Showing papers in "Proteomics in 2010"

Matrigel: A complex protein mixture required for optimal growth of cell culture

Abstract: Numerous cell types require a surface for attachment to grow and proliferate. Certain cells, particularly primary and stem cells, necessitate the use of specialized growth matrices along with specific culture media conditions to maintain the cells in an undifferentiated state. A gelatinous protein mixture derived from mouse tumor cells and commercialized as Matrigel is commonly used as a basement membrane matrix for stem cells because it retains the stem cells in an undifferentiated state. However, Matrigel is not a well-defined matrix, and therefore can produce a source of variability in experimental results. In this study, we present an in-depth proteomic analysis of Matrigel using a dynamic iterative exclusion method coupled with fractionation protocols that involve ammonium sulfate precipitation, size exclusion chromatography, and one-dimensional SDS-PAGE. The ability to identify the low mass and abundance components of Matrigel illustrates the utility of this method for the analysis of the extracellular matrix, as well as the complexity of the matrix itself.

A guided tour of the Trans‐Proteomic Pipeline

Abstract: The Trans-Proteomic Pipeline (TPP) is a suite of software tools for the analysis of MS/MS data sets. The tools encompass most of the steps in a proteomic data analysis workflow in a single, integrated software system. Specifically, the TPP supports all steps from spectrometer output file conversion to protein-level statistical validation, including quantification by stable isotope ratios. We describe here the full workflow of the TPP and the tools therein, along with an example on a sample data set, demonstrating that the setup and use of the tools are straightforward and well supported and do not require specialized informatic resources or knowledge.

Scaffold: A bioinformatic tool for validating MS/MS-based proteomic studies

Abstract: Over-reporting of unreliable protein identifications has reduced the accuracy and reproducibility of MS/MS-based proteomic studies. In this work, we demonstrate the analysis workflow used by a bioinformatic tool called Scaffold, which attempts to increase the confidence in protein identification reports through the use of several statistical methods. In addition, this work describes an advanced protein grouping method used by Scaffold to further reduce falsely reported protein identifications, particularly when using large or otherwise sequence redundant protein databases.

Prediction of the human membrane proteome

Abstract: Membrane proteins are key molecules in the cell, and are important targets for pharmaceutical drugs. Few three-dimensional structures of membrane proteins have been obtained, which makes computatio ...

Plant secretome: unlocking secrets of the secreted proteins.

Abstract: Plant secretomics is a newly emerging area of the plant proteomics field. It basically describes the global study of secreted proteins into the extracellular space of plant cell or tissue at any given time and under certain conditions through various secretory mechanisms. A combination of biochemical, proteomics and bioinformatics approaches has been developed to isolate, identify and profile secreted proteins using complementary in vitro suspension-cultured cells and in planta systems. Developed inventories of secreted proteins under normal, biotic and abiotic conditions revealed several different types of novel secreted proteins, including the leaderless secretory proteins (LSPs). On average, LSPs can account for more than 50% of the total identified secretome, supporting, as in other eukaryotes, the existence of novel secretory mechanisms independent of the classical endoplasmic reticulum-Golgi secretory pathway, and suggesting that this non-classical mechanism of protein expression is, for as yet unknown reasons, more massively used than in other eukaryotic systems. Plants LSPs, which seem to be potentially involved in the defense/stress responses, might have dual (extracellular and/or intracellular) roles as most of them have established intracellular functions, yet presently unknown extracellular functions. Evidence is emerging on the role of glycosylation in the apical sorting and trafficking of secretory proteins. These initial secretome studies in plants have considerably advanced our understanding on secretion of different types of proteins and their underlying mechanisms, and opened a door for comparative analyses of plant secretomes with those of other organisms. In this first review on plant secretomics, we summarize and discuss the secretome definition, the applied approaches for unlocking secrets of the secreted proteins in the extracellular fluid, the possible functional significance and secretory mechanisms of LSPs, as well as glycosylation of secreted proteins and challenges involved ahead. Further improvements in existing and developing strategies and techniques will continue to drive forward plant secretomics research to building comprehensive and confident data sets of secreted proteins. This will lead to an increased understanding on how cells couple the concerted action of secreted protein networks to their internal and external environments.

Label-free detection techniques for protein microarrays: prospects, merits and challenges.

