~(60)Co-γ射线诱发皖麦50 Glu-1A亚基等位基因突变体差异蛋白质组学研究

研究了皖麦50及~(60)Co-γ射线诱发的Glu-1A突变体的比较蛋白质组学,结果表明,突变体蛋白质含量增加,2-D电泳图上有11个蛋白质斑点表达差异显著,其中3个蛋白点上调,8个蛋白点下调。经MALDI-TOF/TOF质谱鉴定,差异蛋白点涉及44个蛋白质。通过GO分析,参与生物过程的差异蛋白中,18个蛋白质参与代谢过程,18个蛋白质参与细胞过程,8个蛋白质参与刺激响应。参与分子功能差异蛋白中,18个蛋白质参与催化活性,10个蛋白质参与链结,7个肽段为分子功能调解剂,9个肽段为未知蛋白。参与细胞成分的差异蛋白中,12个蛋白质为胞外区蛋白,12个蛋白质为细胞,9个蛋白质为细胞器,2个蛋白质为膜,9个蛋白质为未知蛋白。KEGG通路注解表明,差异蛋白质中,4个蛋白质参与淀粉和蔗糖代谢,3个蛋白质参与氨基糖和核苷酸糖代谢,1个参与丙酮循环,1个参与半胱氨酸和甲硫氨酸代谢,1个参与丙酮酸代谢,1个参与乙醛酸和二羧酸代谢,1个参与光合生物碳固定,1个参与碳代谢信息通路。     

全基因组预测目标基因的新方法及其应用

运用隐马尔可夫模型, 利用Perl编程, 以几种模式生物的蛋白质数据库为基础, 构建了目标基因的全基因组预测的新方法。该方法具有高通量, 准确度高且操作简易等优点, 特别在多结构域蛋白家族预测上更显优势。应用该方法对几种模式生物的全基因组PPR和TPR蛋白家族进行了预测, 其中粳稻日本晴中含有536个PPR蛋白、199个TPR蛋白; 籼稻9311中含有519个PPR蛋白、177个TPR蛋白; 拟南芥中含有735个PPR蛋白、292个TPR蛋白; 红藻中6个PPR蛋白、32个TPR蛋白; 蓝细菌以及古细菌中没有PPR蛋白, 但蓝细菌含有10个TPR蛋白, 古细菌有4个TPR蛋白, 并对所得结果进行了进一步生物信息学分析。     

Characterization of the proteins of guinea-pig hair and hair-follicle tissue

This work forms a part of a study of the mechanism and control of protein synthesis in the hair follicle and concerns the characterization of the proteins of hair-follicle tissue and for comparative reasons those of the hair itself. 1. Five different groups of reduced carboxymethylated proteins were delineated from both tissues; these were: group 1A proteins, which appeared to be aggregates of the group 2 proteins; group 1B proteins, soluble at pH4.4, which were thought to originate from the medulla and inner-rootsheath layers; group 2 proteins, which were defined as the main low-sulphur keratin proteins insoluble at pH4.4; group 3 proteins, the precise origin of which is not known; and the group 4 proteins, which were defined as the main high-sulphur keratin proteins soluble at pH4.4. 2. With the single exception of the group 1B proteins, the types and properties of all hair and hair-follicle proteins were identical as far as could be determined by use of such criteria as multiplicity of components, molecular charge, molecular weight and amino acid composition. 3. Two significant quantitative differences were noted: in follicle extracts there were more group 2 proteins but less group 3 and group 4 proteins than in hair extracts; and secondly, in the follicle group 4 proteins, there were more proteins of lowest molecular weight and S-carboxymethylcysteine content, but fewer proteins of the highest molecular weight and S-carboxymethylcysteine conent than in the hair group 4 proteins. 4. These quantitative differences are discussed in terms of the mechanism of synthesis of the keratin proteins. 5. Follicle group 1B proteins are postulated to have arisen from the trichohyalin droplets of the developing medulla and inner-root-sheath layers of the follicle and may be precursors of the proteins of the mature medulla and inner root sheath.     

