Proteins from Rhinella jimi parotoid gland secretion: A comprehensive analytical approach.

Toad skin secretions are sources of complex mixtures of bioactive compounds, such as proteins and peptides. Rhinella jimi species is a common toad in the Brazilian northeast, considered by only a few known studies. The experimental design was applied to optimize the protein extraction method from R. jimi parotoid gland secretions. The optimum condition was using 100 mmol L-1 Tris-HCl buffer pH 7.2 under vortexing for 5 min. The FTIR analysis combined with PCA revealed high-protein purity of the extracts, confirming the success of the proposed extraction method. The total protein concentration by the Bradford method was 102.4 and 66.5 mg g-1 on toad poisons from Teresina and Picos, respectively. The comparative proteomic analysis using HPLC-SEC-DAD and 1D SDS-PAGE revealed significant differences in protein abundance. HMW biomolecules showed greater abundance in toads from Teresina, while LMW protein species were more abundant in toads from Picos. The significant difference in amphibian proteome can be attributed to the edaphoclimatic conditions of their habitat. The cytotoxicity of the protein extract from Teresina was higher on the tumor cell lines 4T1 and CT26.WT. These new findings are fundamental for future studies the on identity and biological activity of biomolecules from this noble sample.

Expanding information on the bioaccessibility and bioavailability of iron and zinc in biofortified cowpea seeds.

This work presents new findings on the nutritional quality of recently introduced biofortified and non-biofortified cowpea cultivars as well as some common beans. ICP-MS was used for the measurements. Biofortified cowpea cultivars showed high levels of Fe and Zn, greater than 60 and 40 mg kg-1 dry weight, respectively. The in vitro digestion protocol enabled simultaneous evaluation of bioaccessibility and bioavailability. Fe levels in cowpea cultivars were ca. 2.5-fold higher than in common beans. Cowpea seeds also had higher Zn levels, reaching 50.1% bioaccessibility and 44.2% bioavailability. Cooking improved the availability of micronutrients in bean seeds. The cooked biofortified Aracê cowpea showed a high Zn bioavailability above 60%. Consumption of 50 g of Aracê would contribute 27% and 48% of the Fe and Zn DRI for 1-3-year-old children. The new cowpea cultivars biofortified are a potential vehicle for improving the Fe and Zn status in groups in which the micronutrient deficiency is prevalent.

Comparative study of transgenic and non-transgenic maize (Zea mays) flours commercialized in Brazil, focussing on proteomic analyses.

Genetically modified foods are a major concern around the world due to the lack of information concerning their safety and health effects. This work evaluates differences, at the proteomic level, between two types of crop samples: transgenic (MON810 event with the Cry1Ab gene, which confers resistance to insects) and non-transgenic maize flour commercialized in Brazil. The 2-D DIGE technique revealed 99 differentially expressed spots, which were collected in 2-D PAGE gels and identified via mass spectrometry (nESI-QTOF MS/MS). The abundance of protein differences between the transgenic and non-transgenic samples could arise from genetic modification or as a result of an environmental influence pertaining to the commercial sample. The major functional category of proteins identified was related to disease/defense and, although differences were observed between samples, no toxins or allergenic proteins were found.

Metal ions bound to the human milk immunoglobulin A: metalloproteomic approach.

The presence of calcium, iron, and zinc bound to human milk secretory IgA (sIgA) was investigated. The sIgA components were first separated by two-dimensional polyacrylamide gel electrophoresis and then identified by electrospray ionization-tandem mass spectrometry (ESI MS MS). The metal ions were detected by flame atomic absorption spectrometry after acid mineralization of the spots. The results showed eight protein spots corresponding to the IgA heavy chain constant region. Another spot was identified as the transmembrane secretory component. Calcium was bound to both the transmembrane component and the heavy chain constant region, while zinc was bound to the heavy chain constant region and iron was not bound with the identified proteins. The association of a metal ion with a protein is important for a number of reasons, and therefore, the findings of the present study may lead to a better understanding of the mechanisms of action and of additional roles that sIgA and its components play in human milk.

Comparative proteomics and metallomics studies in Arabidopsis thaliana leaf tissues: evaluation of the selenium addition in transgenic and nontransgenic plants using two-dimensional difference gel electrophoresis and laser ablation imaging.

The main goal of this work is to evaluate some differential protein species in transgenic (T) and nontransgenic (NT) Arabidopsis thaliana plants after their cultivation in the presence or absence of sodium selenite. The transgenic line was obtained through insertion of CaMV 35S controlling nptII gene. Comparative proteomics through 2D-DIGE is carried out in four different groups (NT × T; NT × Se-NT (where Se is selenium); Se-NT × Se-T, and T × Se-T). Although no differential proteins are achieved in the T × Se-T group, for the others, 68 differential proteins (by applying a regulation factor ≥1.5) are achieved, and 27 of them accurately characterized by ESI-MS/MS. These proteins are classified into metabolism, energy, signal transduction, disease/defense categories, and some of them are involved in the glycolysis pathway-Photosystems I and II and ROS combat. Additionally, laser ablation imaging is used for evaluating the Se and sulfur distribution in leaves of different groups, corroborating some results obtained and related to proteins involved in the glycolysis pathway. From these results, it is possible to conclude that the genetic modification also confers to the plant resistance to oxidative stress.

