Proteomic fingerprinting of mistletoe (Viscum album L.) via combinatorial peptide ligand libraries and mass spectrometry analysis.

Combinatorial peptide ligand libraries (CPLLs), coupled to mass spectrometry (MS) analysis, have been used to investigate in depth the proteome of Viscum album L. (VA), commonly named European mistletoe, in order to provide a first proteomic fingerprinting. For this purpose, the proteins were captured via CPLLs at two different pH values (acidic and neutral). A total of 648 non-redundant proteins were identified by using two different databases. The two pH values, chosen for bead incubations, have contributed to increment the capture ability: 56% and 31% of CPLLs species were respectively recognized at pH7.2 and at pH2.2. Finally the biological function of identified proteins was evaluated in order to understand their role on human health and the potential benefits of mistletoe extracts in medicine. SIGNIFICANCE: Viscum album L. (VA) extracts are recently used as supporting medicine for cancer therapy, improving patients' survival and increasing their quality of life in medicine. These anticancer effects are investigated and they are probably due to mistletoe's capability to favor tumor cell's death and to modulate the immune system. Although the increasing interest in VA medical benefits, the role of its components in human health remains unclear. In order to exploit this aspect, it is important to comprehensively study proteins present in Viscum album L. (VA) extracts. Nevertheless, since plant 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 to reduce the dynamic protein concentration range to enable the identification of a higher amount of proteins than employing conventional methods. In this work, a total of 648 non-redundant proteins were identified: 56% and 31% of CPLLs species were respectively recognized at pH7.2 and at pH2.2. This deep proteome identification was useful to investigate the biological functions of proteins in order to evaluate their potential role in human health.

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.

An in depth proteomic analysis based on ProteoMiner, affinity chromatography and nano-HPLC-MS/MS to explain the potential health benefits of bovine colostrum.

Bovine colostrum (BC), the initial milk secreted by the mammary gland immediately after parturition, is widely used for several health applications. We here propose an off-target method based on proteomic analysis to explain at molecular level the potential health benefits of BC. The method is based on the set-up of an exhaustive protein data bank of bovine colostrum, including the minor protein components, followed by a bioinformatic functional analysis. The proteomic approach based on ProteoMiner technology combined to a highly selective affinity chromatography approach for the immunoglobulins depletion, identified 1786 proteins (medium confidence; 634 when setting high confidence), which were then clustered on the basis of their biological function. Protein networks were then created on the basis of the biological functions or health claims as input. A set of 93 proteins involved in the wound healing process was identified. Such an approach also permits the exploration of novel biological functions of BC by searching in the database the presence of proteins characterized by innovative functions. In conclusion an advanced approach based on an in depth proteomic analysis is reported which permits an explanation of the wound healing effect of bovine colostrum at molecular level and allows the search of novel potential beneficial effects.

The secrets of Oriental panacea: Panax ginseng.

The Panax ginseng root proteome has been investigated via capture with combinatorial peptide ligand libraries (CPLL) at three different pH values. Proteomic characterization by SDS-PAGE and nLC­MS/MS analysis, via LTQ-Orbitrap XL, led to the identification of a total of 207 expressed proteins. This quite large number of identifications was achieved by consulting two different plant databases: P. ginseng and Arabidopsis thaliana. The major groups of identified proteins were associated to structural species (19.2%), oxidoreductase (19.5%), dehydrogenases (7.6%) and synthases (9.0%). For the first time, an exploration of protein­protein interactions was performed by merging all recognized proteins and building an interactomic map, characterized by 196 nodes and 1554 interactions. Finally a peptidomic analysis was developed combining different in-silico enzymatic digestions to simulate the human gastrointestinal process: from 661 generated peptides, 95 were identified as possible bioactives and in particular 6 of them were characterized by antimicrobial activity. The present report offers new insight for future investigations focused on elucidation of biological properties of P. ginseng proteome and peptidome. BIOLOGICAL SIGNIFICANCE: Ginseng is a traditional oriental herbal remedy whose use is very diffused in all the world for its numerous pharmacological effects. However, the exact mechanism of action of ginseng components, both ginsenosides and proteins, is still unidentified. So the common use of ginseng requires strict investigations to assess both its efficiency and its safety. Although many reports have been published regarding the pharmacological effects of ginseng, little is known about the biochemical pathways of root. Proteomics analysis could be useful to elucidate the physiological pathways. In this manuscript, an integrated approach to proteomics and peptidomics will usher in exploration of Panax ginseng proteins and proteolytic peptides, obtained by in-silico gastrointestinal digestion, characterized by antimicrobial action. The present research would pave the way for better knowledge of metabolic functions connected with ginseng proteome and provide with new information necessary to understand better antimicrobial activity of P. ginseng.