Abstract: Protein microarrays, on which thousands of discrete proteins are printed, provide a valuable platform for functional analysis of the proteome. They have been widely used for biomarker discovery and to study protein-protein interactions. The accomplishments of DNA microarray technology, which had enabled massive parallel studies of gene expression, sparked great interest for the development of protein microarrays to achieve similar success at the protein level. Protein microarray detection techniques are often classified as being label-based and label-free. Most of the microarray applications have employed labelled detection such as fluorescent, chemiluminescent and radioactive labelling. These labelling strategies have synthetic challenges, multiple label issues and may exhibit interference with the binding site. Therefore, development of sensitive, reliable, high-throughput, label-free detection techniques are now attracting significant attention. Label-free detection techniques monitor biomolecular interactions and simplify the bioassays by eliminating the need for secondary reactants. Moreover, they provide quantitative information for the binding kinetics. In this article, we will review several label-free techniques, which offer promising applications for the protein microarrays, and discuss their prospects, merits and challenges.

De novo sequencing of peptides by MS/MS

Abstract: The current status of de novo sequencing of peptides by MS/MS is reviewed with focus on collision cell MS/MS spectra. The relation between peptide structure and observed fragment ion series is discussed and the exhaustive extraction of sequence information from CID spectra of protonated peptide ions is described. The partial redundancy of the extracted sequence information and a high mass accuracy are recognized as key parameters for dependable de novo sequencing by MS. In addition, the benefits of special techniques enhancing the generation of long uninterrupted fragment ion series for de novo peptide sequencing are highlighted. Among these are terminal (18)O labeling, MS(n) of sodiated peptide ions, N-terminal derivatization, the use of special proteases, and time-delayed fragmentation. The emerging electron transfer dissociation technique and the recent progress of MALDI techniques for intact protein sequencing are covered. Finally, the integration of bioinformatic tools into peptide de novo sequencing is demonstrated.

The proteome of lysosomes

Abstract: Lysosomes are organelles of eukaryotic cells that are critically involved in the degradation of macromolecules mainly delivered by endocytosis and autophagocytosis. Degradation is achieved by more than 60 hydrolases sequestered by a single phospholipid bilayer. The lysosomal membrane facilitates interaction and fusion with other compartments and harbours transport proteins catalysing the export of catabolites, thereby allowing their recycling. Lysosomal proteins have been addressed in various proteomic studies that are compared in this review regarding the source of material, the organelle/protein purification scheme, the proteomic methodology applied and the proteins identified. Distinguishing true constituents of an organelle from copurifying contaminants is a central issue in subcellular proteomics, with additional implications for lysosomes as being the site of degradation of many cellular and extracellular proteins. Although many of the lysosomal hydrolases were identified by classical biochemical approaches, the knowledge about the protein composition of the lysosomal membrane has remained fragmentary for a long time. Using proteomics many novel lysosomal candidate proteins have been discovered and it can be expected that their functional characterisation will help to understand functions of lysosomes at a molecular level that have been characterised only phenomenologically so far and to generally deepen our understanding of this indispensable organelle.

Proteomic analysis identifies highly antigenic proteins in exosomes from M. tuberculosis-infected and culture filtrate protein-treated macrophages.

Abstract: Exosomes are small 30-100 nm membrane vesicles released from hematopoietic and nonhematopoietic cells and function to promote intercellular communication. They are generated through fusion of multivesicular bodies with the plasma membrane and release of interluminal vesicles. Previous studies from our laboratory demonstrated that macrophages infected with Mycobacterium release exosomes that promote activation of both innate and acquired immune responses; however, the components present in exosomes inducing these host responses were not defined. This study used LC-MS/MS to identify 41 mycobacterial proteins present in exosomes released from M. tuberculosis-infected J774 cells. Many of these proteins have been characterized as highly immunogenic. Further, since most of the mycobacterial proteins identified are actively secreted, we hypothesized that macrophages treated with M. tuberculosis culture filtrate proteins (CFPs) would release exosomes containing mycobacterial proteins. We found 29 M. tuberculosis proteins in exosomes released from CFP-treated J774 cells, the majority of which were also present in exosomes isolated from M. tuberculosis-infected cells. The exosomes from CFP-treated J774 cells could promote macrophage and dendritic cell activation as well as activation of naive T cells in vivo. These results suggest that exosomes containing M. tuberculosis antigens may be alternative approach to developing a tuberculosis vaccine.

