Ammonium sulfate-based prefractionation improved proteome coverage and detection of carbonylated proteins in Arabidopsis thaliana leaf extract.

MAIN CONCLUSION: Ammonium sulfate is well known to salt out proteins at high concentrations. The study revealed that it can serve to increase by 60% the total number of identified carbonylated proteins by LC-MS/MS. Protein carbonylation is a significant post-translational modification associated with reactive oxygen species signaling in animal and plant cells. However, the detection of carbonylated proteins involved in signaling is still challenging, as they only represent a small subset of the proteome in the absence of stress. In this study, we investigated the hypothesis that a prefractionation step with ammonium sulphate will improve the detection of the carbonylated proteins in a plant extract. For this, we extracted total protein from the Arabidopsis thaliana leaves and subjected the extract to stepwise precipitation with ammonium sulfate to 40%, 60%, and 80% saturation. The protein fractions were then analyzed by liquid chromatography-tandem mass spectrometry for protein identification. We found that all the proteins identified in the non-fractionated samples were also found in the prefractionated samples, indicating no loss was incurred during the prefractionation. About 45% more proteins were identified in the fractionated samples compared to the non-fractionated total crude extract. When the prefractionation steps were combined with the enrichment of carbonylated proteins labeled with a fluorescent hydrazide probe, several carbonylated proteins, which were unseen in the non-fractionated samples, became visible in the prefractionated samples. Consistently, the prefractionation method allowed to identify 63% more carbonylated proteins by mass spectrometry compared to the number of carbonylated proteins identified from the total crude extract without prefractionation. These results indicated that the ammonium sulfate-based proteome prefractionation can be used to improve proteome coverage and identification of carbonylated proteins from a complex proteome sample.

Proteome-wide analysis of hydrogen peroxide-induced protein carbonylation in Arabidopsis thaliana.

Introduction: Protein carbonylation is a non-enzymatic and irreversible post-translational modification that occurs naturally in living organisms under the direct or indirect effect of reactive oxygen species (ROS). In animals, signaling pathways involving numerous carbonylated proteins have been identified, highlighting the dual role of these molecules in ROS signal transduction. In plants, studies on phytohormone signaling (auxin, methyl jasmonate, abscisic acid) have shown that reactive carbonyl species (RCS: acrolein, malondialdehyde, 4-hydroxynonenal, etc.), derived from the action of ROS on lipids, play important roles in secondary root formation and stomatal closure. However, the carbonylated proteins involved in these signaling pathways remain to be identified. Methods: In this study, we analyzed proteins responsive to carbonylation by exogenous hydrogen peroxide (H2O2) by profiling the carbonyl proteome extracted from Arabidopsis thaliana leaves after H2O2 treatment. Carbonylated proteins were enriched at the peptide level and analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Results and discussion: We identified 35 and 39 uniquely carbonylated proteins in the untreated and the H2O2-treated plant samples, respectively. In comparison to the control treatment, gene ontology enrichment analysis revealed that most of the carbonylated proteins identified in the H2O2-treated plant samples are related to sulfate adenylyl transferases and amidophosphoribosyl transferases involved in the immune system response, defense response, and external stimulus-response. These results indicated that exogenous H2O2 caused a change in the pattern of protein carbonylation in A. thaliana leaves. Protein carbonylation may thus influence the plant transcriptome and metabolism in response to H2O2 and ROS-triggering external stimuli.

Fabrication of label-free and eco-friendly ROS optical sensor with potent antioxidant properties for sensitive hydrogen peroxide detection in human plasma.

Herein, biogenic silver nanoparticles, Cafi-AgNPs was produced based on Cassia fistula-phenolic-rich extract (Cafi) only, without any toxic chemical reagent or organic solvent. Cafi bioactives were characterized using UHPLC-ESI-QTOF-MS/MS analysis. The as-synthesized nanoparticles were characterized using physico-chemical techniques including UV-vis, TEM, SEM, EDX, FTIR, DLS, Zeta potential, XRD, TGA and DGA. In addition, their antioxidant properties and cytocompatibility on erythrocytes and HEK-293 cells were examined. Results show that Cafi mediated the successful synthesis of stable well-dispersed AgNPs. Cafi-AgNPs demonstrated potent reducing and radical scavenging activities against ABTS˙+, DPPH˙ and NO˙. Furthermore, Cafi-AgNPs was compatible with human erythrocytes and HEK-293 cells. Based on the superior surface plasmonic and biological attributes of Cafi-AgNPs, its potential in H2O2 sensing was evaluated. The proposed sensor demonstrated satisfactory analytical performances with linearity of 10-200 µM, detection limit of 3.0 µM for H2O2, and was successfully applied in the detection of H2O2 in human plasma.

