Rohana Liyanage

Ellagitannin Composition of Blackberry As Determined by HPLC-ESI-MS and MALDI-TOF-MS

Blackberries ( Rubus sp.) were evaluated by high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) to identify the ellagitannins present in flesh, torus (receptacle tissue), and seeds. Most ellagitannins were present (or detectable) only in seed tissues. Ellagitannins identified by HPLC-ESI-MS in the seeds included pedunculagin, casuarictin/potentillin, castalagin/vescalagin, lambertianin A/sanguiin H-6, lambertianin C, and lambertianin D. For several of the ellagitannins, isomeric separation was also obtained. The MALDI-TOF-MS analysis was primarily utilized to evaluate and identify high molecular mass (>1000 Da) ellagitannins. The MALDI analysis verified the presence of the ellagitannins identified by HPLC-ESI-MS including lambertianin A/sanguiin H-6, lambertianin C, and lambertianin D, but the analysis also indicated the presence of several other compounds that were most likely ellagitannins based on the patterns observed in the masses (i.e., loss or addition of a gallic acid moiety to a known ellagitannin). This study determined the presence of several possible isomeric forms of ellagitannins previously unidentified in fruit and presents a possible analytical HPLC method for the analysis of the major ellagitannins present in the fruit.

Human cancer cell proliferation inhibition by a pentapeptide isolated and characterized from rice bran

Food-derived bioactive peptides promote functional activity against diseases and present as nutraceutical agents. The purpose of our research was to isolate and fully characterize peptide(s) derived from rice bran having anti-cancer properties. Gastrointestinal juices resistant peptide fractions were initially generated from heat stabilized de-fatted rice bran from which <5 kDa fraction was shown to inhibit proliferation of cancer cells. Based on these published findings the <5 kDa peptide fraction was selected for further characterization to obtain single pure peptide(s) with enhanced anti-cancer properties. Purification and characterization from the fraction was done employing chromatography and mass spectrometric techniques. Cancer cell viability was measured using a cell titer assay that uses a tetrazolium dye [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; (MTS)] and the electron coupling reagent, phenazine methosulfate. Ion-exchange chromatography elutes that showed anti-cancer properties were further purified to liberate pure peptide. The pure peptide at 600-700 microg/mL dose caused 84% inhibition to colon cancer cells (Caco-2, HCT-116) growth, 80% to breast cancer cells (MCF-7, MDA-MB-231) growth and 84% to liver cancer cells (HepG-2) growth. Mass spectrometry analysis and de novo sequencing revealed the sequence of Glu-Gln-Arg-Pro-Arg for the peptide with a molecular mass of 685.378 Da. A novel pentapeptide was isolated from rice bran to possess cancer growth inhibitory properties on colon, breast, lung and liver cancer cells. This peptide could serve as a nutraceutical agent against cancer.

Processing and storage effect on berry polyphenols: challenges and implications for bioactive properties.

Anthocyanins and tannins in blueberries, blackberries and black raspberries are susceptible to degradation during processing, with juices showing the greatest losses due to physical removal of skins and seeds. Anthocyanins and procyanidins are also degraded in processed products stored at ambient temperature with losses accompanied by increased polymeric pigments (PPs). Using chokeberry as a model, formation of PPs occurred in both pasteurized and aged juices and pasteurized juice contained a greater proportion of low molecular weight PPs than aged juice, while aged juice contained a greater proportion of higher molecular PPs. Formation of PP accounts for some of the losses of anthocyanins and procyanidins during processing and storage, but the complete fate of anthocyanins remains unclear. In this review we highlight the steps in processing where significant losses of polyphenols occur, and discuss potential mechanisms responsible for losses, methods to mitigate losses, and implications on bioactive properties.

