Proteome analysis of the silkworm (Bombyx mori. L) colleterial gland during different development stages

The silkworm, Bombyx mori, colleterial gland developed very slowly until 2 days before emergence, then markedly enlarged due to the accumulation of a glue-like substances (mainly including 85% water and 11% proteins). However, the No glue (Ng) mutant female moth secreted only very little glue-like substance and laid loose eggs naturally. High-resolution two-dimensional polyacrylamide gel electrophoresis, followed by computer-assisted analysis, was used to screen the secretory region of colleterial gland protein patterns during different development stages to find quantitative and qualitative difference in protein expression during the pupae and moth stages. More than 700 protein spots were resolved in different developmental stages from the secretory region of the glands and most of the proteins were distributed in the mass range from 30 to 70 kD with pH 4-8. Through comparison and analysis, it was found that 3 proteins were only expressed in the later pupae stage (one or two days before emergence) and moth stage. Furthermore, these proteins were not expressed in the Ng mutant especially actin. There was a great variation of some protein expression volume during the development. Protein spots that changed more than 1.5-fold in expression level (relative to day 9), including 6 spots that were down-regulated and 2 spots that were up-regulated in expression were excised for identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Results indicated that actins that participated or regulated the exocytosis of colleterial gland and other differentially expressed proteins might be related to colleterial gland development or the secretion of a glue-like substance.     

Subproteomic analysis of the mitochondrial proteins in rats 24 h after partial hepatectomy

A 70% partial hepatectomy (70%PHx) induces cell proliferation until the original mass of the liver is restored. Mitochondria are involved directly in the process of liver regeneration (LR); however, their role in the early phase of LR is not clear. In an attempt to identify mitochondrial proteins that are correlated with the early phase of LR, we obtained a mitochondrial fraction via Nycodenz(R) density gradient centrifugation and subcellular proteomic analysis was performed. The mitochondrial proteins were separated by two-dimensional gel electrophoresis and identified by mass spectrometry. Compared to the sham-operation control group, 3 proteins were up-regulated and 22 proteins were down-regulated at 24 h after 70%PHx. We identified 22 differentially expressed proteins that were associated with carbohydrate metabolism, lipid metabolism, the respiratory chain and oxidation-phosphorylation, biotransformation and other metabolic pathways. Prohibitin, a proliferation-regulating protein that was down-regulated at 24 h after PHx, was analyzed by applying RNAi (PHBi) with BRL-3A. This demonstrated a decreased mitochondrial membrane potential, implying a potential role in maintaining mitochondrial integrity. These results indicated that hepatic mitochondrial adaptations to LR after 70%PHx affect various cellular metabolic pathways, which advances our knowledge of the role of mitochondria in the early phase of LR.     

Integrated Seed Proteome and Phosphoproteome Analyses Reveal Interplay of Nutrient Dynamics,Carbon–Nitrogen Partitioning,and Oxidative Signaling in Chickpea

Nutrient dynamics in storage organs is a complex developmental process that requires coordinated interactions of environmental, biochemical, and genetic factors. Although sink organ developmental events have been identified, understanding of translational and post‐translational regulation of reserve synthesis, accumulation, and utilization in legumes is limited. To understand nutrient dynamics during embryonic and cotyledonary photoheterotrophic transition to mature and germinating autotrophic seeds, an integrated proteomics and phosphoproteomics study in six sequential seed developmental stages in chickpea is performed. MS/MS analyses identify 109 unique nutrient‐associated proteins (NAPs) involved in metabolism, storage and biogenesis, and protein turnover. Differences and similarities in 60 nutrient‐associated phosphoproteins (NAPPs) containing 93 phosphosites are compared with NAPs. Data reveal accumulation of carbon–nitrogen metabolic and photosynthetic proteoforms during seed filling. Furthermore, enrichment of storage proteoforms and protease inhibitors is associated with cell expansion and seed maturation. Finally, combined proteoforms network analysis identifies three significant modules, centered around malate dehydrogenase, HSP70, triose phosphate isomerase, and vicilin. Novel clues suggest that ubiquitin–proteasome pathway regulates nutrient reallocation. Second, increased abundance of NAPs/NAPPs related to oxidative and serine/threonine signaling indicates direct interface between redox sensing and signaling during seed development. Taken together, nutrient signals act as metabolic and differentiation determinant governing storage organ reprogramming.     

