Circulating myeloid calcifying cells have antiangiogenic activity via thrombospondin-1 overexpression.

Myeloid calcifying cells (MCCs) represent a subpopulation of human monocytes with procalcific potential and are characterized by coexpression of osteocalcin (OC) and bone alkaline phosphatase (BAP). Herein, an in-depth proteomic investigation of MCCs based on fluorescence-activated cell sorting, protein extraction and digestion, isobaric tag for relative and absolute quantitation labeling, fractionation, and analysis on matrix-assisted laser desorption/ionization-time of flight/time of flight and LTQ Orbitrap mass spectrometers identified and quantified more than 700 proteins and revealed pathways activated in OC(+)BAP(+) MCCs compared with those in OC(-)BAP(-) cells. Among proteins referable to angiogenesis, the thrombospondin-1 pathway was markedly up-regulated in MCCs vs. control cells. Up-regulation of the thrombospondin-1 pathway was confirmed by a genome-wide transcriptional analysis. Using in vitro and in vivo angiogenesis assays, we found that freshly isolated MCCs and cultured MCCs display an antiangiogenic function by means of both paracrine activity (conditioned medium) and altered spatial localization in cocultures with endothelial cells. Thrombospondin-1 inhibition by antibody-mediated neutralization or gene knockdown restored the angiogenic activity of OC(+)BAP(+) MCCs toward normal values and abolished the antiangiogenic effects of MCC conditioned medium. These data indicate that circulating MCCs exert antiangiogenic activity by virtue of their overexpression of thrombospondin-1. The study highlights the successful identification and validation of a pathogenic pathway by a gold standard proteomic/transcriptomic analysis of blood cells.

Biochemical and quantitative proteomics investigations in Arabidopsis ggt1 mutant leaves reveal a role for the gamma-glutamyl cycle in plant's adaptation to environment.

The existence of a gamma-glutamyl cycle consisting of intracellular GSH synthesis, extrusion to the apoplastic space and recovery by gamma-glutamyl transferase (GGT)-assisted degradation into its constituent amino acids, has been demonstrated in plants. To address the significance of this cycle in plant cells, we performed integrated biochemical, immunocytochemical, and quantitative proteomics analyses in the Arabidopsis thaliana ggt1 knockout mutant (lacking apoplastic GGT1 isoform) and its corresponding wild-type (WT). The ggt1 knockout leaves exhibited an increased ascorbate and GSH content, increased apoplastic GSH content, and enhanced protein carbonylations in the low-molecular weight range compared to WT. The combined iTRAQ and LC-MS/MS-based quantitative proteomics approach identified 70 proteins (out of 1013 identified proteins) whose abundance was significantly different in leaves of ggt1 mutant compared to WT, with a fold change ≥1.5. Mining of the proteome data for GSH-associated genes showed that disruption of gamma-glutamyl cycle in ggt1 knockout-leaves was associated with the induction of genes encoding four GSTs in the phi class (GSTF2, GSTF6, GSTF9, and GSTF10), a GSH peroxidase (GPX1), and glyoxylase II. Proteins with a lower abundance compared to the WT are involved in chloroplast functions, carbohydrate/maltose metabolism, and vegetative storage protein synthesis. Present findings suggest that GGT1 plays a role in redox signaling. The disruption of the gamma-glutamyl cycle in the ggt1 mutant results in pleiotropic effects related to biotic and abiotic stress response, antioxidant metabolism, senescence, carbohydrate metabolism, and photosynthesis, with strong implications for plant adaptation to the environment.

Quantitative analysis of the naringenin-inducible proteome in Rhizobium leguminosarum by isobaric tagging and mass spectrometry.

The rhizobium-legume interaction is a critical cornerstone of crop productivity and environmental sustainability. Its potential improvement relies on elucidation of the complex molecular dialogue between its two partners. In the present study, the proteomic patterns of gnotobiotic cultures of Rhizobium leguminosarum bv. viciae 3841 grown for 6 h in presence or absence of the nod gene-inducing plant flavonoid naringenin (10 µM) were analyzed using the iTRAQ approach. A total of 1334 proteins were identified corresponding to 18.67% of the protein-coding genes annotated in the sequenced genome of bv. viciae 3841. The abundance levels of 47 proteins were increased upon naringenin treatment showing fold change ratios ranging from 1.5 to 25 in two biological replicates. Besides the nod units, naringenin enhanced the expression of a number of other genes, many of which organized in operons, including ß(1-2) glucan production and secretion, succinoglycan export, the RopA outer membrane protein with homology to an oligogalacturonide-specific porin motif, other enzymes for carbohydrate and amino acid metabolism, and proteins involved in the translation machinery. Data were validated at the transcriptional and phenotypic levels by RT-PCR and an assay of secreted sugars in culture supernatants, respectively. The current approach provides not only a high-resolution analysis of the prokaryotic proteome but also unravels the rhizobium molecular dialogue with legumes by detecting the enhanced expression of several symbiosis-associated proteins, whose flavonoid-dependency had not yet been reported.

