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A number of fungal and oomycete plant pathogens of major economic importance feed on their hosts by means of haustoria, which they place inside living plant cells. The underlying mechanisms are poorly understood, partly due to difficulty in preparing haustoria. We have therefore developed a procedure for isolating haustoria from the barley powdery mildew fungus (Blumeria graminis f.sp. hordei, Bgh). We subsequently aimed to understand the molecular mechanisms of haustoria through a study of their proteome. Extracted proteins were digested using trypsin, separated by LC, and analysed by MS/MS. Searches of a custom Bgh EST sequence database and the NCBI-NR fungal protein database, using the MS/MS data, identified 204 haustoria proteins. The majority of the proteins appear to have roles in protein metabolic pathways and biological energy production. Surprisingly, pyruvate decarboxylase (PDC), involved in alcoholic fermentation and commonly abundant in fungi and plants, was absent in our Bgh proteome data set. A sequence encoding this enzyme was also absent in our EST sequence database. Significantly, BLAST searches of the recently available Bgh genome sequence data also failed to identify a sequence encoding this enzyme, strongly indicating that Bgh does not have a gene for PDC.

Mycobacterium leprae has undergone extensive degenerative evolution, with a large number of pseudogenes. It is also the organism with the greatest divergence between gene annotations from independent institutes. Therefore, M. leprae is a good model to verify the currently predicted coding sequence regions between different annotations, to identify new ones and to investigate the expression of pseudogenes. We submitted a total extract of the bacteria isolated from Armadillo to Gel-LC-MS/MS using a linear quadrupole ion trap-Orbitrap mass spectrometer. Spectra were analyzed using the Leproma (1614 genes and 1133 pseudogenes) and TIGR (5446 genes) databases and a database containing the full genome translation. We identified a total of 1046 proteins, including five proteins encoded by previously predicted pseudogenes, which upon closer inspection appeared to be proper genes. Only 11 of the additional annotations by TIGR were verified. We also identified six tryptic peptides from five proteins from regions not considered to be coding sequences, in addition to peptides from two unannotated gene candidates that overlap with other genes. Our data show that the Leproma annotation of M. leprae is quite accurate, and there were no peptide observations corresponding to true pseudogenes, except for a new gene candidate, overlapping with an essential enolase on the complementary strand.

The development of insulin resistance in the obese is associated with chronic, low-grade inflammation. We aimed to identify novel links between obesity, insulin resistance and the inflammatory response by comparing C57BL/6 with type I interleukin-1 receptor knockout (IL-1RI-/-) mice, which are protected against diet-induced insulin resistance. Mice were fed a high-fat diet for 16 wk. Insulin sensitivity was measured and proteomic analysis was performed on adipose, hepatic and skeletal muscle tissues. Despite an equal weight gain, IL-1RI-/- mice had lower plasma glucose, insulin and triacylglycerol concentrations, compared with controls, following dietary treatment. The higher insulin sensitivity in IL-1RI-/- mice was associated with down-regulation of antioxidant proteins and proteasomes in adipose tissue and hepatic soluble epoxide hydrolase, consistent with a compromised inflammatory response as well as increased glycolysis and decreased fatty acid [beta]-oxidation in their muscle. Their lower hepatic triacylglycerol concentrations may reflect decreased flux of free fatty acids to the liver, decreased hepatic fatty acid-binding protein expression and decreased lipogenesis. Correlation analysis revealed down-regulation of classical biomarkers of ER stress in their adipose tissue, suggesting that disruption of the IL-1RI-mediated inflammatory response may attenuate cellular stress, which was associated with significant protection from diet-induced insulin resistance, independent of obesity.

Effective inhibition of the proteasome by high doses of proteasome inhibitors induces apoptotic cell death. In contrast, partial proteasome inhibition by low inhibitor doses mediates a protective cellular stress response. The early targets and mediators of these dose-dependent effects of proteasome inhibitors are unknown. Primary human umbilical cord vein endothelial cells were treated with low and high doses of the proteasome inhibitor MG132 for 2 h. In a combined 2-DE and MS approach, we identified more than 20 new targets of proteasome inhibition. These proteins are involved in cell cycle regulation, signaling, cytoskeletal rearrangement, and cellular stress response. Accompanying Affymetrix analysis revealed that these proteins are not regulated on the transcriptional level but are mainly stabilized by proteasome inhibition. The proteasome-dependent accumulation of the anti-oxidative sensor proteins DJ-1, peroxiredoxin-1 and -6 was accompanied by dose-dependent induction of oxidative stress after 2 h of proteasome inhibition and contributed to the differential transcriptional stress response to low- and high-dose proteasome inhibition: Whereas low-dose proteasome inhibition induces a transcriptional profile reminiscent of a physiological stress response that preconditions and protects endothelial cells from oxidative stress, high inhibitor doses induce massive transcriptional dysregulation and pronounced oxidative stress triggering apoptosis.

