正常精子和圆头精子差异表达蛋白的分离与鉴定

圆头精子症是一种表现为顶体缺失的男性不育症.为了研究正常精子和圆头精子的蛋白质组成差异,用固相pH梯度双向凝胶电泳和质谱分析等蛋白质组学方法,分离了30份正常和3份圆头精子标本的蛋白质.对其中16个在正常精子中有高丰度表达而在圆头精子中缺失,和1个在圆头精子中表达明显下调的蛋白质点,以及在圆头精子中存在而在正常精子中缺失的蛋白质簇W和点X,进行了肽质指纹分析和蛋白质鉴定,获得8个点的肽质量指纹图,经MS-Fit软件搜索SWISS-PROT数据库来鉴定其身分.发现其中3个点与高尔基体相关、2个点与蛋白酶体相关、2个点为锌指蛋白,对其功能和与圆头精子形成的可能关系进行了初步探讨.     

Tackling the plant proteome: practical approaches, hurdles and experimental tools

The study of complex biological questions through comparative proteomics is becoming increasingly attractive to plant biologists as the rapidly expanding plant genomic and expressed sequence tag databases provide improved opportunities for protein identification. This review focuses on practical issues associated with comparative proteomic analysis, including the challenges of effective protein extraction and separation from plant tissues, the pros and cons of two-dimensional gel-based analysis and the problems of identifying proteins from species that are not recognized models for functional genomic studies. Specific points are illustrated using data from an ongoing study of the tomato and pepper fruit proteomes.     

Novel Techniques for Identification and Characterization of Proteins Loaded on Gels in Femtomole Amounts

A combination of techniques is presented allowing gel-purified protein identification in the femtomole range using matrix-assisted-laser-desorption-ionization mass spectrometry. The proteins are detected in the primary gel by a sensitive negative staining procedure, transferred, and concentrated in a secondary gel matrix. There, they are digested in the presence of H₂ ¹⁸O and their sequences are predicted (1) by peptide mass fingerprinting, (2) by comparing the post-source-decay (PSD) spectra with theoretical spectra of candidate isobaric peptides using a computer algorithm called MassFrag, and (3) by a manual readout of the ¹⁸O/¹⁶O-labeled fragmentation ions in the PSD spectra.     

应用蛋白质组学技术筛选胃癌SGC-7901细胞中的let-7a功能相关蛋白

为探讨let-7a表达下调在胃癌发病中的机制,高通量地检测了与let-7a功能相关的蛋白质.首先采用基因克隆技术稳定过表达SGC-7901细胞系的let-7a基因,然后用蛋白质组学技术研究稳定过表达该基因对SGC-7901细胞蛋白质表达谱的影响.通过对SGC-7901/let-7a细胞的蛋白质表达谱改变的研究,并用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)分析鉴定了10个差异表达蛋白质.这些差异表达蛋白质可能是let-7a功能相关蛋白质,其中抗氧化蛋白2、胰岛素样生长因子结合蛋白2、二硫化蛋白异构酶A2、四氢叶酸合成酶、细胞周期素依赖性激酶抑制蛋白1、Rho-GTPsae激活蛋白4表达上调,Skp2蛋白、血小板黏附蛋白CD41、纤维连接蛋白、Cks1蛋白表达下调.部分差异表达蛋白质如细胞周期素依赖性激酶抑制蛋白1、Skp2蛋白和纤维连接蛋白经蛋白质印迹分析进行了验证.在SGC-7901/let-7a中鉴定的10个差异表达蛋白质涉及到细胞周期的调控、分子基因表达调控、细胞黏附、细胞代谢等众多事件,它们可能作为let-7a功能相关蛋白质,为阐明let-7a表达下调在胃癌发病中的机制提供了重要线索.     