Changes in the proteome of Salmonella enterica serovar Thompson as stress adaptation to sublethal concentrations of thymol

Abstract: Thymol is a natural biocide and component of some essential oils from herbs. Its inhibitory effect on the growth of different microorganisms is well documented. The precise targets of the antibacterial action of thymol is not yet been fully established, the action seems to take place in different ways. The strain Salmonella enterica serovar Thompson MCV1 was grown in the presence of a sublethal concentration (0.01%) of thymol. The proteins extracted from treated and untreated cells were subjected to 2-D PAGE, followed by in-gel spot digestion and subsequent MALDI-TOF analysis. The analysis of gels showed many proteins that were either upregulated or downregulated by the presence of thymol, with significant changes in proteins belonging to different functional classes. In particular, the thioredoxin-1 was not expressed in the treated cells, indicating that its absence could be a consequence of the stress caused by the presence of thymol. On the other hand, different chaperon proteins were upregulated or de novo synthesis such as GroEL and DnaK, key proteins in the protection mechanism toward thermal stress. Outer membrane proteins were upregulated in treated cells; indeed the bacterial envelope stress response is trigged by the accumulation of misfolded outer membrane proteins. Moreover, the thymol seems to impair the citrate metabolic pathway, as well as many enzymes involved in the synthesis of ATP. Definitely, thymol plays a role in altering very different pathways of cell metabolism.

2-picoline-borane: a non-toxic reducing agent for oligosaccharide labeling by reductive amination.

Abstract: Analysis of N-glycans is often performed by LC coupled to fluorescence detection. The N-glycans are usually labeled by reductive amination with a fluorophore containing a primary amine to allow fluorescence detection. Moreover, many of the commonly applied labels also allow improved mass spectrometric detection of oligosaccharides. For reductive amination, the amine group of the label reacts with the reducing-end aldehyde group of the oligosaccharide to form a Schiff base, which is reduced to a secondary amine. Here, we propose the use of 2-picoline-borane as the reducing agent, as a non-toxic alternative to the extensively used, but toxic sodium cyanoborohydride. Using dextran oligosaccharides and plasma N-glycans, we demonstrate similar labeling efficacies for 2-picoline-borane and sodium cyanoborohydride. Therefore, 2-picoline-borane is a non-toxic alternative to sodium cyanoborohydride for the labeling of oligosaccharides.

Proteomics uncovers extreme heterogeneity in the Staphylococcus aureus exoproteome due to genomic plasticity and variant gene regulation

Abstract: Sequencing of at least 13 Staphylococcus aureus isolates has shown that genomic plasticity impacts significantly on the repertoire of virulence factors. However, genome sequencing does not reveal which genes are expressed by individual isolates. Here, we have therefore performed a comprehensive survey of the composition and variability of the S. aureus exoproteome. This involved multilocus sequence typing, virulence gene, and prophage profiling by multiplex PCR, and proteomic analyses of secreted proteins using 2-DE. Dissection of the exoproteomes of 25 clinical isolates revealed that only seven out of 63 identified secreted proteins were produced by all isolates, indicating a remarkably high exoproteome heterogeneity within one bacterial species. Most interesting, the observed variations were caused not only by genome plasticity, but also by an unprecedented variation in secretory protein production due to differences in transcriptional and post-transcriptional regulation. Our data imply that genomic studies on virulence gene conservation patterns need to be complemented by analyses of the extracellular protein pattern to assess the full virulence potential of bacterial pathogens like S. aureus. Importantly, the extensive variability of secreted virulence factors in S. aureus also suggests that development of protective vaccines against this pathogen requires a carefully selected combination of invariably produced antigens.

Environmental proteomics: analysis of structure and function of microbial communities.

