应用iTRAQ技术分析滩羊二毛期不同花穗型裘皮差异蛋白

为了解二毛期时滩羊串子花型裘皮与其它类型裘皮中蛋白质的差异,本试验采用iTRAQ技术及LC-MS/MS蛋白质组学研究方法,对串子花型、软大花型、绿豆丝型及其它不规则型花穗裘皮蛋白质进行鉴定和筛选,并运用Proteome Discoverer l.4软件进行定量分析,结合数据库搜索,鉴定出具有显著表达差异的蛋白,同时应用生物学技术对其进行GO和Pathway分析。结果显示4类花穗型裘皮共检测出2 886个蛋白,其中有135个、142个、113个差异蛋白分别存在于软大花型与串子花型、绿豆丝型与串子花型、其它不规则型与串子花型3个对比组中。对有表达差异的蛋白进行分析,发现膜联蛋白与血管内皮生长因子可能与软大花型毛股形成相关,KAP3和KAP6可能与滩羊串子花型毛股结构相关。研究发现滩羊不同二毛裘皮蛋白水平上的差异,可为选育优良的滩羊串子花型二毛裘皮提供理论基础。     

基于iTRAQ技术筛选水稻根尖甲基汞胁迫响应差异蛋白及其生物信息学分析

为了解水稻(Oryza sativa L.)对甲基汞胁迫响应的分子机制,采用两优302为实验材料,利用同位素相对标记与绝对定量技术(i TRAQ),联合液相色谱-串联质谱技术(LCMS/MS),筛选水稻甲基汞胁迫下的根尖蛋白组差异表达蛋白,并结合生物信息学对差异蛋白进行分析。结果表明,对照组与处理组共定量了3508个蛋白质,在差异倍数(fold change)≥1.20或≤0.83,且P<0.05条件下,筛选出88个差异蛋白,其中32个蛋白表达上调,56个蛋白表达下调,其中15个差异蛋白(包括类萌发素蛋白和脂氧合酶等12个已知蛋白和3个未知蛋白)具有与金属离子结合相关属性。基因本体(gene ontology,GO)分子功能提示,差异性蛋白主要涉及催化活性、结合和转运活性等生物学过程,京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路分析显示,差异蛋白显著富集于内质网蛋白加工、氧化磷酸化、淀粉与蔗糖代谢和苯丙素生物合成等代谢通路。研究结果为今后调控水稻中MeHg的吸收提供依据。     

iTRAQ标记技术与差异蛋白质组学的生物标志物研究

结合多维液相色谱和串联质谱分析,iTRAQ技术已成为差异蛋白质组学定量研究的主要工具之一。而寻找和发现区别于正常生理状态下的疾病特异表达蛋白质,有利于阐明疾病的发病机理,对疾病的预防、诊断、预后和疗效监测具有重要作用,并有助于用作新靶点来开发临床治疗药物。本文重点就该技术在医学领域中进行差异蛋白质组分析并寻找标记蛋白质的研究进行综述。     

基于iTRAQ技术分析模拟空间诱变酵母菌株的差异蛋白表达

目的：探究模拟空间诱变处理后,生长速率提高的酿酒酵母细胞的诱变机制。方法：采用同位素标记相对和绝对定量（iTRAQ）技术对模拟空间诱变处理前后的酿酒酵母进行定量蛋白质组学研究,探讨对照组与诱变组在蛋白质组表达水平上的差异,以及受影响的生物功能。结果：共鉴定到764个差异蛋白,占检测蛋白总数的15.54%。其中,上调差异表达蛋白378个、下调差异表达蛋白386个,主要涉及细胞核、线粒体、核糖体和质膜等重要细胞器和细胞结构。结论：提高重要代谢的效率、降低DNA修复功能与环境防御能力,可能是模拟空间诱变提高酿酒酵母细胞生长速率的重要机制之一。     