The human erythrocyte proteome: analysis by ion trap mass spectrometry

This report describes an analysis of the red blood cell proteome by ion trap tandem mass spectrometry in line with liquid chromatography. Mature red blood cells lack all internal cell structures and consist of cytoplasm within a plasma membrane envelope. To maximize outcome, total red blood cell protein was divided into two fractions of membrane-associated proteins and cytoplasmic proteins. Both fractions were divided into subfractions, and proteins were identified in each fraction separately through tryptic digestion. Membrane protein digests were collected from externally exposed proteins, internally exposed proteins, "spectrin extract" mainly consisting of membrane skeleton proteins, and membrane proteins minus spectrin extract. Cytoplasmic proteins were divided into 21 fractions based on molecular mass by size exclusion chromatography. The tryptic peptides were separated by reverse-phase high-performance liquid chromatography and identified by ion trap tandem mass spectrometry. A total of 181 unique protein sequences were identified: 91 in the membrane fractions and 91 in the cytoplasmic fractions. Glyceraldehyde-3-phosphate dehydrogenase was identified with high sequence coverage in both membrane and cytoplasmic fractions. Identified proteins include membrane skeletal proteins, metabolic enzymes, transporters and channel proteins, adhesion proteins, hemoglobins, cellular defense proteins, proteins of the ubiquitin-proteasome system, G-proteins of the Ras family, kinases, chaperone proteins, proteases, translation initiation factors, and others. In addition to the known proteins, there were 43 proteins whose identification was not determined.     

An Electrophoretic Analysis of the Seed Protein Body Proteins from Pinus Nigra

Protein bodies (PBs) of European black pine (Pinus nigra Arn.) were isolated from mature seeds. Extracted soluble matrix proteins and crystalloid proteins PBs proteins were investigated by SDS-PAGE electrophoresis in presence and absence of 2-mercaptoethanol. The proteins of molecular masses 16, 17, 18, 61 and 65 kDa were presented only in crystalloid protein samples. Only 15 kDa protein was present in soluble matrix proteins and not in crystalloid proteins. Another protein bands were present in both soluble matrix and crystalloid proteins. 20, 37, 38, 39 and 48 kDa proteins were strongly visible among crystalloid proteins. Bands of 23 and 32 kDa were more visible in soluble matrix protein samples. Different composition in crystalloid proteins was found in absence of 2-mercaptoethanol: no proteins with molecular mass 71 kDa and more proteins in soluble matrix. In case of crystalloid proteins we detected 7 protein bands in interval from 71 to 212 kDa.     

Isolation and characterization of the major proteins of ram seminal plasma

Mammalian seminal plasma contains among others, two major families of proteins, namely spermadhesins and those proteins that contain fibronectin type II domains. Spermadhesins are the major proteins of boar and stallion seminal plasma and homologous proteins have been identified in the bull. These proteins appear to be involved in capacitation and sperm-egg interaction. In bovine seminal plasma, proteins containing fibronectin type II domains are the major proteins and are designated BSP proteins. These proteins play a role in sperm capacitation. In this study, we present the isolation and characterization of the major proteins of ram seminal plasma. Precipitated proteins from Suffolk ram seminal plasma were loaded onto a gelatin-Agarose column. The unadsorbed (fraction A) and retarded proteins (fraction B) were removed by washing the column with phosphate buffered-saline and the adsorbed proteins (fraction C) were eluted with 5 M urea. SDS-PAGE of fraction B indicated the presence of a 15.5 kDa protein, which is the major protein of ram seminal plasma (approximately 45% of total protein by weight) and was identified as a spermadhesin by N-terminal sequencing. SDS-PAGE analysis of fraction C revealed the presence of four proteins, which represented approximately 20% of total ram seminal plasma proteins by weight, and were identified as proteins of the BSP family and named RSP proteins. These RSP proteins were designated RSP-15 kDa, RSP-16 kDa, RSP-22 kDa, and RSP-24 kDa. Only RSP-15 kDa and -16 kDa proteins cross-reacted with antibodies against BSP proteins. Ram spermadhesin and RSP proteins interact with heparin but only RSP proteins bind to hen's egg yolk low-density lipoprotein. In conclusion, spermadhesin is the major protein of ram seminal plasma and other major proteins belong to the BSP protein family.     