Abstract: Prokaryotic and eukaryotic microorganisms make a vital contribution to biogeochemical cycles by decomposing virtually all natural compounds and thereby exert a lasting effect on biosphere and climate. The rapidly growing number of metagenomic sequences together with revolutionary advances in bioinformatics and protein analyses have opened completely new horizons to investigate the molecular basis of such complex processes. Proteomics has contributed substantially to our understanding of individual organisms at the cellular level as it offers excellent possibilities to probe many protein functions and responses simultaneously. However, it has not yet been widely applied in microbial ecology, although most proteins have an intrinsic metabolic function which can be used to relate microbial activities to the identity of defined organisms in multispecies communities. Albeit still in its infancy, environmental proteomics enables simple protein cataloging, comparative and semi-quantitative proteomics, analyses of protein localization, discovery of post-translational modifications, and even determination of amino-acid sequences and genotypes by strain-resolved proteogenomics. This review traces the historical development of environmental proteomics and summarizes milestone publications in the field. In conclusion, we briefly discuss current limitations of microbial community proteomics but also the potential of emerging technologies to shape the future of metaproteome analyses.

The Botrytis cinerea early secretome.

Abstract: The extracellular proteome, or secretome, of phytopathogenic fungi is presumed to be a key element of their infection strategy. Especially interesting constituents of this set are those proteins secreted at the beginning of the infection, during the germination of conidia on the plant surfaces or wounds, since they may play essential roles in the establishment of a successful infection. We have germinated Botrytis cinerea conidia in conditions that resemble the plant environment, a synthetic medium enriched with low molecular weight plant compounds, and we have collected the proteins secreted during the first 16 h by a double precipitation protocol. 2-D electrophoresis of the precipitated secretome showed a spot pattern similar for all conditions evaluated and for the control medium without plant extract. The proteins in 16 of these spots were identified by PMF and corresponded to 11 different polypeptides. Alternative determination of secretome composition by LC-MS/MS of tryptic fragments rendered a much larger number, 105 proteins, which included all previously identified by PMF. All proteins were functionally classified according to their putative function in the infection process. Key features of the early secretome include a large number of proteases, the abundance of proteins involved in the degradation of plant defensive barriers, and plenty of proteins with unknown function.

Profiling the surfacome of Staphylococcus aureus.

Abstract: Staphylococcus aureus is a widespread opportunistic pathogen that can cause a wide variety of life-threatening diseases. Especially for the colonization of human tissues and the development of invasiveness, surface-exposed proteins are of major importance. In the present studies, we optimized a proteolytic shaving approach to identify those surface-exposed protein domains - the surfacome - of S. aureus that are accessible to extracellular bio-macromolecules, for example in the host milieu. Subsequently, this approach was applied to define the surfacomes of four strains with different genetic backgrounds. This resulted in the identification of 96 different proteins. Surprisingly, the overlap between the surfacomes of the four different strains was below 10% and each strain displayed its own characteristic set of surface-exposed proteins. The data were also evaluated at the peptide level and here we observed a similar phenomenon. From 190 unique peptides only five were commonly found in the four strains. Besides well known cell wall proteins, we also identified some essential proteins, several yet uncharacterized exported proteins and predicted intracellular proteins. These results show for the first time that the cell surface of different S. aureus strains is not only highly variable, but also that the displayed proteins are very heterogeneous.

The secreted and surface proteomes of the adult stage of the carcinogenic human liver fluke Opisthorchis viverrini.

Abstract: Infection with the human liver fluke, Opisthorchis viverrini, is a serious public health problem in Thailand, Laos and nearby locations in Southeast Asia. Both experimental and epidemiological evidence strongly implicate liver fluke infection in the etiology of one of the liver cancer subtypes, cholangiocarcinoma (CCA). To identify parasite proteins critical for liver fluke survival and the etiology of CCA, OFFGEL electrophoresis and multiple reaction monitoring were employed to characterize 300 parasite proteins from the O. viverrini excretory/secretory products and, utilizing selective labeling and sequential solubilization, from the host-exposed tegument. The excretory/secretory included a complex mixture of proteins that have been associated with cancers, including proteases of different mechanistic classes and orthologues of mammalian growth factors and anti-apoptotic proteins. Also identified was a cysteine protease inhibitor which, in other helminth pathogens, induces nitric oxide production by macrophages, and, hence may contribute to malignant transformation of inflamed cells. More than 160 tegumental proteins were identified using sequential solubilization of isolated teguments, and a subset of these was localized to the surface membrane of the tegument by labeling living flukes with biotin and confirming surface localization with fluorescence microscopy. These included annexins, which are potential immuno-modulators, and orthologues of the schistosomiasis vaccine antigens Sm29 and tetraspanin-2. Novel roles in pathogenesis were suggested for the tegument-host interface since more than ten surface proteins had no homologues in the public databases. The O. viverrini proteins identified here provide an extensive catalogue of novel leads for research on the pathogenesis of opisthorchiasis and the development of novel interventions for this disease and CCA, as well as providing a scaffold for sequencing the genome of this fluke.