According to the CPLL proteome sheriffs, not all aperitifs are created equal!

Combinatorial peptide ligand libraries (CPLLs) have been adopted for investigating the proteome of a popular aperitif in Northern Italy, called "Amaro Branzi", stated to be an infusion of a secret herbal mixture, of which some ingredients are declared on the label, namely Angelica officinalis, Gentiana lutea and orange peel, sweetened by a final addition of honey. In order to assess the genuineness of this commercial liqueur, we have prepared extracts of the three vegetable ingredients, assessed their proteomes, and compared them to the one found in the aperitif. The amaro's proteome was identified via prior capture with CPLLs at two different pH values (2.2 and 4.8). Via mass spectrometry analysis of the recovered fractions, after elution of the captured populations in 4% boiling SDS, we could confirm the presence of the following: six proteins originating from honey, 11 from orange peels, 29 from G. lutea and 46 from A. officinalis (including shared species), plus 33 species which could not be attributed to the other secret ingredients, due to paucity of genomic data on plant proteins, for a total of 93 unique gene products (merging shared proteins). This fully confirmed the genuineness of the product. Considering that most of these species could be present in trace amounts, undetectable by conventional techniques, the CPLL methodology, due to its ability to enhance the signal of trace components up to 3 to 4 orders of magnitude, could represent a powerful tool for investigating the genuineness and natural origin of commercial beverages in order to protect consumers from adulterated products.

Artichoke and Cynar liqueur: two (not quite) entangled proteomes.

Combinatorial peptide ligand libraries (CPLLs) have been adopted to investigate the proteome of artichoke extracts, of a home-made alcoholic infusion and of the Italian Cynar liqueur. The aim of study was not only to perform the deepest investigation so far of the artichoke proteome but also to assess the genuineness of the commercial aperitif via a three-pronged attack. First, different extraction techniques have been used for the characterization of the artichoke's proteome, secondly a home-made infusion has been analyzed and finally the proteome of the commercial drink was checked. The artichoke proteome has been evaluated via prior capture with CPLLs at four different pH (2.2, 4.0, 7.2 and 9.3) values. Via mass spectrometry analysis of the recovered fractions, after elution of the captured populations in 4% boiling SDS, we could identify a total of 876 unique gene products in the artichoke extracts, 18 in the home-made infusion and no proteins at all in the Italian Cynar liqueur, casting severe doubts on the procedure stated by the manufacturer (that should be made by an infusion of artichoke leaves plus thirteen different herbs). This could be the starting point for investigating the genuineness and natural origin of commercial drinks in order to protect consumers from adulterated products.

Allergomic study of cypress pollen via combinatorial peptide ligand libraries.

Although Cupressus sempervirens (Cups) pollen represents one of the main aeroallergens in southern Europe, only two Cups allergens have yet been identified and reported: Cup s 1 and Cup s 3. The aim of this study was to identify allergens in cypress pollen using an immuno-proteomic approach. A sequential pollen protein extraction was developed and supplemented by a combinatorial peptide ligand library (CPLL) treatment to select low-abundance species. Control extracts and CPLL eluates have then been resolved by 1-DE and 2-DE gel electrophoresis, blotted and confronted with sera from cypress allergic patients. Extracted proteins including IgE-binding components were identified using nanoLC-MS/MS analysis. A total of 108 unique gene products were identified analyzing the eluates and control loaded onto 1-DE SDS-PAGE. Forty proteins were identified in control samples and 68 supplementary species upon CPLL treatment. Out of the 12 IgE-binding proteins characterized in 2-DE gels, 9 were already reported as allergens in various sources including the two major known allergens of Cupressaceae (groups 1 and 2). Three IgE-binding proteins, not previously reported as allergens, are newly described. The improvement in protein extraction combined with the enrichment of low-abundance species allowed us to extend the repertoire of potential cypress pollen allergens.