Seed oil bodies (OBs) are intracellular particles storing lipids as food or biofuel reserves in oleaginous plants. Since Brassica napus OBs could be easily contaminated with protein bodies and/or myrosin cells, they must be purified step by step using floatation technique in order to remove non-specifically trapped proteins. An exhaustive description of the protein composition of rapeseed OBs from two double-zero varieties was achieved by a combination of proteomic and genomic tools. Genomic analysis led to the identification of sequences coding for major seed oil body proteins, including 19 oleosins, 5 steroleosins and 9 caleosins. Most of these proteins were also identified through proteomic analysis and displayed a high level of sequence conservation with their Arabidopsis thaliana counterparts. Two rapeseed oleosin orthologs appeared acetylated on their N-terminal alanine residue and both caleosins and steroleosins displayed a low level of phosphorylation.

Lymph node metastasis (LNM) is recognized as an important factor involved in the tumor malignancy progression. Our previous study has indicated that the hepatocarcinoma cell line with 75% of LNM (Hca-F)-cell-induced neoplasia and the hepatocarcinoma cell line with 25% of LNM-induced neoplasia are accompanied with high (75%) and low (25%) incidences of LNM. In the current study, 62 and 54 protein spots were observed up-regulated and down-regulated in Hca-F cell relative to the hepatocarcinoma cell line with 25% of LNM by 2-D DIGE. Totally, 113 unique proteins were identified by HPLC-nano ESI-MS/MS analysis. The expression levels of Annexin A7, Ulch3, and ER protein 29 were validated by Western blotting analyses. The abnormally regulated proteins were categorized and annotated by protein analysis through evolutionary relationships analysis with the aid of the database for annotation, visualization and integrated discovery tool. Seventeen gene candidates concordantly expressed both at mRNA and protein levels. By making a challenge, we detected expression levels of Annexin A7 in primary gastric cancer (GC) and primary GC cancer tissues with LNMs by immunohistochemisty. Higher ratio of positive and strong expressions Annexin A7 in GC might correlate with the tumor progression. The repression of Annexin A7 inhibits the mobility and invasion abilities of Hca-F cell, increases the apoptosis rate of Hca-F cell. Current study narrows and provides certain specific protein candidates potentially playing important roles in LNM-associated cancers.

The application of proteomic techniques to neuroscientific research provides an opportunity for a greater understanding of nervous system structure and function. As increasing amounts of neuroproteomic data become available, it is necessary to formulate methods to integrate these data in a meaningful way to obtain a more comprehensive picture of neuronal subcompartments. Furthermore, computational methods can be used to make biologically relevant predictions from large proteomic data sets. Here, we applied an integrated proteomics and systems biology approach to characterize the presynaptic (PRE) nerve terminal. For this, we carried out proteomic analyses of presynaptically enriched fractions, and generated a PRE literature-based protein-protein interaction network. We combined these with other proteomic analyses to generate a core list of 117 PRE proteins, and used graph theory-inspired algorithms to predict 92 additional components and a PRE complex containing 17 proteins. Some of these predictions were validated experimentally, indicating that the computational analyses can identify novel proteins and complexes in a subcellular compartment. We conclude that the combination of techniques (proteomics, data integration, and computational analyses) used in this study are useful in obtaining a comprehensive understanding of functional components, especially low-abundance entities and/or interactions in the PRE nerve terminal.

