Tryptophan and Cysteine Oxidation Products Dominate in α-Lactalbumin-Derived Peptides Analyzed with LC-MSn.

α-Lactalbumin (α-La), a major milk whey protein, is comprised of several amino acids prone to metal-catalyzed oxidation (MCO) typical in processing and during storage of foods. New tools are needed for the detection of characteristic oxidation products especially from tryptophan and cysteine that often remain unrecognized when using the traditional methods of carbonyl formation monitoring. In this study, the oxidative changes in α-La were investigated through tryptic digestion and collection of 3 descriptive peptides fitted into a metal-catalyzed oxidation (Fenton reaction) model. The peptide samples were oxidized at +37 °C for 14 d and explored with liquid chromatography-quadrupole ion trap-mass spectrometer (LC-MSn ). The fractionated α-La peptides were valyl-glycyl-isoleucyl-asparaginyl-tyrosyl-tryptophyl-leucyl-alanyl-histidyl-lysine (VGINYWLAHK), leucyl-aspartyl-glutaminyl-tryptophyl-leucyl-cysteinyl-glutamyl-lysine (LDQWLCEK), and tryptophyl+16 -leucyl-alanyl-histidyl-lysyl-alanyl-leucyl-cysteine (W+16 LAHKALC). Oxidation of several amino acids, such as cysteine, histidine, lysine, and tryptophan was observed. In the peptide LDQWLCEK, cysteine was rapidly trioxidized to sulfonic acid, followed by other amino acid side chains as secondary oxidation sites. Tryptophan oxidation was more pronounced in the peptides W+16 LAHKALC and VGINYWLAHK, and also formation of the harmful N-formylkynurenine was observed. As a conclusion, several stable and promising oxidation markers are proposed for α-La, which could be implemented in the evaluation of quality and safety of dairy protein-containing products.

Ellagitannin-rich cloudberry inhibits hepatocyte growth factor induced cell migration and phosphatidylinositol 3-kinase/AKT activation in colon carcinoma cells and tumors in Min mice.

Berries have been found to inhibit colon carcinogenesis in animal models, and thus represent a potential source of compounds for prevention and treatment of colorectal cancer. The mechanistic basis for their effects is not well understood. We used human colon carcinoma cells and Min mice to investigate the effects of ellagitannin-rich cloudberry (Rubus chamaemorus) extract on cancer cell migration and underlying cell signaling. Intrinsic and hepatocyte growth factor (HGF) -induced cell motility in human HT29 and HCA7 colon carcinoma cells was assessed carrying out cell scattering and scratch wound healing assays using time-lapse microscopy. Activation of Met, AKT, and ERK in cell lines and tumors of cloudberry-fed Min mice were determined using immunoprecipitation, Western blot and immunohistochemical analyses. Cloudberry extract significantly inhibited particularly HGF-induced cancer cell migration in both cell lines. Cloudberry extract inhibited the Met receptor tyrosine phosphorylation by HGF and strongly suppressed HGF-induced AKT and ERK activation in both HT29 and HCA7 cells. Consistently, cloudberry feeding (10% w/w freeze-dried berries in diet for 10 weeks) reduced the level of active AKT and prevented phosphoMet localization at the edges in tumors of Min mice. These results indicate that cloudberry reduces tumor growth and cancer cell motility by inhibiting Met signaling and consequent activation of phosphatidylinositol 3-kinase/AKT in vitro and in tumors in vivo. As the Met receptor is recognized to be a major target in cancer treatment, our results suggest that dietary phytochemicals may have therapeutic value in reducing cancer progression and metastasis.

Validated Method for the Characterization and Quantification of Extractable and Nonextractable Ellagitannins after Acid Hydrolysis in Pomegranate Fruits, Juices, and Extracts.

Pomegranates are one of the main highly valuable sources of ellagitannins. Despite the potential health benefits of these compounds, reliable data on their content in pomegranates and derived extracts and food products is lacking, as it is usually underestimated due to their complexity, diversity, and lack of commercially available standards. This study describes a new method for the analysis of the extractable and nonextractable ellagitannins based on the quantification of the acid hydrolysis products that include ellagic acid, gallic acid, sanguisorbic acid dilactone, valoneic acid dilactone, and gallagic acid dilactone in pomegranate samples. The study also shows the occurrence of ellagitannin C-glycosides in pomegranates. The method was optimized using a pomegranate peel extract. To quantify nonextractable ellagitannins, freeze-dried pomegranate fruit samples were directly hydrolyzed with 4 M HCl in water at 90 °C for 24 h followed by extraction of the pellet with dimethyl sulfoxide/methanol (50:50, v/v). The method was validated and reproducibility was assessed by means of an interlaboratory trial, showing high reproducibility across six laboratories with relative standard deviations below 15%. Their applicability was demonstrated in several pomegranate extracts, different parts of pomegranate fruit (husk, peels, and mesocarp), and commercial juices. A large variability has been found in the ellagitannin content (150-750 mg of hydrolysis products/g) and type (gallagic acid/ellagic acid ratios between 4 and 0.15) of the 11 pomegranate extracts studied.

Protein-phenolic interaction of tryptic digests of ß-lactoglobulin and cloudberry ellagitannin.

LC-ESI-MS was applied to investigate interaction reactions between a dimeric ellagitannin, sanguiin H-6, isolated from cloudberries (Rubus chamaemorus) and peptides of ß-lactoglobulin (ß-Lg). Three peptides, LIVTQTMK (m/z 934), ALPMHIR (m/z 838), and IPAVFK (m/z 674) were isolated from enzymatic (trypsin) digestion of ß-Lg. Oxidation of the peptides with and without sanguiin H-6 was monitored by LC-ESI-MS for up to 7 days. Sanguiin H-6 showed radical scavenging activities toward oxidation of the selected peptides. An interaction product was found with sanguiin H-6 and peptide LIVTQTMK by using MS and supported by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). An observable (haze) but unstable interaction product of sanguiin H-6 was seen with peptide ALPMHIR, but no detectable interaction products were seen with peptide IPAVFK. A higher proportion of sanguiin H-6 toward the amount of peptide might allow for further characterization of these interaction products.

A novel LC-MS application to investigate oxidation of peptides isolated from ß-lactoglobulin.

Oxidation of ß-lactoglobulin (ß-Lg), a typical milk whey protein, was investigated by oxidizing its three tryptic peptides after separation and fractionation by preparative HPLC. Oxidation was performed with H2O2 and Fe(3+) in piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) buffer containing ascorbic acid, by keeping the samples in an oven of +37 °C for 14 days. Changes in the oxidized peptides were then analyzed with LC-ESI-QIT-MS. The peptides chosen were Ala-Leu-Pro-Met-His-Ile-Arg (ALPMHIR), Leu-Ile-Val-Thr-Gln-Thr-Met-Lys (LIVTQTMK) and Val-Leu-Val-Leu-Asp-Thr-Asp-Tyr-Lys (VLVLDTDYK), all containing amino acids of oxidative interest. Especially methionine (M) was prone to oxidize as well as dioxidize, along with tyrosine (Y), histidine (H) and/or proline (P). The ions m/z 854 [ALPMHIR + O], m/z 950 [LIVTQTMK + O] and m/z 966 [LIVTQTMK + 2O] are considered very promising indicators of ß-Lg oxidation. Consequently, this study proposes a novel approach in peptide oxidation research through monitoring the oxidation markers identified with the LC-MS(n).