Comparative studies focusing on transgenic through cp4EPSPS gene and non-transgenic soybean plants: an analysis of protein species and enzymes.

This work evaluates the activity of a few key enzymes involved in combating reactive oxygen species (ROS), such as ascorbate peroxidase (EC 1.11.1.11), catalase (EC 1.11.1.6), glutathione reductase (EC 1.6.4.2), and superoxide dismutase (EC 1.15.1.1), as well as the concentration of malondialdehyde and hydrogen peroxide in transgenic and non-transgenic soybean leaves. Additionally, differential protein species from leaves of both genotypes were evaluated by applying a regulation factor of ≥1.8 to further corroborate the hypothesis that genetic modification itself can be a stress factor for these plants. For this task, transgenic soybean plants were obtained from seeds modified with the cp4EPSPS gene. The results revealed higher activities of all evaluated enzymes in transgenic than in non-transgenic soybean leaves (ranging from 13.8 to 70.1%), as well as higher concentrations of malondialdehyde and hydrogen peroxide in transgenic soybean leaves, clearly indicating a condition of oxidative stress established in the transgenic genotype. Additionally, 47 proteins were differentially abundant when comparing the leaves of both plants, with 26 species accurately identified, including the protein involved in the genetic modification (CP4EPSPS). From these results, it is possible to conclude that the plant is searching for a new equilibrium to maintain its metabolism because the stress condition is being maintained within levels that can be tolerated by the plant. BIOLOGICAL SIGNIFICANCE: The present paper is the first one in the literature where are shown translational aspects involving plant stress and the genetic modification for soybean involving the cp4 EPSPS gene. The main biological importance of this work is to make possible the demystification of the genetic modification, allowing answers for some questions that still remain unknown, and enlarge our knowledge about genetically modified organisms. This article is part of a Special Issue entitled: Translational Plant Proteomics.

Iron insertion and hematite segregation on Fe-doped TiO2 nanoparticles obtained from sol-gel and hydrothermal methods.

Iron-doped TiO(2) (Fe:TiO(2)) nanoparticles were synthesized by the sol-gel method (with Fe/Ti molar ratio corresponding to 1, 3, and 5%), followed by hydrothermal treatment, drying, and annealing. A similar methodology was used to synthesize TiO(2) and α-Fe(2)O(3) nanoparticles. For comparison, a mixture hematite/titania, with Fe/Ti = 4% was also investigated. Characterization of the samples using Rietveld refinement of X-ray diffraction data revealed that TiO(2) consisted of 82% anatase and 18% brookite; for Fe:TiO(2), brookite increased to 30% and hematite was also identified (0.5, 1.0, and 1.2 wt % for samples prepared with 1, 3, and 5% of Fe/Ti). For hematite/titania mixture, Fe/Ti was estimated as 4.4%, indicating the Rietveld method reliability for estimation of phase composition. Because the band gap energy, estimated as 3.2 eV for TiO(2), gradually ranged from 3.0 to 2.7 eV with increasing Fe content at Fe:TiO(2), it can be assumed that a Fe fraction was also inserted as dopant in the TiO(2) lattice. Extended X-ray absorption fine structure spectra obtained for the Ti K-edge and Fe K-edge indicated that absorbing Fe occupied a Ti site in the TiO(2) lattice, but hematite features were not observed. Hematite particles also could not be identified in the images obtained by transmission electron microscopy, in spite of iron identification by elemental mapping, suggesting that hematite can be segregated at the grain boundaries of Fe:TiO(2).

New insights on proteomics of transgenic soybean seeds: evaluation of differential expressions of enzymes and proteins.

This work reports the evaluation of differentially expressed enzymes and proteins from transgenic and nontransgenic soybean seeds. Analysis of malondialdehyde, ascorbate peroxidase (EC 1.11.1.11), glutathione reductase (EC 1.6.4.2), and catalase (EC 1.11.1.6) revealed higher levels (29.8, 30.6, 71.4, and 35.3%, respectively) in transgenic seeds than in nontransgenic seeds. Separation of soybean seed proteins was done by two-dimensional polyacrylamide gel electrophoresis, and 192 proteins were identified by matrix-assisted laser desorption/ionization (MALDI) quadrupole time-of-flight (QTOF) mass spectrometry (MS) and electrospray ionization (ESI) QTOF MS. Additionally, the enzyme CP4 EPSPS, involved in the genetic modification, was identified by enzymatic digestions using either trypsin or chymotrypsin and ESI-QTOF MS/MS for identification. From the proteins identified, actin fragment, cytosolic glutamine synthetase, glycinin subunit G1, and glycine-rich RNA-binding protein were shown to be differentially expressed after analysis using the two-dimensional difference gel electrophoresis technique, and applying a regulator factor of 1.5 or greater.

Two-dimensional difference gel electrophoresis applied for analytical proteomics: fundamentals and applications to the study of plant proteomics.

The present review reports the principles, fundamentals and some applications of two-dimensional difference gel electrophoresis for analytical proteomics based on plant proteome analysis, also emphasizing some advantages of 2-D DIGE over 2-D PAGE techniques. Some fluorescent protein labeling reagents, methods of protein labeling, models of 2-D DIGE experiments, and some limitations of this technique are presented and discussed in terms of 2-D DIGE plant proteomes. Finally, some practical applications of this technique are pointed out, emphasizing its potentialities in plant proteomics.