NAG7基因转染HNE1细胞后下调蛋白质的鉴定及其意义(英文)

NAG7基因是我室克隆的与鼻咽癌相关的肿瘤抑制候选基因 .将NAG7编码框的cDNA片段克隆至pGEM3.1(+ )的表达载体 ,经脂质体转染入HNE1细胞 ,经G4 18筛选 ,并运用PCR技术证实 .建立含NAG7基因稳定转染的细胞系 ,抽提细胞总蛋白质 ,双向凝胶电泳分离蛋白质 ,对表达下调的蛋白质点进行质谱分析 ,获得的肽质指纹经SWISS PROT数据库分析以鉴定蛋白质点 .鉴定出的 7个下调蛋白质包括纤溶酶原、收缩蛋白、Ras 相关蛋白Rab 36及ARF 相关蛋白等 .通过对蛋白质性质和功能的分析 ,发现这些蛋白质参与了细胞信号的转导、蛋白质的转运及细胞代谢等众多事件 .因此 ,NAG7基因很可能是通过介导这些蛋白质的表达下调而发挥其功能     

Identification of proteins with altered expression in colorectal cancer by means of 2D-proteomics

Modern proteomic techniques make it possible to identify numerous changes in protein expression in tumors as compared to normal tissues. Although proteomics is currently widely used, identification of proteins differentially expressed in particular types of cancer remains a challenging task. The goal of our study was to detect novel protein markers of colorectal cancer using comparative proteomics of protein extracts obtained from primary tumors and adjacent normal tissues. Coloreetal cancer is nearly asymptomatic at the early stages, which calls for development of fast and sensitive methods for molecular diagnostics. Proteomes of 11 paired specimens of primary colorectal tumors and adjacent histologically normal tissues were studied using comparative 2D PAGE. Altogether, 16 proteins with altered expression levels were detected, including 13 proteins with increased levels and three proteins with decreased levels in tumor tissues. These proteins were identified using MALDI-TOF mass spectrometry. The proteins GPD1, RRBP1 (increased levels), HNRNPH1, and SERPINB6 (decreased levels) have been associated with colorectal cancer for the first time.     

The structure and genetic control of a new class of disulphide-linked proteins in wheat endosperm

Summary Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of unreduced total protein extracts from the endosperm of hexaploid wheat revealed three high molecular weight protein bands (triplet bands) in a zone of heavy background streaking. Electrophoretic examination of 135 hexaploid cultivars showed at least five different patterns of these triplet bands. Nine durum wheat cultivars showed a single band only. Analysis of nullisomic-tetrasomic and ditelocentric lines of Chinese Spring wheat revealed that the slowest moving band (Tri-1) of the triplet was controlled by gene(s) on chromosome arm 1DS and the fastest moving band (Tri-3) by 1AS. The band with intermediate mobility (Tri-2) was found to be a hybrid aggregate of the subunits controlled by 1DS and 1AS. Using a non-reducing/reducing form of 2-dimensional (2-D) electrophoresis, these triplet bands were shown to be heterotetramers of four subunits designated D (M.W. 58,000), (22,000), A (52,000) and (23,000) where Tri-1=DD, Tri-2 = DA and Tri-3 = AA. With very low concentrations of 2-mercaptoethanol (ME), the tetramers dissociated into dimeric subunit pairs (D, A), the monomers being observed with higher concentrations of ME. The structure of these subunit pairs resembles that of the subunit pairs in the globulin storage proteins of oats and some legumes. The 2-D method employed in this study was useful also for separating low molecular weight (LMW) subunits of glutenin from the monomeric gliadins which have similar electrophoretic mobility in 1-D separation. It was shown that at least four of these LMW glutenin subunits are controlled by genes on 1DS and 1AS and at least one subunit is controlled by gene(s) on 1BS. This electrophoretic separation method has proven useful in understanding the aggregation behaviour of the seed proteins of wheat.     

Proteomics Identification and Validation of Desmocollin‐1 and Catechol‐O‐Methyltransferase as Proteins Associated with Breast Cancer Cell Migration and Metastasis

Biological treatment of many cancers currently targets membrane bound receptors located on a cell surface. To identify novel membrane proteins associated with migration and metastasis of breast cancer cells, a more migrating subpopulation of MDA‐MB‐231 breast cancer cell line is selected and characterized. A high‐resolution quantitative mass spectrometry with SILAC labeling is applied to analyze their surfaceome and it is compared with that of parental MDA‐MB‐231 cells. Among 824 identified proteins (FDR < 0.01), 128 differentially abundant cell surface proteins with at least one transmembrane domain are found. Of these, i) desmocollin‐1 (DSC1) is validated as a protein connected with lymph node status of luminal A breast cancer, tumor grade, and Her‐2 status by immunohistochemistry in the set of 96 primary breast tumors, and ii) catechol‐O‐methyltransferase is successfully verified as a protein associated with lymph node metastasis of triple negative breast cancer as well as with tumor grade by targeted data extraction from the SWATH‐MS data of the same set of tissues. The findings indicate importance of both proteins for breast cancer development and metastasis and highlight the potential of biomarker validation strategy via targeted data extraction from SWATH‐MS datasets.     