Protein abundances are more conserved than mRNA abundances across diverse taxa

Abstract: Proteins play major roles in most biological processes; as a consequence, protein expression levels are highly regulated. While extensive post-transcriptional, translational and protein degradation control clearly influence protein concentration and functionality, it is often thought that protein abundances are primarily determined by the abundances of the corresponding mRNAs. Hence surprisingly, a recent study showed that abundances of orthologous nematode and fly proteins correlate better than their corresponding mRNA abundances. We tested if this phenomenon is general by collecting and testing matching large-scale protein and mRNA expression data sets from seven different species: two bacteria, yeast, nematode, fly, human, and rice. We find that steady-state abundances of proteins show significantly higher correlation across these diverse phylogenetic taxa than the abundances of their corresponding mRNAs (p=0.0008, paired Wilcoxon). These data support the presence of strong selective pressure to maintain protein abundances during evolution, even when mRNA abundances diverge.

Fish proteome analysis: model organisms and non-sequenced species.

Abstract: In the last decade, proteomic technologies have been increasingly used in fish biology research. Proteomics has been applied primarily to investigate the physiology, development biology and the impact of contaminants in fish model organisms, such as zebrafish (Danio rerio), as well as in some commercial species produced in aquaculture, mainly salmonids and cyprinids. However, the lack of previous genetic information on most fish species has been a major drawback for a more general application of the different proteomic technologies currently available. Also, many teleosts of interest in biological research and with potential application in aquaculture hold unique physiological characteristics that cannot be directly addressed from the study of small laboratory fish models. This review describes proteomic approaches that have been used to investigate diverse biological questions in model and non-model fish species. We will also evaluate the current possibilities to integrate fish proteomics with other "omic" approaches, as well as with additional complementary techniques, in order to address the future challenges in fish biology research.

Proteomic changes in maize roots after short-term adjustment to saline growth conditions

Abstract: It is of fundamental importance to understand adaptation processes leading to salt resistance. The initial effects on maize roots in the first hour after the adjustment to saline conditions were monitored to elucidate initial responses. The subsequent proteome change was monitored using a 2-D proteomic approach. We found several new salt-inducible proteins, whose expression has not been previously reported to be modulated by salt. A set of phosphoproteins in maize was detected but only ten proteins were phosphorylated and six proteins were dephosphorylated after the application of 25 mM NaCl for 1 h. Some of the phosphorylated maize proteins such as fructokinase, UDP-glucosyl transferase BX9, and 2-Cys-peroxyredoxine were enhanced, whereas an isocitrate-dehydrogenase, calmodulin, maturase, and a 40-S-ribosomal protein were dephosphorylated after adjustment to saline conditions. The initial reaction of the proteome and phosphoproteome of maize after adjustment to saline conditions reveals members of sugar signalling and cell signalling pathways such as calmodulin, and gave hint to a transduction chain which is involved in NaCl-induced signalling. An alteration of 14-3-3 proteins as detected may change plasma membrane ATPase activity and cell wall growth regulators such as xyloglucane endotransglycosylase were also found to be changed immediately after the adjustment to salt stress.

Gilthead sea bream liver proteome altered at low temperatures by oxidative stress

Abstract: Gilthead sea bream exposed to the cold show multiple physiological alterations, particularly in liver. A typical cold-stress response was reproduced in gilthead sea bream acclimated to 20 degrees C (Warm group) when the water temperature was lowered to 8 degrees C (Cold group). After 10 days, thiobarbituric acid reactive substances in the liver had increased by 50%, and nitric oxide had increased twofold. This indicates that lipid peroxidation and oxidative stress had occurred. Protein profiles of liver from fish in warm and cold environments were obtained by 2-DE. Quantification of differential expression by matching spots showed that a total of 57 proteins were altered significantly. Many proteins were downregulated following cold exposure, including actin, the most abundant protein in the proteome; enzymes of amino acid metabolism; and enzymes with antioxidant capacity, such as betaine-homocysteine-methyl transferase, glutathione-S-transferase and catalase. Some proteins associated with protective action were upregulated at low temperatures, including peroxiredoxin, thioredoxin and lysozyme; as well as enzymes such as aldehyde dehydrogenase and adenosin-methionine synthetase. However, the upregulation of proteases, proteasome activator protein and trypsinogen-like protein indicated an increase in proteolysis. Increases in elongation factor-1alpha, the GAPDH oxidative form, tubulin and Raf-kinase inhibitor protein indicated oxidative stress and the induction of apoptosis. These data indicate that cold exposure induced oxidative damage in hepatocytes.