Calcium-dependent regulation of genes for plant nodulation in Rhizobium leguminosarum detected by iTRAQ quantitative proteomic analysis.

Rhizobia, the nitrogen-fixing bacterial symbionts of legumes, represent an agricultural application of primary relevance and a model of plant-microbe molecular dialogues. We recently described rhizobium proteome alterations induced by plant flavonoids using iTRAQ. Herein, we further extend that experimentation, proving that the transient elevation in cytosolic calcium is a key signaling event necessary for the expression of the nodulation (nod) genes. Ca(2+) involvement in nodulation is a novel issue that we recently flagged with genetic and physiological approaches and that hereby we demonstrate also by proteomics. Exploiting the multiple combinations of 4-plex iTRAQ, we analyzed Rhizobium leguminosarum cultures grown with or without the nod gene-inducing plant flavonoid naringenin and in the presence or absence of the extracellular Ca(2+) chelator EGTA. We quantified over a thousand proteins, 189 of which significantly altered upon naringenin and/or EGTA stimulation. The expression of NodA, highly induced by naringenin, is strongly reduced when calcium availability is limited by EGTA. This confirms, from a proteomic perspective, that a Ca(2+) influx is a necessary early step in flavonoid-mediated legume nodulation by rhizobia. We also observed other proteins affected by the different treatments, whose identities and roles in nodulation and rhizobium physiology are likewise discussed.

A note on protein expression changes in chicken breast muscle in response to time in transit before slaughtering.

Aims of the research were to devise a proteome map of the chicken Pectoralis superficialis muscle, as resolved by two-dimensional gel electrophoresis, and to characterize protein expression changes in the soluble protein fraction in commercial conditions due to age and to time in transit before slaughtering. Broilers were reared under commercial conditions until they reached a mean 1.8 kg and 36 d, or 2.6 kg and 46 d of age. Transport to the slaughterhouse took 90 or 220 minutes. Transport-induced stress was assessed from blood metabolites and leukocyte cell counts, revealing significant changes in albumin, glucose and triglyceride concentrations, in heterophils and leukocyte counts for chickens in transit for longer, and in glucose depending mainly on age. The sarcoplasmic protein fractions were extracted from a total of 39 breast muscle samples, collected 15 min post mortem, for analysis by two-dimensional electrophoresis. Image and statistical analyses enabled us to study the qualitative and quantitative differences between the samples. Twelve up- or down-regulated protein spots were detected (P < 0.05): 8 related to the age effect, 2 to time in transit, and 2 to the interaction between the two. Age and time in transit influenced the avian proteome regulating the biological processes linked to the cellular housekeeping functions, related mainly to metabolism, cell division and control of apoptosis. Principal component analysis clustering was used to assess differences between birds. Age difference discriminated between the chickens analyzed better than time in transit, which seemed to have less general impact on the proteome fraction considered here. Isolating and identifying the proteins whose expression changes in response to transport duration and age shed some light on the biological mechanisms underlying growth and stress-related metabolism in chickens. Our results, combined with a further characterization of the chicken proteome associated with commercial chicken slaughtering management, will hopefully inspire alternative strategies and policies, and action to reduce the impact of stress related to time in transit.

High confidence and sensitivity four-dimensional fractionation for human plasma proteome analysis.

Reducing the complexity of plasma proteome through complex multidimensional fractionation protocols is critical for the detection of low abundance proteins that have the potential to be the most specific disease biomarkers. Therefore, we examined a four dimension profiling method, which includes low abundance protein enrichment, tryptic digestion and peptide fractionation by IEF, SCX and RP-LC. The application of peptide pI filtering as an additional criterion for the validation of the identifications allows to minimize the false discovery rate and to optimize the best settings of the protein identification database search engine. This sequential approach allows for the identification of low abundance proteins, such as angiogenin (10(-9) g/L), pigment epithelium growth factor (10(-8) g/L), hepatocyte growth factor activator (10(-7) g/L) and thrombospondin-1 (10(-6) g/L), having concentrations similar to those of many other growth factors and cytokines involved in disease pathophysiology.