Identification of neutrophil gelatinase-associated lipocalin (NGAL) as a discriminatory marker of the hepatocyte-secreted protein response to IL-1beta: a proteomic analysis

The liver is the major source of proteins used throughout the body for various functions. Upon injury or infection, an acute phase response (APR) is initiated in the liver that is primarily mediated by inflammatory cytokines such as interleukin-1beta (IL-1beta) and interleukin-6. Among others, the APR is characterized by an altered protein synthetic profile. We used two-dimensional gel electrophoresis to study the dynamics of changes in protein synthesis in hepatocytes exposed to these inflammatory cytokines. Protein profiles were quantified using image analysis and further analyzed using multivariate statistical methods. Our results indicate that IL-1beta and IL-6 each induces secreted protein responses with distinct dynamics and dose-dependence. Parallel stimulation by IL-1beta and IL-6 results in a protein pattern indistinguishable from the IL-1beta pattern, indicating a dominant effect of IL-1beta over IL-6 at the doses tested. Multidimensional scaling (MDS) of correlation distances between protein secretion levels revealed two protein pairs that are robustly co-secreted across the various cytokine stimulation conditions, suggesting shared regulatory pathways. Finally, we also used multivariate alternating conditional expectation (MACE) to identify transformation functions that discriminated the cytokine-stimulated and untreated hepatocyte-secreted protein profiles. Our analysis indicates that the expression of neutrophil gelatinase-associated lipocalin (NGAL) was sufficient to discriminate between IL-1beta and IL-6 stimulation. The combination of proteomics and multivariate analysis is expected to provide new information on the cellular regulatory networks involved in generating specific cellular responses.     

Identification of proteins associated with malting quality in a subset of wild barley introgression lines

Malted barley is an important ingredient used in the brewing and distilling industry worldwide. In this study, we used a proteomics approach to investigate the biochemical function of previously identified quantitative trait loci (QTLs) on barley chromosomes 1H and 4H that influence malting quality. Using a subset of barley introgression lines containing wild barley (Hordeum vulgare ssp. spontaneum) alleles at these QTLs, we validated that wild barley alleles at the chromosome 1H QTL reduced overall malting quality, whereas wild barley alleles at the chromosome 4H QTL improved the malting quality parameters α-amylase activity, VZ45, and Kolbach index compared to the control genotype Scarlett. 2DE was used to detect changes in protein expression during the first 72 h of micromalting associated with these QTLs. In total, 16 protein spots showed a significant change in expression between the introgression lines and Scarlett, of which 14 were successfully identified with MS. Notably, the wild barley alleles in the line containing the chromosome 4H QTL showed a sixfold increased expression of a limit dextrinase inhibitor. The possible role of the identified proteins in malting quality is discussed. The knowledge gained will assist ongoing research toward cloning the genes underlying these important QTL.     

Direct Targeting of Proteins from the Cytosol to Organelles: The ER versus Endosymbiotic Organelles

In eukaryotic cells consisting of many different types of organelles, targeting of organellar proteins is one of the most fundamental cellular processes. Proteins belonging to the endoplasmic reticulum (ER), chloroplasts and mitochondria are targeted individually from the cytosol to their cognate organelles. As the targeting to these organelles occurs in the cytosol during or after translation, the most crucial aspect is how specific targeting to these three organelles can be achieved without interfering with other targeting pathways. For these organelles, multiple mechanisms are used for targeting proteins, but the exact mechanism used depends on the type of protein and organelle, the location of targeting signals in the protein and the location of the protein in the organelle. In this review, we discuss the various mechanisms involved in protein targeting to the ER, chloroplasts and mitochondria, and how the targeting specificity is determined for these organelles in plant cells .     