Characterization of the major hnRNP proteins from Drosophila melanogaster

To better understand the role(s) of hnRNP proteins in the process of mRNA formation, we have identified and characterized the major nuclear proteins that interact with hnRNAs in Drosophila melanogaster. cDNA clones of several D. melanogaster hnRNP proteins have been isolated and sequenced, and the genes encoding these proteins have been mapped cytologically on polytene chromosomes. These include the hnRNP proteins hrp36, hrp40, and hrp48, which together account for the major proteins of hnRNP complexes in D. melanogaster (Matunis et al., 1992, accompanying paper). All of the proteins described here contain two amino-terminal RNP consensus sequence RNA-binding domains and a carboxyl-terminal glycine-rich domain. We refer to this configuration, which is also found in the hnRNP A/B proteins of vertebrates, as 2 x RBD-Gly. The sequences of the D. melanogaster hnRNP proteins help define both highly conserved and variable amino acids within each RBD and support the suggestion that each RBD in multiple RBD-containing proteins has been conserved independently and has a different function. Although 2 x RBD-Gly proteins from evolutionarily distant organisms are conserved in their general structure, we find a surprising diversity among the members of this family of proteins. A mAb to the hrp40 proteins crossreacts with the human A/B and G hnRNP proteins and detects immunologically related proteins in divergent organisms from yeast to man. These data establish 2 x RBD-Gly as a prevalent hnRNP protein structure across eukaryotes. This information about the composition of hnRNP complexes and about the structure of hnRNA-binding proteins will facilitate studies of the functions of these proteins.     

The mosaic of proteins synthesized by Salmonella typhimurium

A profusion of proteins with heterologous serological specificities was synthesized by S. typhimurium grown on artificial media; accordingly, sera prepared in rabbits with these proteins displayed an abundance of antibodies reacting, in agar gel, against numerous heterologous proteins. the absorption of the sera with different Enterobacterial proteins proved that the S. typhimurium proteins are a mixture of specific proteins, and common E. coli and Salmonellae determinants; in addition, a group of strongly cross-precipitating proteins common to S. typhimurium and S. choleraesuis and to S. typhimurium and S. kentucky were identified that were not present in the proteins common to S. enteritidis, S. typhi and E. coli, or in the S. paratyphi A proteins used absorptions. The specific proteins of S. typhimurium were synthesized on artificial media in, apparently, smaller amounts than the common proteins; their role in the protection of mice against infection with their natural pathogen was, however, proof of their specificity and contrasted with the ineffectiveness, in protecting the mice, of the common proteins.     

LC-MS/MS analysis of apical and basolateral plasma membranes of rat renal collecting duct cells