The proteins of the grape (Vitis vinifera L.) seed endosperm: fractionation and identification of the major components.

In the present study, grape (Vitis vinifera L.) seed endosperm proteins were characterized after sequential fractionation, according to a modified Osborne procedure. The salt-soluble fraction (albumins and globulins) comprised the majority (58.4%) of the total extracted protein. The protein fractions analysed by SDS-PAGE showed similar bands, indicating different solubility of the same protein components. SDS-PAGE in non-reducing and reducing conditions revealed the polypeptide composition of the protein bands. The main polypeptides, which were similar in all the grape varieties analysed, were identified by LC-MS/MS as homologous to the 11S globulin-like seed storage proteins of other plant species, while a monomeric 43 kDa protein presented high homology with the 7S globulins of legume seeds. The results provide new insights about the identity, structure and polypeptide composition of the grape seed storage proteins.

Proteins and enzymatic activities in Erbaluce grape berries with different response to the withering process.

During the off-vine natural withering process of Erbaluce (white) grapes to obtain "Erbaluce Caluso" Passito wine, some berries change in color from green-yellow to blue. This phenomenon appears at different extents in different years and might be related to several parameters, such as temperature and humidity during withering, grape composition and Botrytis cinerea loading. To better understand the mechanism involved in color variation, the metabolic changes corresponding to this event were studied. At the end of the withering process berries with different colors were separated using a reflectance spectrophotometer, obtaining three color classes identified as "green" (L*=40.3, a*=-0.56, b*=15.20), "gold" (L*=37.7, a*=5.01, b*=14.12) and "blue" (L*=28.6, a*=0.89, b*=-0.67). The three groups of berries had different water contents, the blue berries containing about 30% less water than the green ones. Samples were crushed and the juices were analyzed. The juice yield for blue berries was less than 50% of that of the other two classes, confirming their higher dehydration level. Protein extraction from de-seeded berries was carried out using two different protocols, the first involving a treatment with phenol (to remove polyphenolic substances) and the second based on an extraction with a mild detergent (to recover the proteins to be used for enzymatic analyses). No trace of laccase activity was found in any of the samples, although DNA analysis, by quantitative PCR, suggested the presence of B. cinerea infection in the blue grapes. Chitinase activity of the blue berries was only 30% of that of the other two samples, as confirmed also by zymographic analysis on electrophoretic gels. The same was found also for esterase activity, which was lower (of about 85%) in the blue berries, which, in contrast, showed the highest beta-glucosidase activity. The electrophoretic analysis of the protein extracts revealed strong differences among the samples. Compared to the green and gold ones, the blue berries showed a complete absence of bands corresponding to chitinases and the appearance of unusual protein bands. Mass spectrometry analysis showed that these latter proteins were of grape origin, the main of them corresponding to ß-1,3-glucanase, although no significant increase in glucanase activity was measured in the corresponding protein extract.

Immunochemical and mass spectrometry detection of residual proteins in gluten fined red wine.

Recently, wheat gluten has been proposed as technological adjuvant in order to clarify wines. However, the possibility that residual gluten proteins remain in treated wines cannot be excluded, representing a hazard for wheat allergic or celiac disease patients. In this work, commercial wheat glutens, in both partially hydrolyzed (GBS-P51) and nonhydrolyzed (Gluvital 21000) forms, were used as fining agents in red wine at different concentrations. Beside immunoenzymatic analyses using anti-gliadin, anti-prolamin antibodies and pooled sera of wheat allergic patients, a method based on liquid chromatography coupled to mass spectrometry has been proposed to detect residues of gluten proteins. Residual gluten proteins were detected by anti-prolamin antibodies, anti-gliadin antibodies and sera-IgE only in the wine treated with GBS-P51 at concentration 50, 150, and 300 g/hL, respectively, whereas no residual proteins were detected by these systems in the wine treated with Gluvital 21000. In contrast liquid chromatography-mass spectrometry analyses allowed the detection of proteins in red wines fined down to 1 g/hL of Gluvital 21000 and GBS-P51. Our results indicate that MS methods are superior to immunochemical methods in detecting gluten proteins in wines and that adverse reactions against gluten treated wines cannot be excluded.

High abundance proteins depletion vs low abundance proteins enrichment: comparison of methods to reduce the plasma proteome complexity.