The mechanisms underlying white matter changes in psychiatric disease are not known. We aimed to characterise the differential protein expression in deep white matter from the dorsolateral prefrontal cortex from 35 schizophrenia, 35 bipolar disorder, and 35 control subjects, from the Stanley Array Collection. We used 2-D DIGE to profile for protein expression changes in the brain. We found 70 protein spots to be significantly differentially expressed between disease and control subjects (ANCOVA, p<0.05), 46 of which were subsequently identified by LC-MS/MS. The proteins identified included novel disease candidates as well as proteins that have previously been reported as abnormal in schizophrenia, thus reinforcing their association with the disease. Furthermore, we confirmed the direction of change for three proteins using ELISA, namely neurofilament-light, amphiphysin II, and Rab-GDP-[alpha], in a subset of the Stanley Array Collection. In addition, altered expression of neurofilament-light, amphiphysin II, and Rab-GDP-[alpha] was not observed in the cortex of mice chronically treated with haloperidol, making it less likely that these alterations are a consequence of neuroleptic medication. The data presented here strongly suggest disruption of the cytoskeleton and its associated signal transduction proteins in schizophrenia, and to a lesser extent in bipolar disorder.

The assembly of primordial follicles early in ovarian development and subsequent transition to primary follicles are critical processes in ovarian biology. Inappropriate coordination of these processes contributes to ovarian pathologies such as premature ovarian failure and infertility. To better understand the molecular mechanisms involved in primordial follicle assembly and development, 2-D PAGE and MALDI-TOF/TOF technologies were used to construct a comparative proteome profile of the immature rat ovary at specific time-points (0, 24, 48, and 72 h postpartum). A total of 154 differential protein spots corresponding to 134 different proteins were definitively identified between any two time-points. Further cluster analysis showed four expression patterns, and each pattern correlated with specific cell processes that occur during early ovarian development. Seven proteins were randomly selected to verify expression patterns using Western blotting, and subsequently immunohistochemistry was performed to further investigate their cellular localization. Additionally, detailed functional analyses of these differentially expressed proteins were performed. Elucidation of how these changes in protein expression level coordinate primordial follicles assembly and development is intended to provide a better understanding of these critical biological processes early in ovarian development and will provide potential therapeutic molecular targets to regulate ovarian function and treat ovarian disease.

We report on a novel strategy for identification of specific sulfation motifs in chondroitin/dermatan sulfate (CS/DS) chain derived from decorin (Dcn), based on enzyme cleavage and multistage MS (MSn). Released CS/DS chains were digested with chondroitin B and in parallel with AC I lyases to obtain oligosaccharides of known hexuronic acid (HexA) epimerization. The depolymerized chains were separated by gel filtration, and collected di- and hexasaccharides were analyzed by ESI MSn. MS2 on bisulfated 4,5-[Delta]-HexAGalNAc revealed an additional sulfate ester group at 4,5-[Delta]-HexA. MS2 data provided evidence upon GlcA sulfation in Dcn due to the fact that 4,5-[Delta]-HexA derived from GlcA after chondroitin AC I lyase treatment. Hexasaccharide screening in the MS1 mode indicated direct correlation between the sulfate distribution and HexA epimerization. MSn performed on ions that, according to mass calculation, correspond to pentasulfated [4,5-[Delta]-HexAGalNAc(GlcAGalNAc)2], trisulfated [4,5-[Delta]-HexAGalNAc(GlcAGalNAc)2] with IdoA-derived 4,5-[Delta]-HexA at the nonreducing end, tetrasulfated [4,5-[Delta]-HexAGalNAc(IdoAGalNAc)2] and monosulfated [4,5-[Delta]-HexAGalNAc(IdoAGalNAc)2] with GlcA-derived 4,5-[Delta]-HexA at the nonreducing end rendered fragmentation patterns confirming the presence of over-, regular, and under-sulfated regions as well as structural motifs having both types of HexA sulfated within Dcn CS/DS.

Protein sumoylation is an important reversible post-translational modification on proteins, and orchestrates a variety of cellular processes. Recently, computational prediction of sumoylation sites has attracted much attention for its cost-efficiency and power in genomic data mining. In this work, we developed SUMOsp 2.0, an accurate computing program with an improved group-based phosphorylation scoring algorithm. Our analysis demonstrated that SUMOsp 2.0 has greater prediction accuracy than SUMOsp 1.0 and other existing tools, with a sensitivity of 88.17% and a specificity of 92.69% under the medium threshold. Previously, several large-scale experiments have identified a list of potential sumoylated substrates in Saccharomyces cerevisiae and Homo sapiens; however, the exact sumoylation sites in most of these proteins remain elusive. We have predicted potential sumoylation sites in these proteins using SUMOsp 2.0, which provides a great resource for researchers and an outline for further mechanistic studies of sumoylation in cellular plasticity and dynamics. The online service and local packages of SUMOsp 2.0 are freely available at: .

