Multiple protective effect of peptides released from Olea europaea and Prunus persica seeds against oxidative damage and cancer cell proliferation.

The long exposition to reactive species results in oxidative stress which has been related with the development of cancer and other serious diseases. Olea europaea and Prunus persica seeds present a high protein content and preliminary results demonstrated their high potency to obtain bioactive peptides. The protective effect against oxidative damage exerted by peptides released from Olea europaea and Prunus persica seeds has been evaluated in this work. Seed hydrolysates showed protection against oxidation through four different mechanisms: inhibition of the formation of hydroxyl radicals, scavenging of free radicals, reduction of oxidizing compounds, and inhibition of lipid peroxidation. Moreover, seed hydrolysates also reduced the oxidative stress induced by an oxidizing agent on human cancer cells. Despite protection evaluated by individual mechanisms seemed to be significantly affected by the seed genotype, overall protection of seed hydrolysates was not so different. Seeds hydrolysates were not cytotoxic on normal cells but they demonstrated antiproliferative effect on human cancer cells (HeLa, PC-3, and HT-29). Peptides in all seed hydrolysates were sequenced by RP-HPLC-ESI-Q-TOF. Eighteen common peptides were observed among olive seed hydrolysates while a wider variability was observed among Prunus seed hydrolysates.

Proof of concept of a "greener" protein purification/enrichment method based on carboxylate-terminated carbosilane dendrimer-protein interactions.

Protein sample preparation is a critical and an unsustainable step since it involves the use of tedious methods that usually require high amount of solvents. The development of new materials offers additional opportunities in protein sample preparation. This work explores, for the first time, the potential application of carboxylate-terminated carbosilane dendrimers to the purification/enrichment of proteins. Studies on dendrimer binding to proteins, based on protein fluorescence intensity and emission wavelengths measurements, demonstrated the interaction between carboxylate-terminated carbosilane dendrimers and proteins at all tested pH levels. Interactions were greatly affected by the protein itself, pH, and dendrimer concentration and generation. Especially interesting was the interaction at acidic pH since it resulted in a significant protein precipitation. Dendrimer-protein interactions were modeled observing stable complexes for all proteins. Carboxylate-terminated carbosilane dendrimers at acidic pH were successfully used in the purification/enrichment of proteins extracted from a complex sample. Graphical Abstract Images showing the growing turbidity of solutions containing a mixture of proteins (lysozyme, myoglobin, and BSA) at different protein:dendrimer ratios (1:0, 1:1, 1:8, and 1:20) at acidic pH and SDS-PAGE profiles of the corresponsing supernatants. Comparison of SDS-PAGE profiles for the pellets obtained during the purification of proteins present in a complex sample using a conventional "no-clean" method based on acetone precipitation and the proposed "greener" method using carboxylate-terminated carbosilane dendrimer at a 1:20 protein:dendrimer ratio.

Identification of plum and peach seed proteins by nLC-MS/MS via combinatorial peptide ligand libraries.