We used biotinylation and streptavidin affinity chromatography to label and enrich proteins from apical and basolateral membranes of rat kidney inner medullary collecting ducts (IMCDs) prior to LC-MS/MS protein identification. To enrich apical membrane proteins and bound peripheral membrane proteins, IMCDs were perfusion-labeled with primary amine-reactive biotinylation reagents at 2 degrees C using a double barreled pipette. The perfusion-biotinylated proteins and proteins bound to them were isolated with CaptAvidin-agarose beads, separated with SDS-PAGE, and sliced into continuous gel pieces for LC-MS/MS protein identification (LTQ, Thermo Electron Corp.). 17 integral and glycosylphosphatidylinositol (GPI)-linked membrane proteins and 44 non-integral membrane proteins were identified. Immunofluorescence confocal microscopy confirmed ACVRL1, H(+)/K(+)-ATPase alpha1, NHE2, and TauT expression in the IMCDs. Basement membrane and basolateral membrane proteins were biotinylated via incubation of IMCD suspensions with biotinylation reagents on ice. 23 integral and GPI-linked membrane proteins and 134 non-integral membrane proteins were identified. Analyses of non-integral membrane proteins preferentially identified in the perfusion-biotinylated and not in the incubation-biotinylated IMCDs revealed protein kinases, scaffold proteins, SNARE proteins, motor proteins, small GTP-binding proteins, and related proteins that may be involved in vasopressin-stimulated AQP2, UT-A1, and ENaC regulation. A World Wide Web-accessible database was constructed of 222 membrane proteins (integral and GPI-linked) from this study and prior studies.     

2-DE技术中疏水性和碱性蛋白质的研究进展

双向凝胶电泳(2-DE)具有高分辨率、高通量等特点,已被广泛地用于蛋白质组的分离．但是它在分离疏水性蛋白质和碱性蛋白质时却遇到了极大的挑战．然而,疏水性与碱性蛋白质在全蛋白质中占相当大的比例,且具有很重要的生物学意义．因而,近年来,越来越多的研究者将目标瞄准这些蛋白质,并且取得了一些令人鼓舞的进展：用亚细胞预分离技术,顺序提取法等方法来富集疏水性蛋白质,用一些新的有效的增溶剂如硫脲,ASB一14等来改善疏水性蛋白质的溶解,应用这些技术2一DE可分辨出总平均疏水值达O．80的蛋白质;在碱性蛋白质分离方面,通过等电聚焦预处理,使用窄pH梯度胶条等大大地改善了碱性蛋白质在2-DE中的分离,能分辨出等电点达11．7的蛋白质．现对2-DE技术中疏水性和碱性蛋白质分离的研究进展进行综述．     

Global landscape of structural proteins of infectious spleen and kidney necrosis virus

Infectious spleen and kidney necrosis virus (ISKNV), the type species of the genus Megalocytivirus in the family Iridoviridae, causes severe damage to mandarin fish cultures in China. Little is known about the proteins of ISKNV virions. In this study, a total of 38 ISKNV virion-associated proteins were identified by four different workflows with systematic and comprehensive proteomic approaches. Among the 38 identified proteins, 21 proteins were identified by the gel-based workflows (one-dimensional [1-D] and two-dimensional [2-D] gel electrophoresis). Fifteen proteins were identified by 1-D gel electrophoresis, and 16 proteins were identified by 2-D gel electrophoresis, with 10 proteins identified by both methods. Another 17 proteins were identified only by liquid chromatography (LC)-based workflows (LC-matrix-assisted laser desorption ionization [MALDI] and linear trap quadrupole [LTQ]-Orbitrap). Among these 17 LC-identified proteins, 5 proteins were identified uniquely by the LC-MALDI workflow, whereas another 6 proteins were identified only by the LTQ-Orbitrap workflow. These results underscore the importance of incorporation of multiple approaches in identification of viral proteins. Based on viral genomic sequence, genes encoding these 38 viral proteins were cloned and expressed in vitro. Antibodies were produced against these 38 proteins to confirm the ISKNV structural proteins by Western blotting. Of the newly identified proteins, ORF 056L and ORF 118L were identified and confirmed as two novel viral envelope proteins by Western blotting and immunoelectron microscopy (IEM). The ISKNV proteome reported here is currently the only characterized megalocytivirus proteome. The systematic and comprehensive identification of ISKNV structural proteins and their localizations in this study will facilitate future studies of the ISKNV assembly process and infection mechanism.     