Protein glycation and oxidation inhibitory activity of Centella asiatica phenolics (CAP) in glucose-mediated bovine serum albumin glycoxidation.

The study aimed to evaluate the inhibitory effects of Centella asiatica phenolics (CAP) on bovine serum albumin glycoxidation in a BSA-glucose model in vitro. The impact of the phenolic extract on the formation of total fluorescent advanced glycation end products (AGEs) and Amadori adducts were determined. Dityrosine, N-formylkynurenine, kynurenine and protein-carbonyls were quantified as markers of protein oxidation. Protein structural perturbations were determined by Congo red binding and FTIR analysis. Chemical characterization and CAP phytoconstituent profile was obtained by colorimetric and UHPLC-ESI-qTOF-MS analysis, respectively. Our data show that CAP attenuated the formation of fluorescent AGEs (38.5%), Dityrosine (44.6%), N-formylkynurenine (42.9%), Amadori products, and resisted structural alterations of BSA subjected to glycation. These effects could be due to the antioxidant and radical scavenging activities of CAP mediated by the presence of phenolics and triterpenoids. The results collectively suggest that CAP possesses antiglycative properties with potentials for nutraceutical applications.

The catalytic kinetics of chicken transthyretin towards human Aß1-42.

The novel property of transthyretin (TTR) as a protease has been proposed to be significant. However, the study of TTR proteolysis properties has not been completely elucidated. Herein, we first report the catalytic activity of chicken TTR from plasma determined by using fluorescently labeled amyloid beta 1-42 peptide (Aß1-42), and compared it with human TTR (human TTR) from plasma and recombinant Crocodylus porosus TTR. The enzyme kinetic study revealed that the affinity for Aß1-42 of chicken TTR and C. porosus TTR (KM values were 12.72 ±â€¯0.27 µM and 16.21 ±â€¯0.02 µM, respectively) were significantly lower than human TTR (KM was 43.05 ±â€¯0.39 µM). In addition, the catalytic efficiency of chicken TTR (Kcat/KM was 310,386.87 ±â€¯13,627.12 M-1 s-1) was 4.3 and 5.5 folds higher than those of C. porosus TTR and human TTR (Kcat/KM were 72,893.80 ±â€¯355.74 M-1 s-1 and 56,519.12 ±â€¯5009.50 M-1 s-1, respectively), respectively. These results does not only indicated the relationship between structure and the proteolytic activity of TTR, but also suggested a potential development of TTR as a therapeutic anti-Aß agent.

Centella asiatica phenolic extract-mediated bio-fabrication of silver nanoparticles: characterization, reduction of industrially relevant dyes in water and antimicrobial activities against foodborne pathogens.

In this article, we have reported an environmentally benign and cost-effective method for the synthesis of monodispersed silver nanoparticles (AgNPs), based on Centella asiatica phenolic extracts (CAPE). The presence of phenolics was confirmed by ultra high-performance liquid chromatography coupled with electrospray ionization quadrupole time of flight mass spectrometry (UHPLC-ESI-qTOF-MS). Colloidal AgNPs synthesized under different concentrations of silver nitrate were monitored with a UV-vis spectrophotometer. Maximum absorption spectra intensity was found to range between 430-440 nm, during a synthesis time of 90 minutes at room temperature. The as-synthesized CAPE-AgNPs, was subjected to various instrumental characterizations such as, transmission electron microscopy (TEM), X-ray powder diffraction (XRD), energy dispersive X-ray spectroscopy (EDS), Fourier transform infrared (FTIR) spectroscopy, dynamic light scattering (DLS) and zeta potential. At the optimized synthesis conditions, spherical and monodispersed CAPE-AgNPs were obtained, with an absorption maximum at 430 nm. The crystalline CAPE-AgNPs had a face-centered-cubic (fcc) crystallographic structure, possessing average sizes estimated from TEM, to be between 20-25 nm diameter, a hydrodynamic diameter from DLS of about 90 nm and a zeta potential value of -28.7 mV. FTIR results validated the presence of phenolics on the surfaces of CAPE-AgNPs. The anti-microbial capacity of CAPE-AgNPs was further demonstrated on different pathogenic bacterial strains with satisfactory performances. As a result of the high surface area to volume ratio of CAPE-AgNPs, it was investigated as a catalyst towards the reduction of prominent environmental pollutants, 4 nitrophenol (4 NP), Congo red (CR) and methylene blue (MB). Pseudo first order kinetics were obtained with rate constants of 3.9 × 10-3 s-1 for 4 NP, 54.7 × 10-3 min-1 for MB and 5.6 × 10-3 s-1 for CR. The catalytic performance and antimicrobial activities of CAPE-AgNPs suggest its potential application in wastewater treatment and control of pathogenic microbes.