on the sperm domains, which indirectly gives a clue about its putative function. Validation of 3 proteins, namely MMSDH, NDUFS1, and UQCRC2, using antibodies very elegantly demonstrates that the strategy has been very effective. This comprehensive data of domain-specific epididymal sperm proteins will be useful in development of newer targets for posttesticular contraception and diagnostic markers for infertility.