白桦叶绿体RNA结合蛋白的蛋白质组学分析

在高等植物叶绿体中,RNA结合蛋白在转录后RNA处理、运输以及mRNA的稳定等方面发挥重要作用.本项研究使用多聚腺苷酸(polyA）吸附柱或单链DNA(ssDNA)吸附柱富集白桦叶绿体的polyA结合蛋白或RNA结合蛋白,并通过MALDI-TOF-MS以及ESI MS/MS进行鉴定,13个叶绿体蛋白质得到了鉴定.按照Swiss Prot数据库的注释,这些蛋白质的功能主要包括4个相关种类,分别为NAD结合蛋白、RNA结合蛋白、DNA结合蛋白和ATP结合蛋白.使用这些方法还鉴定出包括转录因子的4个高丰度蛋白.这些结果加深了对树木中叶绿体RNA结合蛋白的全面了解,可以将其应用于其他树木叶绿体中RNA 蛋白质的相互作用的研究.     

StaRProtein,A Web Server for Prediction of the Stability of Repeat Proteins

Repeat proteins have become increasingly important due to their capability to bind to almost any proteins and the potential as alternative therapy to monoclonal antibodies. In the past decade repeat proteins have been designed to mediate specific protein-protein interactions. The tetratricopeptide and ankyrin repeat proteins are two classes of helical repeat proteins that form different binding pockets to accommodate various partners. It is important to understand the factors that define folding and stability of repeat proteins in order to prioritize the most stable designed repeat proteins to further explore their potential binding affinities. Here we developed distance-dependant statistical potentials using two classes of alpha-helical repeat proteins, tetratricopeptide and ankyrin repeat proteins respectively, and evaluated their efficiency in predicting the stability of repeat proteins. We demonstrated that the repeat-specific statistical potentials based on these two classes of repeat proteins showed paramount accuracy compared with non-specific statistical potentials in: 1) discriminate correct vs. incorrect models 2) rank the stability of designed repeat proteins. In particular, the statistical scores correlate closely with the equilibrium unfolding free energies of repeat proteins and therefore would serve as a novel tool in quickly prioritizing the designed repeat proteins with high stability. StaRProtein web server was developed for predicting the stability of repeat proteins.     

Changes in chorion proteins induced by the exudate released from the egg cortex at the time of fertilization in the teleost, Oryzias latipes

The chorion of unfertilized medaka Oryzias latipes eggs consists of two major proteins (77–73 and 49 kDa) and a minor 150 kDa protein. Upon fertilization, these major chorion proteins are polymerized to insoluble high molecular weight proteins via the temporary formation of several new proteins (132, 114, 62 and 61 kDa). Increasing chorion toughness is closely related to the formation of high molecular weight proteins and the increasing insolubility of the chorion proteins. The changes in chorion proteins and hardening could be induced in vitro in isolated chorions by an egg exudate, which includes cortical alveolar contents. The effects of temperature and pH on the egg exudate-induced changes in chorion proteins were examined in the present study. The major proteins could be digested by proteolytic enzymes. The 49 kDa protein was PAS-positive. Analysis with polyclonal antibodies against the major proteins demonstrated that the temporarily formed 62 and 61 kDa proteins were derived from the 77–73 kDa protein and that higher molecular weight proteins, newly formed in the process of chorion hardening, contained the same epitopes as did the 77–73 and 49 kDa proteins. The results suggest that the changes in chorion proteins of the medaka egg at the time of fertilization can be induced by an enzyme(s) released from the egg cortex into the perivitelline space.     

Design and discovery of metamorphic proteins

Metamorphic proteins are single amino acid sequences that reversibly interconvert between multiple, dramatically different native structures, often with distinct functions. Since the discovery of the first metamorphic proteins in the early 2000s, several additional metamorphic proteins have been identified, and it was suggested that up to 4% of proteins in the PDB may switch folds. Metamorphic proteins have been found to share common features such as marginal thermostability and inconsistencies in predicted secondary structures. Outstanding challenges in the field include the search for more metamorphic proteins and the design of new proteins that switch folds. Identification of novel metamorphic proteins in nature will improve therapeutic targeting of fold-switching proteins involved in human pathology and will enhance the design of protein-based therapies. Designed fold switching proteins have applications as biosensors, molecular switches, molecular machines, and self-assembling systems.     