Inside human aortic stenosis: a proteomic analysis of plasma

Valvular aortic stenosis (AS) produces a slowly progressive obstruction in left ventricular outflow track. For this reason, aortic valve replacement is warranted when the valvular stenosis is hemodinamically significant, becoming the most common worldwide cause of aortic valve surgery. Recent epidemiologic studies have revealed an association between degenerative AS and cardiovascular risk factors for atherosclerosis, althought reducing the exposure to such factors and statin therapies both fail to delay or reverse the pathology. Hence, a deeper understanding of the pathophysiology of this disease is required to identify appropriate preventive measures. A proteomic analysis of plasma will permit to know and identify the changes in protein expression induced by AS in this tissue. Using two-dimensional difference gel electrophoresis (2D-DIGE) followed by mass spectrometry (MS), we compared the crude (not pre-fractioned) and pre-fractioned plasma from AS patients and control subjects. We sought to identify plasma proteins whose expression is modified in AS. In addition we investigated if crude plasma presented some alterations in the more abundant proteins since to date, has never been studied before. We also further investigated the link between this disease and atherosclerosis with a view to identifying new potential markers and therapeutic targets.     

Tangible benefits of the aphid Acyrthosiphon pisum genome sequencing for aphid proteomics: Enhancements in protein identification and data validation for homology-based proteomics

Homology-driven proteomics promises to reveal functional biology in insects with sparse genome sequence information. A proteomics study comparing plant virus transmission competent and refractive genotypes of the aphid Schizaphis graminum isolated numerous candidate proteins involved in virus transmission, but limited genome sequence information hampered their identification. The complete genome of the pea aphid, Acyrthosiphon pisum, released in 2008, enabled us to double the number of protein identifications beyond what was possible using available EST libraries and other insect sequences. This was concomitant with a dramatic increase of the number of MS and MS/MS peptide spectra matching the genome-derived protein sequence. LC-MS/MS proved to be the most robust method of peptide detection. Cross-matching spectral data to multiple EST sequences and error tolerant searching to identify amino acid substitutions enhanced the percent coverage of the Schizaphis graminum proteins. 2-D electrophoresis provided the protein pI and MW which enabled the refinement of the candidate protein selection and provided a measure of protein abundance when coupled to the spectral data. Thus, the homology-based proteomics pipeline for insects should include efforts to maximize the number of peptide matches to the protein to increase certainty in protein identification and relative protein abundance.     

Allergome: the characterization of allergens based on a 2D gel electrophoresis approach

Type I hypersensitivity reactions are in constant progression in industrialized countries. The physiopathologic mechanism of these diseases implicates the production of specific immunoglobulin (Ig)E to allergenic molecules, their binding to the Fcε receptor on the surface of mast cells and basophils, and the release of inflammatory mediators when allergens are introduced into the body and crosslink with the IgE bound to the cell surface. An allergen is defined as a molecule that induces the production of, and binds to, IgE. The identification of the allergenic molecules is an important goal to improve diagnosis and treatment of allergy. This characterization aims to extract proteins from the allergenic source, to analyze IgE specificity by immunoblotting and to identify the proteins that bind IgE.     

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.     

Structural Analysis of Metal Sites in Proteins: Non-heme Iron Sites as a Case Study

In metalloproteins, the protein environment modulates metal properties to achieve the required goal, which can be protein stabilization or function. The analysis of metal sites at the atomic level of detail provided by protein structures can thus be of benefit in functional and evolutionary studies of proteins. In this work, we propose a structural bioinformatics approach to the study of metalloproteins based on structural templates of metal sites that include the PDB coordinates of protein residues forming the first and the second coordination sphere of the metal. We have applied this approach to non-heme iron sites, which have been analyzed at various levels. Templates of sites located in different protein domains have been compared, showing that similar sites can be found in unrelated proteins as the result of convergent evolution. Templates of sites located in proteins of a large superfamily have been compared, showing possible mechanisms of divergent evolution of proteins to achieve different functions. Furthermore, template comparisons have been used to predict the function of uncharacterized proteins, showing that similarity searches focused on metal sites can be advantageously combined with typical whole-domain comparisons. Structural templates of metal sites, finally, may constitute the basis for a systematic classification of metalloproteins in databases.     