基于iTRAQ技术分析五倍子作用白念珠菌后的差异蛋白表达

为了探讨五倍子抗白念珠菌的作用机制,提取4 μg/mL五倍子提取液作用后的白念珠菌（SC5314）总蛋白,采用同位素标记相对和绝对定量（isobaric tags for relative and absolute quantitation,iTRAQ）蛋白质组学技术、液相色谱串联质谱（LC-MS/MS）技术分析和鉴定差异表达的蛋白质,并对差异表达蛋白进行生物信息学分析。经LC-MS/MS鉴定出3 721种蛋白质,其中,差异表达蛋白104种,包括57种表达上调蛋白和47种表达下调蛋白。通过生物信息学分析发现,上述差异蛋白参与了氧化还原反应、过氧化氢分解代谢以及能量代谢等生物学过程。研究表明,五倍子抗白念珠菌的作用机制可能是通过抑制氧化磷酸化,进而影响菌体能量代谢和物质生成,最终导致细胞结构与功能的改变。     

iTRAQ技术在真菌研究中的应用进展

iTRAQ技术是一种新的、功能强大的、可以最多同时比较8种不同样品中蛋白质相对或绝对含量的蛋白质组学方法,结合多维液相色谱和串联质谱分析,iTRAQ技术已成为差异蛋白质组学定量研究的主要工具之一。而真菌的致病作用是多种蛋白质共同参与的真菌?宿主相互作用的复杂过程,因此整体、定量地分析真菌致病过程中的差异表达蛋白质谱,对于研究真菌的致病机制具有重要作用。该文重点就iTRAQ技术在真菌研究中的应用进展进行综述。     

基于iTRAQ技术对植物乳杆菌FS5-5的耐盐特性分析

【目的】对大酱中耐盐性较好的植物乳杆菌进行蛋白质组学研究,为植物乳杆菌盐胁迫应激机制的研究提供实验数据。【方法】本项研究以筛选自东北传统农家大酱的耐盐性较好的植物乳杆菌FS5-5为研究对象,绘制了其在0%、6.0%、7.0%和8.0%(W/V)Na Cl浓度下的生长曲线,并利用i TRAQ技术研究了其在0%、6.0%、7.0%和8.0%(W/V)Na Cl浓度下的蛋白质表达情况。【结果】植物乳杆菌FS5-5在0%、6.0%、7.0%和8.0%(W/V)Na Cl浓度下到达对数生长期中期的时间点分别为5、10、12和12 h;以差异倍数在1.2倍以上且P0.05为筛选条件对6.0%、7.0%和8.0%(W/V)Na Cl浓度下与0%进行差异蛋白质的筛选,共筛选出1271个差异蛋白质。这些差异蛋白质主要参与糖代谢、氨基酸代谢、脂肪酸代谢、核苷酸代谢、应激反应、转运、PTS系统和核糖体代谢等。【结论】植物乳杆菌在高盐浓度下生长与能量合成蛋白质、应激蛋白质以及相容性溶质转运蛋白质的表达上调有密切关系。     

基于iTRAQ蛋白定量技术对多浪羊不同生理时期卵巢候选蛋白生物标志物的筛选

应用同位素标记相对和绝对定量(iTRAQ)技术筛选多浪羊(Dolang sheep)发情期、发情间期和妊娠期差异表达蛋白质,为发情调控与鉴定、早期妊娠诊断等研究提供基础.通过iTRAQ并结合液相色谱串联质谱(LC-MS/MS)技术,鉴定出4845个蛋白,其中显著差异蛋白470个.与发情间期相比,发情期上调蛋白18个,下调蛋白102个,妊娠期上调蛋白20个,下调蛋白60个;与发情期相比,妊娠期上调蛋白50个,下调蛋白24个;上调蛋白中有 cyclin-Y isoform X1,cleavages timulation factor subunit 1 isoform X1,follistatin-related protein 1 isoform X2,nicotinate phosphoribosyltransferase isoform X1,下调蛋白中 hemopexin isoform X1,apolipoprotein A-Ⅱ,nucleophosmin等蛋白与激素生成、发情调控、合子的产生与凋亡、早期胚胎附植及胚胎发育等功能有关,其中相对于发情期和发情间情期,cyclin-Y isoform X1和nicotinate phosphoribosyltrans-ferase isoform X1在妊娠期显著上调.试验通过iTRAQ蛋白质组学技术鉴定出的多浪羊发情及妊娠相关的差异蛋白质,为进一步探索绵羊发情鉴定和早期妊娠诊断候选生物标志物提供了新的思路和方法.     