蛋白质组研究进展

蛋白质组研究技术是后基因组时代的重要研究手段.综述了流感嗜血杆菌、细菌、酵母、线虫、果蝇蛋白质组的研究进展以及蛋白质组研究技术在人类疾病研究中的应用.     

Proteome analysis on differentially expressed proteins of the fat body of two silkworm breeds, Bombyx mori, exposed to heat shock exposure

Proteomes of heat tolerant (multivoltine) and heat susceptible (bivoltine) silkworms (Bombyx mori) in response to heat shock were studied. Detected proteins from fat body were identified by using MALDI-TOF/TOF spectrometer, MS/MS, and MS analysis. Eight proteins, including small heat shock proteins (sHSPs) and HSP70, were expressed similarly in both breeds, while 4 protein spots were expressed specifically in the bivoltine breed and 12 protein spots were expressed specifically in the multivoltine breed. In the present proteomics approach, 5 separate spots of sHSP proteins (HSP19.9, HSP20.1, HSP20.4, HSP20.8, and HSP21.4) were identified. Protein spot intensity of sHSPs was lower in the multivoltine breed than in the bivoltine breed after the 45°C heat shock treatment, while the difference between two breeds was not significant after the 41°C heat shock treatment. These results indicated that some other mechanisms might be engaged in thermal tolerance of multivotine breed except for the expression of sHSP and HSP70. There were visible differences in the intensity of heat shock protein expression between male and female, however, differences were not statistically significant.     

Proteome analysis of chicken embryonic gonads: identification of major proteins from cultured gonadal primordial germ cells

The domestic chicken (Gallus gallus) is an important model for research in developmental biology because its embryonic development occurs in ovo. To examine the mechanism of embryonic germ cell development, we constructed proteome map of gonadal primordial germ cells (gPGCs) from chicken embryonic gonads. Embryonic gonads were collected from 500 embryos at 6 days of incubation, and the gPGCs were cultured in vitro until colony formed. After 7-10 days in culture, gPGC colonies were separated from gonadal stroma cells (GSCs). Soluble extracts of cultured gPGCs were then fractionated by two-dimensional gel electrophoresis (pH 4-7). A number of protein spots, including those that displayed significant expression levels, were then identified by use of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and LC-MS/MS. Of the 89 gPGC spots examined, 50 yielded mass spectra that matched avian proteins found in on-line databases. Proteome map of this type will serve as an important reference for germ cell biology and transgenic research.     

Proteome analysis of silk gland proteins from the silkworm, Bombyx mori

The silk gland of Bombyx mori is an organ specialized for the synthesis and secretion of silk proteins. We report here the resolution of silk gland proteins by 2-DE and the identification of many of those proteins. This was accomplished by dissecting the glands into several sections, with each exhibiting more than 400 protein spots by 2-DE, of which 100 spots were excised and characterized by in-gel digestion followed by PMF. Ninety-three proteins were tentatively identified. These were then categorized into groups involved in silk protein secretion, transport, lipid metabolism, defense, etc. Western blotting of a 2-DE gel using an antibody of the carotenoid binding protein confirmed the presence of this protein in the silk gland. Proteins including fibroin L-chain and P25 were found as multiple isoforms, some of which contained differential amounts of phosphate residues as analyzed by on-probe dephosphorylation. The current analysis contributes to our understanding of proteins expressed by the silk gland not only of the model lepidopteran B. mori, but also to proteins from other silk-producing insects such as Philosamia cynthia ricini.     

叶绿体蛋白质组研究进展

亚细胞蛋白质组学是近年来蛋白组学研究中的一个热点。通过细胞器的纯化和亚细胞组分的分离,降低了样品的复杂性,增大了相应蛋白质组分的富集,有利于由此分离获得的蛋白质的序列分析及功能鉴定。叶绿体蛋白质组为植物亚细胞蛋白质组学研究中相对全面的一部分,利用亚细胞分离结合双向电泳技术系统地鉴定叶绿体中蛋白质组分是获取叶绿体蛋白质信息、确定其功能的重要技术手段。本文就近年来植物叶绿体蛋白质组涵盖的叶绿体内、外被膜、叶绿体基质、类囊体膜和类囊体腔蛋白的研究进行综述,以全面认识叶绿体蛋白的组成、特点及其在叶绿体生理生化代谢网络中的作用。     