Proteins in ecotoxicology – How, why and why not?

Abstract: The growing interest in the application of proteomic technologies to solve toxicology issues and its relevance in ecotoxicology research has resulted in the emergence of "ecotoxicoproteomics". There is a general consensus that ecotoxicoproteomics is a powerful tool to spot early molecular events involved in toxicant responses, which are responsible for the adverse effects observed at higher levels of biological organization, thus contributing to elucidate the mode of action of stressors and to identify specific biomarkers. Ultimately, early-warning indicators can then be developed and deployed in "in situ" bioassays and in environmental risk assessment. The number of field experiments or laboratory trials using ecologically relevant test-species and involving proteomics has been, until recently, insufficient to allow a critical analysis of the real benefits of the application of this approach to ecotoxicology. This article intends to present an overview on the applications of proteomics in the context of ecotoxicology, focusing mainly on the prospective research to be done in invertebrates. Although these represent around 95% of all animal species and in spite of the key structural and functional roles they play in ecosystems, proteomic research in invertebrates is still in an incipient stage. We will review applications of ecotoxicoproteomics by evaluating the technical methods employed, the organisms and the contexts studied, the advances achieved until now and lastly the limitations yet to overcome will be discussed.

Aβ and human amylin share a common toxicity pathway via mitochondrial dysfunction

Abstract: Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM) are leading causes of morbidity and mortality in the elderly. Both diseases are characterized by amyloid deposition in target tissues: aggregation of amylin in T2DM is associated with loss of insulin-secreting β-cells, while amyloid β (Aβ) aggregation in AD brain is associated with neuronal loss. Here, we used quantitative iTRAQ proteomics as a discovery tool to show that both Aβ and human amylin (HA) deregulate identical proteins, a quarter of which are mitochondrial, supporting the notion that mitochondrial dysfunction is a common target in these two amyloidoses. A functional validation revealed that mitochondrial complex IV activity was significantly reduced after treatment with either HA or Aβ, as was mitochondrial respiration. In comparison, complex I activity was reduced only after treatment with HA. Aβ and HA, but not the non-amyloidogenic rat amylin, induced significant increases in the generation of ROS. Co-incubation of HA and Aβ did not produce an augmented effect in ROS production, again suggesting common toxicity mechanisms. In conclusion, our data suggest that Aβ and HA both exert toxicity, at least in part, via mitochondrial dysfunction, thus restoring their function may be beneficial for both AD and T2DM.

Proteomic analysis of rice seedlings infected by Sinorhizobium meliloti 1021.

Abstract: Rhizobial endophytes infect and colonize not only leguminous plants, but several non-leguminous species as well. Using green fluorescent protein tagging technique, it has been shown that Rhizobia infect different varieties of rice species and migrate from plant roots to aerial tissues such as leaf sheaths and leaves. The interaction between them was found to promote the growth of rice. The growth promotion is the cumulative result of enhanced photosynthesis and stress resistance. In addition, indole-3-acetic acid also contributes to the promotion. Gel-based comparative proteomic approaches were applied to analyze the protein profiles of three different tissues (root, leaf sheath and leaf) of Sinorhizobium meliloti 1021 inoculated rice in order to get an understanding about the molecular mechanism. Upon the inoculation of rhizobia, proteins involved in nine different functional categories were either up-regulated or down-regulated. Photosynthesis related proteins were up-regulated only in leaf sheath and leaf, while the up-regulated proteins in root were exclusively defense related. The results implied that there might have been an increase in the import and transport of proteins involved in light and dark reactions to the chloroplast as well as more efficient distribution of nutrients, hence enhanced photosynthesis. Although the initiation of defensive reactions mainly occurred in roots, some different defense mechanisms were also evoked in the aerial tissues.

Comparative proteome analysis of metabolic proteins from seeds of durum wheat (cv. Svevo) subjected to heat stress.