茶足柄瘤蚜茧蜂滞育和非滞育蛹中与能量代谢相关的差异表达蛋白

【目的】本研究旨在从蛋白质组整体层面阐明茶足柄瘤蚜茧蜂Lysiphlebus testaceipes蛹滞育背后的多蛋白调控,重点筛选与能量代谢相关的滞育关联蛋白并分析其功能,有助于更好地理解茶足柄瘤蚜茧蜂蛹滞育的代谢机制。【方法】利用同位素标记相对和绝对定量(isobaric tags for relative and absolute quantification, iTRAQ)技术比较了茶足柄瘤蚜茧蜂滞育蛹与非滞育蛹的蛋白含量;利用GO, KEGG网络数据库等生物信息学方法分析鉴定茶足柄瘤蚜茧蜂滞育蛹与非滞育蛹中差异表达蛋白(differentially expressed proteins, DEPs)。【结果】分析得到茶足柄瘤蚜茧蜂滞蛹与非滞育蛹DEPs有135个,包括滞育蛹中上调表达蛋白有38个,下调表达蛋白有97个。GO和KEGG富集分析表明,与天冬氨酸转运、L-谷氨酸转运、胆碱脱氢酶活性和胆碱生物合成甘氨酸甜菜碱条目以及氧化磷酸化通路相关的蛋白上调表达。【结论】氧化磷酸化通路相关蛋白在茶足柄瘤蚜茧蜂滞育过程中呈显著上调表达,说明能量代谢与该蜂滞育密切相关,并推测氧化磷酸化...     

牛精子蛋白质组学技术平台的建立及冻融前后精子差异蛋白初步分析

本研究通过探索不同的精子蛋白制备方法、水化液成分和优化2D电泳程序以建立牛精子蛋白质组学研究技术平台,同时以牛鲜冻精为实验材料通过差异凝胶电泳寻找冻融前后精子蛋白的改变。结果表明：使用改进的热TRIzol法裂解精子细胞制备蛋白,结合优化的2D电泳技术可建立稳定的牛精子蛋白质组学研究技术平台。差异凝胶电泳揭示牛精子在冻融后有质和量的改变：冻融后缺失的蛋白点有20个,表达下调的有2个,表达上调的有10个。作为一项阶段性的实验成果,本研究建立的2D平台和所发现的冻融引起的差异表达蛋白质点为揭示冷冻损伤机理和性控精液的差异蛋白质组学研究奠定了较好的基础。     

Bayesian Analysis of iTRAQ Data with Nonrandom Missingness: Identification of Differentially Expressed Proteins

iTRAQ (isobaric Tags for Relative and Absolute Quantitation) is a technique that allows simultaneous quantitation of proteins in multiple samples. In this paper, we describe a Bayesian hierarchical model-based method to infer the relative protein expression levels and hence to identify differentially expressed proteins from iTRAQ data. Our model assumes that the measured peptide intensities are affected by both protein expression levels and peptide specific effects. The values of these two effects across experiments are modeled as random effects. The nonrandom missingness of peptide data is modeled with a logistic regression which relates the missingness probability for a peptide with the expression level of the protein that produces this peptide. We propose a Markov chain Monte Carlo method for the inference of model parameters, including the relative expression levels across samples. Our simulation results suggest that the estimates of relative protein expression levels based on the MCMC samples have smaller bias than those estimated from ANOVA models or fold changes. We apply our method to an iTRAQ dataset studying the roles of Caveolae for postnatal cardiovascular function.     