Activation of methane by size-selected iron cluster cations, Fen+ (n=2–15): Cluster-CHx (x=0–3) bond energies and reaction mechanisms

The kinetic energy dependences of the reactions of Fen+ (n=2–15) with CD4 are studied in a guided ion beam tandem mass spectrometer over the energy range of 0–10 eV. All reactions exhibit thresholds and two main products, FenD+ and FenCD2+, are formed. These primary products decompose at higher energies to form secondary and higher order products, FenCD+, FenC+, Fen−1D+, Fen−1CD2+, Fen−1CD+, and Fen−1C+. The cross-section magnitudes for the dehydrogenation products, FenCD2+, are observed to vary considerably as a function of cluster size; subsequent dehydrogenation to form FenC+ becomes more facile for larger clusters. Thresholds for the various primary and secondary reactions are analyzed and bond energies for iron cluster cation bonds to C, CD, CD2, and CD3 are determined. As a function of cluster size, these bond energies rapidly reach relatively constant values, which are argued to lie close to bulk phase values. The relative magnitudes in these bond energies are consistent with simple bond order cons...

Guided ion beam studies of the reaction of Nin + (n=2-16) with D2: Nickel cluster-deuteride bond energies

The kinetic-energy dependences of the reactions of Nin+ (n=2–16) with D2 are studied in a guided ion beam tandem mass spectrometer. The products observed are NinD+ for all clusters and NinD2+ for n=5–16. Reactions for formation of NinD+ are observed to exhibit thresholds, whereas cross sections for formation of NinD2+ (n=5–16) exhibit no obvious barriers to reaction. Rate constants of D2 chemisorption on the cationic clusters are compared with results from previous work on neutral nickel clusters. Nin+–D bond energies as a function of cluster size are derived from threshold analysis of the kinetic-energy dependence of the endothermic reactions, and are compared to previously determined metal–metal bond energies, D0(Nin+–Ni). The bond energies of Nin+–D generally increase as the cluster size increases, and parallel those for Nin+–Ni for many clusters. These trends are explained in terms of electronic and geometric structures for the Nin+ clusters. The bond energies of Nin+–D for larger clusters (n⩾11) are ...

Proteomic analysis of Salmonella enterica serovar Enteritidis following propionate adaptation

BackgroundSalmonella Enteritidis is a highly prevalent and persistent foodborne pathogen and is therefore a leading cause of nontyphoidal gastrointestinal disease worldwide. A variety of stresses are endured throughout its infection cycle, including high concentrations of propionate (PA) within food processing systems and within the gut of infected hosts. Prolonged PA exposure experienced in such milieus may have a drastic effect on the proteome of Salmonella Enteritidis subjected to this stress.ResultsIn this study, we used 2 D gel electrophoresis to examine the proteomes of PA adapted and unadapted S. Enteritidis and have identified five proteins that are upregulated in PA adapted cultures using standard peptide mass fingerprinting by MALDI-TOF-MS and sequencing by MALDI LIFT-TOF/TOF tandem mass spectrometry. Of these five, two significant stress-related proteins (Dps and CpxR) were shown (via qRT-PCR analysis) to be upregulated at the transcriptional level as well. Unlike the wild type when adapted to PA (which demonstrates significant acid resistance), PA adapted S. Enteritidis ∆dps and S. Enteritidis ∆cpxR were at a clear disadvantage when challenged to a highly acidic environment. However, we found the acid resistance to be fully restorable after genetic complementation.ConclusionsThis work reveals a significant difference in the proteomes of PA adapted and unadapted S. Enteritidis and affirms the contribution of Dps and CpxR in PA induced acid resistance.

Thermochemistry of small cationic iron–sulfur clusters

The kinetic energy dependences of the reactions of Fen+ with COS (n=2–6) and CS2 (n=2–5) are studied in a guided-ion beam tandem mass-spectrometer. The main products arise from sulfur transfer and subsequent losses of Fe atoms. In the case of CS2, this reactant also formally replaces one Fe atom of the cluster to form Fen−1CS2+ with losses of further Fe atoms at elevated energies. In addition, the kinetic energy dependences of the reactions of FenS+ (n=2–4) with Xe and CS2 are studied. The former system yields collision-induced dissociations, whereas the latter reagent effects sulfur transfer accompanied by subsequent losses of Fe atoms. Analyses of the cross sections for endothermic reactions yield the bond energies D0(Fen+–S), n=2–5, D0(SFen−1+–Fe), n=2–5, D0(SFen+−S), n=1–3, and D0(S2Fen−1+–Fe), n=2, 3, as well as the ionization energy IE(Fe2S2). These values are derived with explicit consideration of the lifetimes of the energized reaction intermediates. The binding between sulfur and the cluster core...