蛋白质组研究的技术体系及其进展

随着后基因组时代的到来,蛋白质组研究越来越受到国内外科学工作者的密切关注, 我国国家自然科学基金委员会已把蛋白质组研究列为重大科研项目.概述了蛋白质组研究中的基本技术,包括双向凝胶电泳的样品制备和分离、蛋白质的检测、凝胶图像分析、蛋白质的鉴定以及蛋白质数据库构建等,并就蛋白质鉴定的常用方法如氨基酸组成分析方法、蛋白质末端序列分析、肽质量指纹谱作了详细阐述.直观地列出了蛋白质组研究的技术体系流程图,着重介绍了蛋白质组研究的最新技术及其进展.     

蛋白质组研究中肽质量指纹谱鉴定方法的建立及应用

建立了用肽质量指纹谱和数据库检索方法鉴定凝胶电泳分离蛋白南的方法。用标准蛋白质对胶上蛋白５质原位酶切制备肽谱的方法进行了讨论。分析了实际细胞蛋白质样品,获得双向电泳分离的人肺癌细胞蛋白质谱中三个蛋白质点的肽指纹谱。并通过数据７库检索分别鉴定为甘油醛－３－磷酸脱氢酶－２,测在蛋白羟基末端水解同工酶和丙糖磷酸异构酶。     

适于小麦叶片蛋白质组分析的样品提取方法研究

以‘铭贤169'小麦苗期叶片为材料,分别采用传统的TCA/丙酮沉淀法、酚提取-甲醇/醋酸铵沉淀法以及改进的TCA/丙酮沉淀-酚/SDS联合抽提法提取叶片总蛋白,进行双向电泳分离和胶体考染,以建立适用于小麦蛋白质组分析的样品制备方法.结果表明:TCA/丙酮沉淀法较酚提取-甲醇/醋酸铵沉淀法获得的蛋白杂质较少,在二维电泳图谱中的蛋白点较酚抽提-甲醇/醋酸铵沉淀法提取的蛋白点清晰且多.相比于以上2种提取蛋白样品方法,改进的TCA/丙酮沉淀-酚/SDS联合抽提法提取的小麦叶片蛋白杂质少、二维电泳图谱上的点明显增多、分辨率较高.所选小麦的代表性蛋白点能获得成功鉴定.该方法可推广应用于水稻叶片蛋白质组分析的样品提取.     

Proteins Involved in the Interaction of Potato Tubers with Pectobacterium atrosepticum: a Proteomic Approach to Understanding Partial Resistance

Potato can be severely affected by various pathogens, including Pectobacterium atrosepticum, the cause of bacterial soft rot on tubers and of blackleg on stems. To date, no complete resistance to P. atrosepticum is available, so that only cultivars exhibiting partial resistance can be found. The mechanistic basis of this type of resistance is still poorly understood. A proteomic approach was thus developed to identify pathways specifically activated during the interaction between potato tubers and P. atrosepticum. Protein profiles on silver‐stained gels in the 5–8 pH range were obtained from healthy and infected tubers from two cultivars differing for resistance level and analyzed by 2‐DE and nano‐LC‐MS/MS. Thirteen proteins were differentially up‐regulated in the partially resistant cv. Kerpondy; by contrast, no significant differences in protein profiles of inoculated and control tubers were observed in the susceptible cv. Bintje. Mass spectrometry and database searching showed that these proteins are involved in energetic metabolism (glyceraldehyde‐3‐phosphate dehydrogenase, 2‐phosphoglycerate dehydratase or enolase, fructose biphosphate aldolase and ATPase α subunit), cytoskeleton structure (actin), protein catabolism (cysteine protease inhibitor) and patatins or patatin precursors. Their involvement in defence responses of cv. Kerpondy to P. atrosepticum is discussed. Proteomic appears as an efficient approach to have insight into the mechanisms and pathways leading to potato resistance against P. atrosepticum.     