Identification of Differentially Expressed Proteins and Phosphorylated Proteins in Rice Seedlings in Response to Strigolactone Treatment

Strigolactones (SLs) are recently identified plant hormones that inhibit shoot branching and control various aspects of plant growth, development and interaction with parasites. Previous studies have shown that plant D10 protein is a carotenoid cleavage dioxygenase that functions in SL biosynthesis. In this work, we used an allelic SL-deficient d10 mutant XJC of rice (Oryza sativa L. spp. indica) to investigate proteins that were responsive to SL treatment. When grown in darkness, d10 mutant seedlings exhibited elongated mesocotyl that could be rescued by exogenous application of SLs. Soluble protein extracts were prepared from d10 mutant seedlings grown in darkness in the presence of GR24, a synthetic SL analog. Soluble proteins were separated on two-dimensional gels and subjected to proteomic analysis. Proteins that were expressed differentially and phosphoproteins whose phosphorylation status changed in response to GR24 treatment were identified. Eight proteins were found to be induced or down-regulated by GR24, and a different set of 8 phosphoproteins were shown to change their phosphorylation intensities in the dark-grown d10 seedlings in response to GR24 treatment. Analysis of these proteins revealed that they are important enzymes of the carbohydrate and amino acid metabolic pathways and key components of the cellular energy generation machinery. These proteins may represent potential targets of the SL signaling pathway. This study provides new insight into the complex and negative regulatory mechanism by which SLs control shoot branching and plant development.     

Screening for Differentially Expressed Proteins Relevant to the Differential Diagnosis of Sarcoidosis and Tuberculosis

Background

In this study, we sought to identify differentially expressed proteins in the serum of patients with sarcoidosis or tuberculosis and to evaluate these proteins as markers for the differential diagnosis of sarcoidosis and sputum-negative tuberculosis.

Methods

Using protein microarrays, we identified 3 proteins exhibiting differential expression between patients with sarcoidosis and tuberculosis. Elevated expression of these proteins was verified using the enzyme-linked immunosorbent assay (ELISA) and was further confirmed by immunohistochemistry. Receiver operating characteristic (ROC) curve, logistic regression analysis, parallel, and serial tests were used to evaluate the diagnostic efficacy of the proteins.

Results

Intercellular Adhesion Molecule 1(ICAM-1) and leptin were screened for differentially expressed proteins relevant to sarcoidosis and tuberculosis. Using ROC curves, we found that ICAM-1 (cutoff value: 57740 pg/mL) had an area under the curve (AUC), sensitivity, and specificity of 0.718, 62.3%, and 79.5% respectively, while leptin (cutoff value: 1193.186 pg/mL) had an AUC, sensitivity, and specificity of 0.763, 88.3%, and 65.8%, respectively. Logistic regression analysis revealed that the AUC, sensitivity, and specificity of combined leptin and ICAM-1 were 0.787, 89.6%, and 65.8%, respectively, while those of combined leptin, ICAM-1, and body mass index (BMI) were 0.837, 90.9%, and 64.4%, respectively, which had the greatest diagnostic value. Parallel and serial tests indicated that the BMI-leptin parallel with the ICAM-1 serial was the best diagnostic method, achieving a sensitivity and specificity of 86.5% and 73.1%, respectively. Thus, our results identified elevated expression of ICAM-1 and leptin in serum and granulomas of sarcoidosis patients.

Conclusions

ICAM-1 and leptin were found to be potential markers for the diagnosis of sarcoidosis and differential diagnosis of sarcoidosis and sputum-negative tuberculosis.     

筛选差异表达基因和蛋白质的方法进展

分离和鉴定差异表达基因和蛋白质不仅有助于发现基因和蛋白质的功能,更有助于揭示某些疾病的发生机理.目前筛选差异表达基因的方法主要有差异显示PCR方法(differential display RT-PCR,DDRT-PCR)、消减杂交法(subtractive hybridization,SH)、基因芯片技术(DNA chip technique)和基因表达的系统分析(serial analysis of gene expression,SAGE)等,其中消减杂交法中又先后建立了代表性差异分析技术(representational difference analysis,RDA)、抑制消减杂交法(suppression subtractive hybridization,SSH)和获得全长基因的消减杂交法(full-length-gene-obtainable subtractive hybridization).筛选差异表达蛋白质的方法主要有双向电泳技术(two-dimentional gel electrophoresis)和噬菌体全套抗体库技术(phage display antibody repertoire library technique).这些方法各有特点,各有利弊,研究者可根据自己的需要选择适合于自己的方法.     