Anyone for an aperitif? Yes, but only a Braulio DOC with its certified proteome.

The trace proteome of a Braulio aperitif (a 21% alcohol beverage, named after a mountain in the Val di Stelvio, Italy) has been investigated via capture with combinatorial peptide ligand libraries (CPLL, ProteoMiner). This aperitif is made with an infusion of 13 mountain herbs and berries, among which four are officially indicated in the label: Achillea moschata, juniper (Juniperus communis subsp. alpina) berries, absinthe (Artemisia absinthium) and gentian (Gentiana alpina) roots. Via capture with CPLLs at pH 7.0 and 2.2 we were able to identify 29 unique gene products, among which the PR5 (parasite resistance) allergen Jun r 3.2, a 25kDa species from Juniperus rigida. Due to the paucity of data on these alpine herbs, it was difficult to attribute these proteins to the specific plant extracts presumably present in this beverage; however most of the species identified indeed belong to alpine herbs and plants, living in a habitat between 1000 and 2000m of elevation. Most of them are enzymes, spanning a Mr range from 10 to 65kDa. It is hoped that such a proteomic signature should help tracking counterfeited products sold on the market.

Ginger Rogers? No, Ginger Ale and its invisible proteome.

The trace proteome of a Ginger drink, stated to be produced with a ginger root extract, has been investigated via capture with combinatorial peptide ligand libraries (ProteoMiner). Although in traces, we could confirm the presence of five grape proteins and one apple protein, but not even the faintest trace of any ginger root proteins. The first two findings are correct, as the producer stated that this beverage had been reinforced with 12% grape juice and 6% apple juice, but the absence of even traces of ginger proteins does not permit the classification of this beverage as a ginger extract on a proteomics scale. However, organoleptic tasting has confirmed the presence of a ginger extract, due to its piquant and tongue-biting taste. Nevertheless, any ginger root extract must be considered as a minor component as compared to the presence of grape and apple juice. At the light of these findings, it is hoped that the competent authorities will in the future make compulsory the proper labelling also of beverages so that all amounts of compounds utilized will be clearly stated in the label, including the presumptive main component.

Going nuts for nuts? The trace proteome of a Cola drink, as detected via combinatorial peptide ligand libraries.

The "invisible" proteome of a Cola drink, stated to be produced with a kola nut extract, has been investigated via capture with combinatorial peptide ligand libraries (CPLL). Indeed, a few proteins in the M(r) 15-20 kDa range could be identified by treating large beverage volumes (1 L) and performing the capture with CPLLs at very acidic pH values (pH 2.2) under conditions mimicking reverse-phase adsorption. Ascertaining the presence of proteins deriving from plant extracts has confirmed the genuineness of such beverage and suggests the possibility of certifying whether soft drinks present on the market are indeed made with vegetable extracts or only with artificial chemical flavoring.

In-depth proteomic analysis of non-alcoholic beverages with peptide ligand libraries. I: Almond milk and orgeat syrup.

Combinatorial peptide ligand libraries, both commercial and home-made, have been adopted to investigate the proteome of non-alcoholic beverages, in order to assess their genuineness and detect also trace proteins, in search of potential allergens. Two such beverages have been studied: almond milk and orgeat syrup. In the first product we have been able to identify 132 unique protein species, the deepest investigation so far of the almond proteome. In the second beverage, a handful of proteins (just 14) have been detected, belonging to a bitter almond extract. In both cases, the genuineness of such products has been verified, as well as the fact that almond milk, judging on the total protein and fat content, must have been produced with 100g ground almonds per litre of beverage, as required by authorities. On the contrary, cheap orgeat syrups produced by local supermarkets and sold as their own brands, where found not to contain any residual proteins, suggesting that they contained only synthetic aromas and no natural plant extracts. This could be the starting point for investigating the myriad of beverages that in the last decades have invaded the shelves of supermarkets the world over, whose genuineness and natural origin have never been properly assessed.