长期中小强度有氧运动对大鼠左心室肌蛋白质组差异表达的影响*

目的:探讨长期中小强度有氧运动对大鼠左室肌蛋白质组差异性表达的影响,筛选出对中小强度有氧运动刺激敏感的目标蛋白质,丰富运动健身的基础理论及为慢性心血管疾病的康复治疗提供新的思路和实验依据。方法:20只雄性SD大鼠随机分为运动组(E组)和对照组(C组)(n=10)。建立大鼠长期中小强度有氧运动跑台训练模型,采用双向凝胶电泳(2-DE)对大鼠左心室肌全蛋白样品进行分离。采用串联飞行时间质谱蛋白质仪对部分分离后差异表达量上调大于5倍或下调超过80%的蛋白质点进行质谱鉴定。结果:与C组比较,E组大鼠心脏重量指数(HWI)增加了32.0%,具有显著性差异(P＜0.05);与C组比较,E组有71个蛋白质点表达上调≥2倍或下调≥50%,对4个表达上调≥5倍或下调大于等于80%的蛋白质点进行质谱鉴定,鉴定出3个蛋白质和1个未知蛋白质。结论:长期中小强度有氧运动后,大鼠心脏发生了良好的适应性改变;大鼠左心室肌蛋白质组发生了明显变化,长期中小强度有氧运动能有效增强大鼠心肌抗氧化能力。     

Subproteomes of soluble and structure-bound Helicobacter pylori proteins analyzed by two-dimensional gel electrophoresis and mass spectrometry

Helicobacter pylori is one of the most common bacterial pathogens and causes a variety of diseases, such as peptic ulcer or gastric cancer. Despite intensive study of this human pathogen in the last decades, knowledge about its membrane proteins and, in particular, those which are putative components of the type IV secretion system encoded by the cag pathogenicity island (PAI) remains limited. Our aim is to establish a dynamic two-dimensional electrophoresis-polyacrylamide gel electrophoresis (2-DE-PAGE) database with multiple subproteomes of H. pylori (http://www.mpiib-berlin.mpg.de/2D-PAGE) which facilitates identification of bacterial proteins important in pathogen-host interactions. Using a proteomic approach, we investigated the protein composition of two H. pylori fractions: soluble proteins and structure-bound proteins (including membrane proteins). Both fractions differed markedly in the overall protein composition as determined by 2-DE. The 50 most abundant protein spots in each fraction were identified by peptide mass fingerprinting. We detected four cag PAI proteins, numerous outer membrane proteins (OMPs), the vacuolating cytotoxin VacA, other potential virulence factors, and few ribosomal proteins in the structure-bound fraction. In contrast, catalase (KatA), gamma-glutamyltranspeptidase (Ggt), and the neutrophil-activating protein NapA were found almost exclusively in the soluble protein fraction. The results presented here are an important complement to genome sequence data, and the established 2-D PAGE maps provide a basis for comparative studies of the H. pylori proteome. Such subproteomes in the public domain will be effective instruments for identifying new virulence factors and antigens of potential diagnostic and/or curative value against infections with this important pathogen.     

Proteomic analysis of inviable salmonid hybrids between female masu salmon Oncorhynchus masou masou and male rainbow trout Oncorhynchus mykiss during early embryogenesis

Early embryos of inviable hybrids between female masu salmon Oncorhynchus masou masou and male rainbow trout Oncorhynchus mykiss at 9, 12, 15 and 20 days after fertilization were examined for protein expression profiles. A total of 44 proteins, mostly down-regulated products of house-keeping genes and those involved in nucleic acid metabolism or chromatin replication, were identified in hybrid embryos by mass spectrometry analysis and protein database searching. The identified down-regulated proteins may be responsible for the inviability in the hybrids.     

A Simplified Thermal Proteome Profiling Approach to Screen Protein Targets of a Ligand