A Statistical Model to Identify Differentially Expressed Proteins in 2D PAGE Gels

Two dimensional polyacrylamide gel electrophoresis (2D PAGE) is used to identify differentially expressed proteins and may be applied to biomarker discovery. A limitation of this approach is the inability to detect a protein when its concentration falls below the limit of detection. Consequently, differential expression of proteins may be missed when the level of a protein in the cases or controls is below the limit of detection for 2D PAGE. Standard statistical techniques have difficulty dealing with undetected proteins. To address this issue, we propose a mixture model that takes into account both detected and non-detected proteins. Non-detected proteins are classified either as (a) proteins that are not expressed in at least one replicate, or (b) proteins that are expressed but are below the limit of detection. We obtain maximum likelihood estimates of the parameters of the mixture model, including the group-specific probability of expression and mean expression intensities. Differentially expressed proteins can be detected by using a Likelihood Ratio Test (LRT). Our simulation results, using data generated from biological experiments, show that the likelihood model has higher statistical power than standard statistical approaches to detect differentially expressed proteins. An R package, Slider (Statistical Likelihood model for Identifying Differential Expression in R), is freely available at http://www.cebl.auckland.ac.nz/slider.php.     

五倍体草莓及其十倍体的叶片差异表达蛋白分析

以二倍体绿色草莓(Fragaria viridis Duch.)为父本、八倍体栽培草莓‘房香’(F.×ananassa‘Fusanoka’)为母本杂交所得的五倍体草莓(株系代号:FxLs-11-37,2n=5x=35)及其染色体加倍而成的十倍体(株系代号:A3,2n=10x=70)草莓为试材,观察记载其部分农艺性状、SPAD值以及净光合速率,利用双向凝胶电泳技术对草莓染色体加倍后叶片蛋白质进行了分析,获得了分辨率高、重复性好的电泳图谱。结果表明:(1)与五倍体FxLs-11-37相比,十倍体A3的株型明显矮化、植株冠径变大、叶长叶宽增大、叶片增厚以及叶色浓绿,其叶片的SPAD值与净光合速率显著高于FxLs-11-37。(2)通过PDQuest软件对图谱分析表明,两者在等电点4~7、分子量14.4~66.2kD范围内蛋白质斑点分布最多,可识别的总蛋白质斑点数超过700个,其中蛋白质表达差异水平在1.5倍以上的有18个,2.0倍以上的有4个。利用MALDI-TOF-MS/MS质谱技术鉴定了这4个差异蛋白质,分别是草莓主要过敏原a 1-E、核内不均一核糖核蛋白1、叶绿体硫辛酰基合酶1、NAD(P)H脱氢酶(醌)FQR1类似蛋白质。这些差异蛋白质主要与抗逆、mRNA转运、物质与能量代谢相关。荧光定量PCR对上述4个蛋白编码基因的转录表达水平检测表明,与蛋白质表达差异的趋势一致。该研究获得了草莓染色体加倍后差异表达的蛋白质,为深入研究提供了线索。     

Identification of Prolificacy‐Related Differentially Expressed Proteins from Sheep (Ovis aries) Hypothalamus by Comparative Proteomics

Reproduction, as a physiologically complex process, can significantly affect the development of the sheep industry. However, a lack of overall understanding to sheep fecundity has long blocked the progress in sheep breeding and husbandry. In the present study, the aim is to identify differentially expressed proteins (DEPs) from hypothalamus in sheep without FecB mutation in two comparison groups: polytocous (PF) versus monotocous (MF) sheep at follicular phase and polytocous (PL) versus monotocous (ML) sheep at luteal phase. Totally 5058 proteins are identified in sheep hypothalamus, where 22 in PF versus MF, and 39 proteins in PL versus ML are differentially expressed, respectively. A functional analysis is then conducted including Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis to reveal the potential roles of these DEPs. The proteins ENSOARP00000020097, ENSOARP00000006714, growth hormone (GH), histone deacetylase 4 (HDAC4), and 5′‐3′ exoribonuclease 2 (XRN2) in PF versus MF, and bcl‐2‐associated athanogene 4 (BAG4), insulin‐like growth factor‐1 receptor (IGF1R), hydroxysteroid 11‐beta dehydrogenase 1 (HSD11B1), and transthyretin (TTR) in PL versus ML appear to modulate reproduction, presumably by influencing the activities of gonadotropin‐releasing hormone (GnRH). This study provides an alternative method to identify DEPs associated with sheep prolificacy from the hypothalamus. The mass spectrometry data are available via ProteomeXchange with identifier PXD013822.     