Over the last years virus-host cell interactions were investigated in numerous studies. Viral strategies for evasion of innate immune response, inhibition of cellular protein synthesis and permission of viral RNA and protein production were disclosed. With quantitative proteome technology, comprehensive studies concerning the impact of viruses on the cellular machinery of their host cells at protein level are possible. Therefore, 2-D DIGE and nanoHPLC-nanoESI-MS/MS analysis were used to qualitatively and quantitatively determine the dynamic cellular proteome responses of two mammalian cell lines to human influenza A virus infection. A cell line used for vaccine production (MDCK) was compared with a human lung carcinoma cell line (A549) as a reference model. Analyzing 2-D gels of the proteomes of uninfected and influenza-infected host cells, 16 quantitatively altered protein spots (at least ±1.7-fold change in relative abundance, p<0.001) were identified for both cell lines. Most significant changes were found for keratins, major components of the cytoskeleton system, and for Mx proteins, interferon-induced key components of the host cell defense. Time series analysis of infection processes allowed the identification of further proteins that are described to be involved in protein synthesis, signal transduction and apoptosis events. Most likely, these proteins are required for supporting functions during influenza viral life cycle or host cell stress response. Quantitative proteome-wide profiling of virus infection can provide insights into complexity and dynamics of virus-host cell interactions and may accelerate antiviral research and support optimization of vaccine manufacturing processes.

The peptide-based quantitation accuracy and precision of LC-ESI (QSTAR Elite) and LC-MALDI (4800 MALDI TOF/TOF) were compared by analyzing identical Escherichia coli tryptic digests containing iTRAQ-labeled peptides of defined abundances (1:1, 2.5:1, 5:1, and 10:1). Only 51.4% of QSTAR spectra were used for quantitation by ProteinPilot Software versus 66.7% of LC-MALDI spectra. The average protein sequence coverages for LC-ESI and LC-MALDI were 24.0 and 18.2% (14.9 and 8.4 peptides per protein), respectively. The iTRAQ-based expression ratios determined by ProteinPilot from the 57 467 ESI-MS/MS and 26 085 MALDI-MS/MS spectra were analyzed for measurement accuracy and reproducibility. When the relative abundances of peptides within a sample were increased from 1:1 to 10:1, the mean ratios calculated on both instruments differed by only 0.7-6.7% between platforms. In the 10:1 experiment, up to 64.7% of iTRAQ ratios from LC-ESI MS/MS spectra failed S/N thresholds and were excluded from quantitation, while only 0.1% of the equivalent LC-MALDI iTRAQ ratios were rejected. Re-analysis of an archived LC-MALDI sample set stored for 5 months generated 3715 MS/MS spectra for quantitation, compared with 3845 acquired originally, and the average ratios differed by only 3.1%. Overall, MS/MS-based peptide quantitation performance of offline LC-MALDI was comparable with on-line LC-ESI, which required threefold less time. However, offline LC-MALDI allows the re-analysis of archived HPLC-separated samples.

Annotation of the human serum N-linked glycome is a formidable challenge but is necessary for disease marker discovery. A new theoretical glycan library was constructed and proposed to provide all possible glycan compositions in serum. It was developed based on established glycobiology and retrosynthetic state-transition networks. We find that at least 331 compositions are possible in the serum N-linked glycome. By pairing the theoretical glycan mass library with a high mass accuracy and high-resolution MS, human serum glycans were effectively profiled. Correct isotopic envelope deconvolution to monoisotopic masses and the high mass accuracy instruments drastically reduced the amount of false composition assignments. The high throughput capacity enabled by this library permitted the rapid glycan profiling of large control populations. With the use of the library, a human serum glycan mass profile was developed from 46 healthy individuals. This paper presents a theoretical N-linked glycan mass library that was used for accurate high-throughput human serum glycan profiling. Rapid methods for evaluating a patient's glycome are instrumental for studying glycan-based markers.