Abstract: In Central and Southern Italy, where durum wheat represents one of the most widely cultivated crops, grain filling occurs during Spring, a period characterized by sudden increases in temperature. Wheat grain proteins are classified into albumins, globulins, and prolamins. The nonprolamin fractions include proteins with metabolic activity or structural function. In order to investigate the consequences of heat stress on the accumulation of nonprolamin proteins in mature durum wheat kernels, the Italian cultivar Svevo was subjected to two thermal regimes (heat stress versus control). The 2-D patterns of nonprolamin proteins were monitored to identify polypeptides affected by heat stress during grain fill. This study shows that heat stress alters significantly the durum wheat seed proteome, although the changes range is only between 1.2- and 2.2-fold. This analysis revealed 132 differentially expressed polypeptides, 47 of which were identified by MALDI-TOF and MALDI-TOF-TOF MS and included HSPs, proteins involved in the glycolysis and carbohydrate metabolism, as well as stress-related proteins. Many of the heat-induced polypeptides are considered to be allergenic for sensitive individuals.

Identification of candidate biomarkers with cancer-specific glycosylation in the tissue and serum of endometrioid ovarian cancer patients by glycoproteomic analysis.

Abstract: Epithelial ovarian cancer is diagnosed less than 25% of the time when the cancer is confined to the ovary, leading to 5-year survival rates of less than 30%. Therefore, there is an urgent need for early diagnostics for ovarian cancer. Our study using glycotranscriptome comparative analysis of endometrioid ovarian cancer tissue and normal ovarian tissue led to the identification of distinct differences in the transcripts of a restricted set of glycosyltransferases involved in N-linked glycosylation. Utilizing lectins that bind to glycan structures predicted to show changes, we observed differences in lectin-bound glycoproteins consistent with some of the transcript differences. In this study, we have extended our observations by the use of selected lectins to perform a targeted glycoproteomic analysis of ovarian cancer and normal ovarian tissues. Our results have identified several glycoproteins that display tumor-specific glycosylation changes. We have verified these glycosylation changes on glycoproteins from tissue using immunoprecipitation followed by lectin blot detection. The glycoproteins that were verified were then analyzed further using existing microarray data obtained from benign ovarian adenomas, borderline ovarian adenocarcinomas, and malignant ovarian adenocarcinomas. The verified glycoproteins found to be expressed above control levels in the microarray data sets were then screened for tumor-specific glycan modifications in serum from ovarian cancer patients. Results obtained from two of these glycoprotein markers, periostin and thrombospondin, have confirmed that tumor-specific glycan changes can be used to distinguish ovarian cancer patient serum from normal serum.

Proteomic analysis of the Echinococcus granulosus metacestode during infection of its intermediate host

Abstract: Cystic hydatid disease (CHD) is caused by infection with the Echinococcus granulosus metacestode and affects both humans and livestock. In this work, we performed a proteomic analysis of the E. granulosus metacestode during infection of its intermediate bovine host. Parasite proteins were identified in different metacestode components (94 from protoscolex, 25 from germinal layer and 20 from hydatid cyst fluid), along with host proteins (58) that permeate into the hydatid cyst, providing new insights into host-parasite interplay. E. granulosus and platyhelminth EST data allowed successful identification of proteins potentially involved in downregulation of host defenses, highlighting possible evasion mechanisms adopted by the parasite to establish infection. Several intracellular proteins were found in hydatid cyst fluid, revealing a set of newly identified proteins that were previously thought to be inaccessible for inducing or modulating the host immune response. Host proteins identified in association with the hydatid cyst suggest that the parasite may bind/adsorb host molecules with nutritional and/or immune evasion purposes, masking surface antigens or inhibiting important effector molecules of host immunity, such as complement components and calgranulin. Overall, our results provide valuable information on parasite survival strategies in the adverse host environment and on the molecular mechanisms underpinning CHD immunopathology.

An integrated proteomics and transcriptomics reference data set provides new insights into the Bradyrhizobium japonicum bacteroid metabolism in soybean root nodules.