Differentially Expressed Outer Membrane Proteins of Vibrio alginolyticus in Response to Six Types of Antibiotics

Vibrio alginolyticus is an opportunistic pathogen that occasionally causes life-threatening infections in individuals and results in great losses in marine aquacultures of crustaceans and fish. Recently, antibiotic-resistant strains of the bacterium from clinical and environmental sources have been reported with increasing frequency. However, few reports were involved in the antibiotic resistance of this bacterium at molecular levels. In the present study, Western blotting was utilized to investigate altered OM proteins of V. alginolyticus in response to six types of antibiotics: erythromycin, kanamycin, tetracycline, streptomycin, nalidixic acid, and chloromycetin. Seventeen OM proteins have been reported here for the first time to be related to antibiotic resistance. They were porins OmpU, OmpN, putative OmpU and LamB; transport proteins VA0802, VA2212 (FadL) and VPA0860; TolC family TolC and VA1631; lipoprotein VA0449; OmpA family VPA1186 and VA0764; iron-regulated proteins OmpV, VPA1435, and VA2602; and receptor protein OmpK; hypothetical protein VA1475. Importantly, VA2212 was up-regulated in response to the five antibiotics except nalidixic acid, and VPA1186 was down-regulated in response to the six antibiotics in antibiotic-stressed bacteria. They might be potentially universal targets for designing the new drugs that inhibit multi-resistant bacteria. These findings suggested that parallel investigations into a bacterium responding to several types of antibiotics would be helpful not only for the further understanding of antibiotic-resistant mechanisms but also for the screening of valuable targets of new drugs controlling antibiotic-resistant bacteria.     

Identification of Differentially Expressed Proteins in Murine Embryonic and Postnatal Cortical Neural Progenitors

Background

The central nervous system (CNS) develops from a heterogeneous pool of neural stem and progenitor cells (NSPC), the underlying differences among which are poorly understood. The study of NSPC would be greatly facilitated by the identification of additional proteins that mediate their function and that would distinguish amongst different progenitor populations.

Methodology/Principal Findings

To identify membrane and membrane-associated proteins expressed by NSPC, we used a proteomics approach to profile NSPC cultured as neurospheres (NS) isolated from the murine cortex during a period of neurogenesis (embryonic day 11.5, E11.5), as compared to NSPC isolated at a peak of gliogenesis (postnatal day 1, P0) and to differentiated E11.5 NS. 54 proteins were identified with high expression in E11.5 NS, including the TrkC receptor, several heterotrimeric G proteins, and the Neogenin receptor. 24 proteins were identified with similar expression in E11.5 and P0 NS over differentiated E11.5 NS, and 13 proteins were identified with high expression specifically in P0 NS compared to E11.5 NS. To illustrate the potential relevance of these identified proteins to neural stem cell biology, the function of Neogenin was further studied. Using Fluorescence Activated Cell Sorting (FACS) analysis, expression of Neogenin was associated with a self-renewing population present in both E11.5 and adult subventricular zone (SVZ) NS but not in P0 NS. E11.5 NS expressed a putative Neogenin ligand, RGMa, and underwent apoptosis when exposed to a ligand-blocking antibody.

Conclusions/Significance

There are fundamental differences between the continuously self-renewing and more limited progenitors of the developing cortex. We identified a subset of differentially expressed proteins that serve not only as a set of functionally important proteins, but as a useful set of markers for the subsequent analysis of NSPC. Neogenin is associated with the continuously self-renewing and neurogenic cells present in E11.5 cortical and adult SVZ NS, and the Neogenin/RGMa receptor/ligand pair may regulate cell survival during development.