Thermal proteome profiling is a powerful energetic‐based chemical proteomics method to reveal the ligand‐protein interaction. However, the costly multiplexed isotopic labeling reagent, mainly Multiplexed isobaric tandem mass tag (TMT), and the long mass spectrometric time limits the wide application of this method. Here a simple and cost‐effective strategy by using dimethyl labeling technique instead of TMT labeling is reported to quantify proteins and by using the peptides derived from the same protein to determine significantly changed proteins in one LC‐MS run. This method is validated by identifying the known targets of methotrexate and geldanamycin. In addition, several potential off‐targets involved in detoxification of reactive oxygen species pathway are also discovered for geldanamycin. This method is further applied to map the interactome of adenosine triphosphate (ATP) in the 293T cell lysate by using ATP analogue, adenylyl imidodiphosphate (AMP‐PNP), as the ligand. As a result, a total of 123 AMP‐PNP‐sensitive proteins are found, of which 59 proteins are stabilized by AMP‐PNP. Approximately 53% and 20% of these stabilized candidate protein targets are known as ATP and RNA binding proteins. Overall, above results demonstrated that this approach could be a valuable platform for the unbiased target proteins identification with reduced reagent cost and mass spectrometric time.     

Proteome analysis of antibody-producing CHO cell lines with different metabolic profiles

Two-dimensional gel electrophoresis and tandem mass spectrometry were used to identify proteins associated with a metabolic shift during fed-batch cultures of two recombinant antibody-producing CHO cell lines. The first cell line underwent a marked change in lactate metabolism during culture, initially producing lactate and then consuming it, while the second cell line produced lactate for a similar duration but did not later consume it. The first cell line displayed a declining specific antibody productivity during culture, correlating to the 2-D gel results and the intracellular antibody concentration determined by HPLC. Several statistical analysis methods were compared during this work, including a fixed fold-change criterion and t-tests using standard deviations determined in several ways from the raw data and mathematically transformed data. Application of a variance-stabilizing transformation enabled the use of a global empirical standard deviation in the t-tests. Most of the protein spots changing in each cell line did not change significantly in the other cell line. A substantial fraction of the changing proteins were glycolytic enzymes; others included proteins related to antibody production, protein processing, and cell structure. Enolase, pyruvate kinase, BiP/GRP78, and protein disulfide isomerase were found in spots that changed over time in both cell lines, and some protein changes differed from previous reports. These data provide a foundation for future investigation of metabolism in industrially relevant mammalian cell culture processes, and suggest that along with differences between cell types, the proteins expressed in cultures with low lactate concentrations may depend on how those conditions were generated.     

SDS-聚丙烯酰氨凝胶电泳与液质联用技术分离和鉴定小鼠巨噬细胞膜蛋白

用膜蛋白分离试剂盒提取巨噬细胞膜蛋白,然后用SDS-聚丙烯酰氨凝胶电泳进行分离。将每个泳道平均切成8份,合并两个泳道同样位置的胶条,分别进行胶内酶解。酶解得到的多肽经脱盐后进入毛细管反相柱进行反相分离,分离后的肽段直接进入电喷离子源质谱仪进行一级和二级质谱分析。质谱数据用SEQUEST软件对小鼠IPI蛋白数据库进行检索,得到一个含有1000多种蛋白的名单,其中包括458种经GOA注释的膜蛋白。对膜蛋白部分进一步分析发现,其中包括CD11b、TNF-a、F4/80、CD14、CD18、CD86、CD44、CD16、Toll样受体等已知表达在巨噬细胞表面的蛋白分子,还包括另外13种CD分子和18种Ras相关GTPase,除了这些已知蛋白之外,还鉴定出若干新蛋白分子,为进一步深入研究巨噬细胞生物学功能提供了目标分子。     

Proteome analysis of rice root proteins regulated by gibberellin

To gain an enhanced understanding of the mechanism by which gibberellins （GAs） regulate the growth and development of plants, it is necessary to identify proteins regulated by GA. Proteome analysis techniques have been applied as a direct, effective, and reliable tool in differential protein expressions. In previous studies, sixteen proteins showed differences in accumulation levels as a result of treatment with GA3, uniconazole, or abscisic acid （ABA）, and/or the differences between the GA-deficient semi-dwarf mutant, Tan-ginbozu, and normal cultivars. Among these proteins, aldolase increased in roots treated with GA3, was present at low levels in Tan-ginbozu roots, and decreased in roots treated with uniconazole or ABA. In a root elongation assay, the growth of aldolase-antisense transgenic rice was half of that of vector control transgenic rice. These results indicate that increases in aldolase activity stimulate the glycolytic in the GA-induced growth of roots. In among GA, aldolase, and root growth. pathway and may play an important role this review, we discuss the relationship among GA, aldolase, and root growth.