Thermodynamics of ammonia activation by iron cluster cations: Guided ion beam studies of the reactions of Fen + (n=2-10,14) with ND3

The kinetic energy dependences of the reactions of Fen+ (n=2–10,14) with ND3 are studied in a guided ion beam tandem mass spectrometer over the energy range of 0–10 eV. Dehydrogenation of ammonia to form FenND+ is found to be efficient and exothermic for n=4 in agreement with previous FT-ICR studies. In contrast to the ICR studies, we also observe exothermic dehydrogenation for n=3 and 5, although these processes are much less efficient than for n=4. Other clusters also undergo this process but exhibit an energy threshold. A multitude of other primary products are observed including Fen−1ND3+ (n=2–4,9,10), FenND2+ (n=1,4–8), and Fen−1ND2+ (n=2–5), which all have reaction efficiencies that depend on cluster size. At high energies, FenN+ and FenD+ are observed along with products corresponding to Fe atom loss from the primary products. Thresholds for the various primary and secondary reactions are analyzed and bond energies for iron cluster cations bound to N, ND, ND2, and ND3 are determined. Comparisons of...

Guided ion beam studies of the reactions of Vn+ (n=2–13) with D2: Cluster–deuteride bond energies as a chemical probe of cluster electronic structure

The kinetic energy dependencies of the reactions of Vn+ (n=2–13) with D2 are studied in a guided ion beam tandem mass spectrometer. Products observed are VnD+ for all clusters and VnD2+ for n=4–13. All reactions are observed to exhibit thresholds, except for formation of VnD2+ for n=4,5,7,9,11–13. The enhanced reactivity of the odd-sized clusters towards D2 chemisorption is nicely correlated with the D0(Vn+–V) bond energies. The odd-number clusters are less stable and more reactive, suggesting that they are open shell, whereas the even-number clusters, which are more stable and less reactive, appear to be closed shell. Threshold analyses of the endothermic reactions lead to Vn+–D binding energies (n=1–13), which reach values comparable to the bulk phase for larger clusters. The Vn+–D bond energies show odd–even oscillations anticorrelated with D0(Vn+–V) for n 5. Magnitude differences in the two series of bond energies suggest that the metal–metal bonding has ap...

Prednisolone-Induced Predisposition to Femoral Head Separation and the Accompanying Plasma Protein Changes in Chickens

Femoral head separation (FHS) is an idiopathic bone problem that causes lameness and production losses in commercial poultry. In a model of prednisolone-induced susceptibility to FHS, the changes in plasma proteins and peptides were analyzed to find possible biomarkers. Plasma samples from control and FHS-susceptible birds were depleted of their high abundance proteins by acetonitrile precipitation and were then subjected to cation exchange and reverse-phase (RP) fractionations. Analysis with matrix assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) showed several differentially expressed peptides, two of which were isolated by RP-HPLC and identified as the fragments of apolipoprotein A-I. The acetonitrile fractionated plasma proteins were subjected to reduction/alkylation and trypsin digestion followed by liquid chromatography and tandem mass spectrometry, which showed the absence of protocadherin 15, vascular endothelial growth factor-C, and certain transcription and ubiquitin-mediated proteolytic factors in FHS-prone birds. It appears that prednisolone-induced dyslipidemia, vascular, and tissue adhesion problems may be consequential to FHS. Validity of these biomarkers in our model and the natural disease must be verified in future using traditional approaches. Biomarker Insights Lameness because of femoral head separation (FHS) is a production and welfare problem in the poultry industry. Selection against FHS requires identification of the birds with subclinical disease with biomarkers from a source such as blood. Prednisolone can induce femoral head problems and predisposition to FHS. Using this experimental model, we analyzed the plasma peptides and proteins from normal and FHS-prone chickens by mass spectrometry to identify differentially expressed peptides and proteins. We found two peptides, both derived from apolipoprotein A-I, quantitatively elevated and two proteins, protocadherin 15 and VEGF-C, that were conspicuously absent in FHS-susceptible birds.