Abstract: Bradyrhizobium japonicum, a gram-negative soil bacterium that establishes an N(2)-fixing symbiosis with its legume host soybean (Glycine max), has been used as a symbiosis model system. Using a sensitive geLC-MS/MS proteomics approach, we report the identification of 2315 B. japonicum strain USDA110 proteins (27.8% of the theoretical proteome) that are expressed 21 days post infection in symbiosis with soybean cultivated in growth chambers, substantially expanding the previously known symbiosis proteome. Integration of transcriptomics data generated under the same conditions (2780 expressed genes) allowed us to compile a comprehensive expression profile of B. japonicum during soybean symbiosis, which comprises 3587 genes/proteins (43% of the predicted B. japonicum genes/proteins). Analysis of this data set revealed both the biases and the complementarity of these global profiling technologies. A functional classification and pathway analysis showed that most of the proteins involved in carbon and nitrogen metabolism are expressed, including a complete set of tricarboxylic acid cycle enzymes, several gluconeogenesis and pentose phosphate pathway enzymes, as well as several proteins that were previously not considered to be present during symbiosis. Congruent results were obtained for B. japonicum bacteroids harvested from soybeans grown under field conditions.

Comparative analysis of soybean plasma membrane proteins under osmotic stress using gel‐based and LC MS/MS‐based proteomics approaches

Abstract: To study the soybean plasma membrane proteome under osmotic stress, two methods were used: a gel-based and a LC MS/MS-based proteomics method. Two-day-old seedlings were subjected to 10% PEG for 2 days. Plasma membranes were purified from seedlings using a two-phase partitioning method and their purity was verified by measuring ATPase activity. Using the gel-based proteomics, four and eight protein spots were identified as up- and downregulated, respectively, whereas in the nanoLC MS/MS approach, 11 and 75 proteins were identified as up- and downregulated, respectively, under PEG treatment. Out of osmotic stress responsive proteins, most of the transporter proteins and all proteins with high number of transmembrane helices as well as low-abundance proteins could be identified by the LC MS/MS-based method. Three homologues of plasma membrane H(+)-ATPase, which are transporter proteins involved in ion efflux, were upregulated under osmotic stress. Gene expression of this protein was increased after 12 h of stress exposure. Among the identified proteins, seven proteins were mutual in two proteomics techniques, in which calnexin was the highly upregulated protein. Accumulation of calnexin in plasma membrane was confirmed by immunoblot analysis. These results suggest that under hyperosmotic conditions, calnexin accumulates in the plasma membrane and ion efflux accelerates by upregulation of plasma membrane H(+)-ATPase protein.

Subsarcolemmal and intermyofibrillar mitochondria proteome differences disclose functional specializations in skeletal muscle

Abstract: Skeletal muscle is a highly specialized tissue that contains two distinct mitochondria subpopulations, the subsarcolemmal (SS) and the intermyofibrillar (IMF) mitochondria. Although it is established that these mitochondrial subpopulations differ functionally in several ways, limited information exists about the proteomic differences underlying these functional differences. Therefore, the objective of this study was to biochemically characterize the SS and IMF mitochondria isolated from rat red gastrocnemius skeletal muscle. We separated the two mitochondrial subpopulations from skeletal muscle using a refined method that provides an excellent division of these unique mitochondrial subpopulations. Using proteomics of mitochondria and its subfractions (intermembrane space, matrix and inner membrane), a total of 325 distinct proteins were identified, most of which belong to the functional clusters of oxidative phosphorylation, metabolism and signal transduction. Although more gel spots were observed in SS mitochondria, 38 of the identified proteins were differentially expressed between the SS and IMF subpopulations. Compared to the SS mitochondrial, IMF mitochondria expressed a higher level of proteins associated with oxidative phosphorylation. This observation, coupled with the finding of a higher respiratory chain complex activity in IMF mitochondria, suggests a specialization of IMF mitochondria toward energy production for contractile activity.

Peptide and protein quantification: a map of the minefield.

Abstract: The increasing popularity of gel-free proteomics technologies has created a strong demand for compatible quantitative analysis methods. As a result, a plethora of different techniques has been proposed to perform gel-free quantitative analysis of proteomics samples. Each of these methods comes with certain strengths and shortcomings, and they often are dedicated to a specific purpose. This review will present a brief overview of the main methods, organized by their underlying concepts, and will discuss the issues they raise with a focus on data processing. Finally, we will list the available software that can help with the data processing from quantitative experiments. We hope that this review will thus enable researchers to find the most appropriate method available for their research objectives, and can also serve as a basis for creating a reliable data processing strategy.