The human salivary proteome: a critical overview of the results obtained by different proteomic platforms

The development of new separation techniques and different mass spectrometry instrumental devices, as well as the great availability of specific reactants, offers ample choice to scientists for carrying out high-throughput proteomic studies and being competitive in the field today. However, the different options available often do not provide comparable results, which can be linked to factors such as the strategy adopted, the nature of the sample and the instrumental availability. In this critical review, the results obtained so far in the study of human saliva by different proteomic approaches will be compared and discussed.     

Influence of Diet on the Proteome of Drosophila Melanogaster as Assessed by Two-Dimensional Gel Electrophoresis and Capillary Liquid Chromatography–Mass Spectrometry: The Hamburger Effect Revisited

Proteomic biomarker discovery has been called into question. Diamandis hypothesized that seemingly trivial factors, such as eating a hamburger, may cause sufficient proteomic change as to confound proteomic differences. This has been termed the hamburger effect. Little is known about the variability of complex proteomes in response to the environment. Two methods—two-dimensional gel electrophoresis (2DGE) and capillary liquid chromatography–electrospray ionization time-of-flight mass spectrometry (LCMS)—were used to study the hamburger effect in two cross-sections of the soluble fruit fly proteome. 2DGE measured abundant proteins, whereas LCMS measured small proteins and peptides. Proteomic differences between males and females were first evaluated to assess the discriminatory capability of the methods. Likewise, wild-type and white-eyed flies were analyzed as a further demonstration that genetically based proteomic differences could be observed above the background analytical variation. Then dietary interventions were imposed. Ethanol was added to the diet of some populations without significant proteomic effect. However, after a 24-h fast, proteomic differences were found using LCMS but not 2DGE. Even so, only three of ~1000 molecular species were altered significantly, suggesting that the influence of even an extreme diet change produced only modest proteomic variability, and that much of the fruit fly proteome remains relatively constant in response to diet. These experiments suggest that proteomics can be a viable approach to biomarker discovery.     

The effect of DTT in protein preparations for proteomic analysis: Removal of a highly abundant plant enzyme, ribulose bisphosphate carboxylase/oxygenase

Rubisco is a major photosynthetic plant enzyme in the chloroplasts, catalyzing a photosynthetic reaction through carboxylation and oxygenation in the leaves. Despite its biological importance, its high abundance causes difficulties in the proper separation of protein mixtures during 2-dimensional gel electrophoresis (2-DE). Here, we resolved those plant soluble proteins by efficiently removing Rubisco. This resulted in a high quality and resolution of 2-DE gels. Rubisco removal was achieved through aggregation in the presence of a high DTT concentration, which subsequently increased the visualization of less abundant proteins and reduced horizontal streaking. This simple method may provide a means for finding more biologically important protein targets via plant proteomics.     

Identification of major Ca(2+)/calmodulin-dependent protein kinase phosphatase-binding proteins in brain: biochemical analysis of the interaction

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) is a unique protein phosphatase that specifically dephosphorylates and regulates multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs). To clarify the physiological significance of CaMKP, we identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fructose bisphosphate aldolase as major binding partners of CaMKP in a soluble fraction of rat brain using the two-dimensional far-Western blotting technique, in conjunction with peptide mass fingerprinting analysis. We analyzed the affinities of these interactions. Wild type CaMKP-glutathione S-transferase (GST) associated with GAPDH in a GST pull-down assay. Deletion analysis suggested that the N-terminal side of the catalytic domain of CaMKP was responsible for the binding to GAPDH. Further, anti-CaMKP antibody coimmunoprecipitated GAPDH in a rat brain extract. GAPDH was phosphorylated by CaMKI or CaMKIV in vitro; however, when CaMKP coexisted, the phosphorylation was markedly attenuated. Under these conditions, CaMKP significantly dephosphorylated CaMKI and CaMKIV, which had been phosphorylated by CaMK kinase, whereas it did not dephosphorylate the previously phosphorylated GAPDH. The results suggest that CaMKP regulates the phosphorylation level of GAPDH in the CaMKP-GAPDH complex by dephosphorylating and deactivating CaMKs that are responsible for the phosphorylation of GAPDH.