UNLABELLED: Plum (Prunus domestica L.) and peach (Prunus persica (L.) Batsch) seed proteins are a source of bioactive peptides. These seeds, though, are usual residues produced during canning and beverage preparation that, in most cases, are irreversibly lost. The recovery and identification of these proteins might be of importance in human nutrition. This work employs the combinatorial peptide ligand libraries (CPLLs) technology as a tool to reduce the proteins dynamic concentration range. The most suitable extraction and CPLL capture conditions have been obtained and applied for the comprehensive identification of seed proteins. The analysis of recovered species by nLC-MS/MS has allowed the identification of 141 and 97 unique gene products from plum and peach seeds, respectively. It was possible to identify 16 proteins belonging to the Prunus genus. Moreover, a high number of histones and seed storage proteins were identified. Additionally, 21 and 14 bioactive peptides previously identified were found within protein sequences in plum and peach seeds, respectively. SIGNIFICANCE: Plums and peaches seeds are cheap sources of proteins that are irretrievably lost after canning and beverage production. Although this kind of residues has been used in animal feed or production of biofuel, they are usually incinerated or sent to landfills, wasting their huge potential. In order to exploit this, it is important to comprehensively study proteins present in plum and peach seeds. Nevertheless, since proteomics analysis is in most cases handicapped by the presence of high-abundance proteins masking the detection of the low-abundance ones, it is important to overcome this challenge. In this sense, combinatorial peptide ligand libraries (CPLLs) have been used in this work to reduce the dynamic protein concentration range to enable the identification of a higher amount of proteins than employing conventional methods. In this work, the better extracting conditions have been optimized and up to 141 and 97 unique gene products from plum and peach seeds have been found, respectively. Moreover, 21 and 14 peptides previously identified as bioactive peptides were ascertained within protein sequences in plum and peach seeds, respectively. For that reason, this research takes the first step in the recovery of these valuable proteins and in the extraction of bioactive peptides, which could be successfully adopted in human nutrition.

HPLC-Q-TOF-MS identification of antioxidant and antihypertensive peptides recovered from cherry (Prunus cerasus L.) subproducts.

The processing of fruits, such as cherries, is characterized by generating a lot of waste material such as fruit stones, skins, etc. To contribute to environmental sustainability, it is necessary to recover these residues. Cherry stones contain seeds with a significant amount of proteins that are underused and undervalued. The aim of this work was to extract cherry seed proteins, to evaluate the presence of bioactive peptides, and to identify them by mass spectrometry. The digestion of cherry seed proteins was optimized, and three different enzymes were employed: Alcalase, Thermolysin, and Flavourzyme. Peptide extracts obtained by the digestion of the cherry seed protein isolate with Alcalase and Thermolysin yielded the highest antioxidant and antihypertensive capacities. Ultrafiltration of hydrolysates allowed obtaining fractions with high antioxidant and antihypertensive capabilities. HPLC-Q-TOF-MS together with bioinformatics tools enabled one to identify peptides in these fractions.

Proteins in olive fruit and oil.

This paper is a comprehensive review grouping the information on the extraction, characterization, and quantitation of olive and olive oil proteins and providing a practical guide about these proteins. Most characterized olive proteins are located in the fruit, mainly in the seed, where different oleosins and storage proteins have been found. Unlike the seed, the olive pulp contains a lower protein content having been described a polypeptide of 4.6 kDa and a thaumain-like protein. Other important proteins studied in olive fruits have been enzymes which could play important roles in olives characteristics. Part of these proteins is transferred from the fruit to the oil during the manufacturing process of olive oil. In fact, the same polypeptide of 4.6 kDa found in the pulp has been described in the olive oil and, additionally, the presence of other proteins and enzymes have also been described. Protein profiles have recently been proposed as an interesting strategy for the varietal classification of olive fruits and oils. Nevertheless, there is still a lot of knowledge without being explored requiring new studies focused on the determination and characterization of these proteins.

Analytical approaches for the characterization and identification of olive (Olea europaea) oil proteins.

Proteins in olive oil have been scarcely investigated probably due to the difficulty of working with such a lipidic matrix and the dramatically low abundance of proteins in this biological material. Additionally, this scarce information has generated contradictory results, thus requiring further investigations. This work treats this subject from a comprehensive point of view and proposes the use of different analytical approaches to delve into the characterization and identification of proteins in olive oil. Different extraction methodologies, including capture via combinational hexapeptide ligand libraries (CPLLs), were tried. A sequence of methodologies, starting with off-gel isoelectric focusing (IEF) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or high-performance liquid chromatography (HPLC) using an ultraperformance liquid chromatography (UPLC) column, was applied to profile proteins from olive seed, pulp, and oil. Besides this, and for the first time, a tentative identification of oil proteins by mass spectrometry has been attempted.