Comparative analysis of SET domain proteins in maize and Arabidopsis reveals multiple duplications preceding the divergence of monocots and dicots

Histone proteins play a central role in chromatin packaging, and modification of histones is associated with chromatin accessibility. SET domain [Su(var)3-9, Enhancer-of-zeste, Trithorax] proteins are one class of proteins that have been implicated in regulating gene expression through histone methylation. The relationships of 22 SET domain proteins from maize (Zea mays) and 32 SET domain proteins from Arabidopsis were evaluated by phylogenetic analysis and domain organization. Our analysis reveals five classes of SET domain proteins in plants that can be further divided into 19 orthology groups. In some cases, such as the Enhancer of zeste-like and trithorax-like proteins, plants and animals contain homologous proteins with a similar organization of domains outside of the SET domain. However, a majority of plant SET domain proteins do not have an animal homolog with similar domain organization, suggesting that plants have unique mechanisms to establish and maintain chromatin states. Although the domains present in plant and animal SET domain proteins often differ, the domains found in the plant proteins have been generally implicated in protein-protein interactions, indicating that most SET domain proteins operate in complexes. Combined analysis of the maize and Arabidopsis SET domain proteins reveals that duplication of SET domain proteins in plants is extensive and has occurred via multiple mechanisms that preceded the divergence of monocots and dicots.     

DING proteins; novel members of a prokaryotic phosphate-binding protein superfamily which extends into the eukaryotic kingdom

PstS proteins are the cell-bound phosphate-binding elements of the ubiquitous bacterial ABC phosphate uptake mechanisms. Primary and tertiary structures, characteristic of pstS proteins, are conserved in proteins, which are expressed in secretory operons and induced by phosphate deprivation, in Pseudomonas species. There are two subsets of these proteins; AP proteins, which are alkaline phosphatases, and DING proteins, named for their N-terminal sequence, which are phosphate-binding proteins. Both form elements of a proposed phosphate-scavenging system in pseudomonads. DING proteins have also been isolated from many eukaryotic sources, and are associated with both normal and pathological functions in mammals. Their phosphate-binding function suggests a role in biomineralization, but the ability to bind other ligands may be related to signal transduction in eukaryotes. Though it has been claimed that all such proteins may originate from pseudomonads, many eukaryotic DING proteins have unique features which are incompatible with a bacterial origin.     

Changes related to the oestrous cycle in the expression of endometrial and oviductal proteins of mice.

Soluble proteins extracted from the endometria and oviducts of normal sexually mature cycling Swiss Webster mice were analysed by two-dimensional high-resolution sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Thirty endometrial and 25 oviductal proteins showed differential expression related to the oestrous cycle. In the endometrium, 19 proteins were maximally expressed in the oestrous phase, and significantly decreased or could not be detected in dioestrus. Eleven additional proteins were more prominent in dioestrus. Most of these endometrial cyclic proteins were acidic. In the oviduct, almost two-thirds of cycle-related, differentially expressed proteins were more strongly expressed in dioestrus and were significantly less prominent or could not be detected in the oestrous phase. In contrast to the endometrial proteins, most of the oviductal cyclic proteins were basic. Fourteen proteins appeared to be identical in both organs, and five of these showed the same cyclic pattern of expression. The remaining cyclic proteins were organ specific and showed uterus- or oviduct-specific changes during the oestrous cycle. Among the cyclic proteins, four endometrial and two oviductal proteins were restricted to oestrus, whereas two endometrial and seven oviductal proteins were restricted to dioestrus. These proteins could serve as markers for specific phases of the oestrous cycle. Our data show that the mouse oestrous cycle is associated with consistent and predictable changes in protein expression in both the endometrium and oviduct.