In eukaryotes, 14-3-3 dimers regulate hundreds of functionally diverse proteins (clients), typically in phosphorylation-dependent interactions. To uncover new clients, 14-3-3 omega (At1g78300) from Arabidopsis was engineered with a "tandem affinity purification" tag and expressed in transgenic plants. Purified complexes were analyzed by tandem MS. Results indicate that 14-3-3 omega can dimerize with at least 10 of the 12 14-3-3 isoforms expressed in Arabidopsis. The identification here of 121 putative clients provides support for in vivo 14-3-3 interactions with a diverse array of proteins, including those involved in: (i) Ion transport, such as a K+ channel (GORK), a Cl- channel (CLCg), Ca2+ channels belonging to the glutamate receptor family (1.2, 2.1, 2.9, 3.4, 3.7); (ii) hormone signaling, such as ACC synthase (isoforms ACS-6, -7 and -8 involved in ethylene synthesis) and the brassinolide receptors BRI1 and BAK1; (iii) transcription, such as 7 WRKY family transcription factors; (iv) metabolism, such as phosphoenol pyruvate carboxylase; and (v) lipid signaling, such as phospholipase D ([beta] and [gamma]). More than 80% (101) of these putative clients represent previously unidentified 14-3-3 interactors. These results raise the number of putative 14-3-3 clients identified in plants to over 300.

Preeclampsia (PE) is a common, potentially life-threatening pregnancy syndrome triggered by placental factors released into the maternal circulation, resulting in maternal vascular dysfunction along with activated inflammation and coagulation. Currently there is no screening test for PE. We sought to identify differentially expressed plasma proteins in women who subsequently develop PE that may perform as predictive biomarkers. In seven DIGE experiments, we compared the plasma proteome at 20 wk gestation in women who later developed PE with an appropriate birth weight for gestational age baby (n=27) or a small for gestational age baby (n=12) to healthy controls with uncomplicated pregnancies (n=57). Of the 49 differentially expressed spots associated with PE-appropriate for gestational age, PE-small for gestational age or both (p<0.05, false discovery rate corrected), 39 were identified by LC-MS/MS. Two protein clusters that accurately (>90%) classified women at risk of developing PE were identified. Immunoblots confirmed the overexpression of fibrinogen [gamma] chain and [alpha]-1-antichymotrypsin in plasma prior to PE. The proteins identified are involved in lipid metabolism, coagulation, complement regulation, extracellular matrix remodeling, protease inhibitor activity and acute-phase responses, indicating novel synergism between pathways involved in the pathogenesis of PE. Our findings are remarkably similar to recently identified proteins complexed to high-density lipoprotein and linked to cardiovascular disease.

Imaging MS is a powerful technique that combines the chemical and spatial analysis of surfaces. It allows spatial localization of multiple different compounds that are recorded in parallel without the need of a label. It is currently one of the rapidly developing techniques in the proteomics toolbox. Different complementary imaging MS methods, i.e. MALDI and secondary ion MS imaging for direct tissue analysis, can be applied on exactly the same tissue sample. This allows the identification of small molecules, peptides and proteins present on the same sample surface. Sample preparation is crucial to obtain high quality, reliable and reproducible complementary molecular images. It is essential to optimize the conditions for each step in the sample preparation protocol, ranging from sample collection and storage to surface modification. In this article, we review and discuss the importance of correct sample treatment in case of MALDI and secondary ion MS imaging experiments and describe the experimental requirements for optimal sample preparation.

Numerous protocols for isolation of mitochondria are available. Here, three methods for the isolation of intact mitochondria from mouse liver tissues are compared with regard to yield, purity and activity. Mitochondria were isolated by sucrose density gradient ultracentrifugation, free-flow electrophoresis or a commercially available kit-based method. Our analyses show that the sophisticated (and most expensive) free-flow electrophoresis method enables isolation of intact mitochondria with an enrichment of approximately 70%. Using the classical density centrifugation method is very laborious and time-consuming, but delivers about 57% intact mitochondria. Using standard laboratory equipment in a quick and simple procedure, the kit provides approximately 50% intact mitochondria, suitable for most standard investigations.

Proteomics has been shown to significantly contribute to the investigation of the pathogenicity of the extremely infectious bacteria Francisella tularensis. In this study, the authors employed iTRAQ quantitative proteomic analysis in order to monitor alterations in proteomes of F. tularensis ssp. holarctica live vaccine strain and F. tularensis ssp. tularensis SCHU S4 associated with the cultivation at different temperatures or in the stationary phase. Correlated production of the identified proteins studied by the exploratory statistical analysis revealed novel candidates for virulence factors that were regulated in a similar manner to the genes encoded in the Francisella Pathogenicity Island. Moreover, the assessment of the adaptation of live vaccine strain and SCHU S4 strain to the examined stimuli uncovered differences in their physiological responses to the stationary phase of growth.