BACKGROUND: To date, the complexity of the plasma proteome exceeds the analytical capacity of conventional approaches to isolate lower abundance proteins that may prove to be informative biomarkers. Only complex multistep separation strategies have been able to detect a substantial number of low abundance proteins (<100 ng/ml). The first step of these protocols is generally the depletion of high abundance proteins by the use of immunoaffinity columns or, alternatively, the enrichment of by the use of solid phase hexapeptides ligand libraries. METHODOLOGY/PRINCIPAL FINDINGS: Here we present a direct comparison of these two approaches. Following either approach, the plasma sample was further fractionated by SCX chromatography and analyzed by RP-LC-MS/MS with a Q-TOF mass spectrometer. The depletion of the 20 most abundant plasma proteins allowed the identification of about 25% more proteins than those detectable following low abundance proteins enrichment. The two datasets are partially overlapping and the identified proteins belong to the same order of magnitude in terms of plasma concentration. CONCLUSIONS/SIGNIFICANCE: Our results show that the two approaches give complementary results. However, the enrichment of low abundance proteins has the great advantage of obtaining much larger amount of material that can be used for further fractionations and analyses and emerges also as a cheaper and technically simpler approach. Collectively, these data indicate that the enrichment approach seems more suitable as the first stage of a complex multi-step fractionation protocol.

The oleic acid complexes of proteolytic fragments of alpha-lactalbumin display apoptotic activity.

The complexes formed by partially folded human and bovine alpha-lactalbumin with oleic acid (OA) have been reported to display selective apoptotic activity against tumor cells. These complexes were named human (HAMLET) or bovine (BAMLET) alpha-lactalbumin made lethal to tumor cells. Here, we analyzed the OA complexes formed by fragments of bovine alpha-lactalbumin obtained by limited proteolysis of the protein. Specifically, the fragments investigated were 53-103 and the two-chain fragment species 1-40/53-123 and 1-40/104-123, these last being the N-terminal fragment 1-40 covalently linked via disulfide bridges to the C-terminal fragment 53-123 or 104-123. The OA complexes were obtained by mixing the fatty acid and the fragments in solution (10-fold and 15-fold molar excess of OA over protein fragment) or by chromatography of the fragments loaded onto an OA-conditioned anion exchange column and salt-induced elution of the OA complexes. Upon binding to OA, all fragments acquire an enhanced content of alpha-helical secondary structure. All OA complexes of the fragment species showed apoptotic activity for Jurkat tumor cells comparable to that displayed by the OA complex of the intact protein. We conclude that the entire sequence of the protein is not required to form an apoptotic OA complex, and we suggest that the apoptotic activity of a protein-OA complex does not imply specific binding of the protein.

Characterization of oligomeric species on the aggregation pathway of human lysozyme.

The aggregation process of wild-type human lysozyme at pH3.0 and 60 degrees C has been analyzed by characterizing a series of distinct species formed on the aggregation pathway, specifically the amyloidogenic monomeric precursor protein, the oligomeric soluble prefibrillar aggregates, and the mature fibrils. Particular attention has been focused on the analysis of the structural properties of the oligomeric species, since recent studies have shown that the oligomers formed by lysozyme prior to the appearance of mature amyloid fibrils are toxic to cells. Here, soluble oligomers of human lysozyme have been analyzed by a range of techniques including binding to fluorescent probes such as thioflavin T and 1-anilino-naphthalene-8-sulfonate, Fourier transform infrared spectroscopy, and controlled proteolysis. Oligomers were isolated after 5 days of incubation of the protein and appear as spherical particles with a diameter of 8-17 nm when observed by transmission electron microscopy. Unlike the monomeric protein, oligomers have solvent-exposed hydrophobic patches able to bind the fluorescent probe 1-anilino-naphthalene-8-sulfonate. Fourier transform infrared spectroscopy spectra of oligomers are indicative of misfolded species when compared to monomeric lysozyme, with a prevalence of random structure but with significant elements of the beta-sheet structure that is characteristic of the mature fibrils. Moreover, the oligomeric lysozyme aggregates were found to be more susceptible to proteolysis with pepsin than both the monomeric protein and the mature fibrils, indicating further their less organized structure. In summary, this study shows that the soluble lysozyme oligomers are locally unfolded species that are present at low concentration during the initial phases of aggregation. The nonnative conformational features of the lysozyme molecules of which they are composed are likely to be the factors that confer on them the ability to interact inappropriately with a variety of cellular components including membranes.