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.     

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.     

Anton Chekhov and Robert Koch Cheek to Cheek: A Proteomic Study

Five different letters and post cards as well as the shirt worn by Anton Chekhov on his death bed, stored in the State Literary‐Memorial Museum‐Reserve A. P. Chekhov Melikhovo (nearby Moscow), have been analyzed by applying EVA (an ethyl vinyl acetate foil studded with crushed strong anion and cation exchangers and with C₈ resins) diskettes to these surfaces. Three different eluates (under acidic and basic conditions and with acetonitrile) were analyzed by high resolution mass spectrometry. The environmental microbiota present on samples and the Mycobacterium tuberculosis strain were described by a meta‐proteomics approach. Eight identified M. tuberculosis proteins confirmed the presence of the bacterium and the cause of Chekhov's death, in addition to several sequenced peptides belonging to other bacterial species. The human plasma proteins and human keratins, detected on a tiny blood spot on the shirt, demonstrated the power of the combined approach.     

Noah's nectar: The proteome content of a glass of red wine

Combinatorial peptide ligand libraries (CPLLs) have been adopted for harvesting and identifying traces of proteins present in red wines. Surprisingly, although it is stated that red wines are in general fined with egg albumin, for all Italian wines investigated (in the areas around Chiari and Verona as well as in the Chianti area) we find that the only fining agent used is bovine casein, just like in white wines. Although the typical levels of casein found range between 45 to 85 μg/L, in one case as little as 3.8 μg/L of casein could be detected, an extremely high level of sensitivity, close to our lower detection limit of 1 μg/L reported for white wines. As a result of such treatments, very small amounts of residual proteins in red wines could be identified: essentially no residual grape proteins (except for thaumatin), but only traces of proteins from Saccharomyces cerevisiae and a few proteins from plant pathogens and fungi (e.g., Botryotinia fuckeliana, Sclerotinia sclerotiorum, Aspergillus aculeatus). Contrary to what has been found in white wines, the best capture efficiency with CPLLs has occurred at pH 7.2 and pH 9.3, with minimal capture at pH 3.3. The fact that such very low levels of fining agents can still be detected in treated red wines should be taken into consideration by winemakers in labelling their products and by EC rulers in issuing proper regulations.     

Permeability changes of connexin32 hemi channels reconstituted in liposomes induced by extremely low frequency, low amplitude magnetic fields

The effect of extremely low frequency and low amplitude magnetic fields on gap junctional permeability was investigated by using reconstituted connexin32 hemi channel in liposomes. Cytochrome c was loaded inside these proteoliposomes and its reduction upon addition of ascorbate in the bulk aqueous phase was adopted as the index of hemi channel permeability. The permeability rate of the hemi channels, expressed as DeltaA/min, was dependent on the incubation temperature of proteoliposomes. The effect of exposures to magnetic fields at different frequencies (7, 13 and 18 Hz) and amplitudes (50, 50 and 70 microT, respectively), and at different temperatures (16, 18 and 24 degrees C) was studied. Only the exposure of proteoliposomes to 18-Hz (B(acpeak) and B(dc)=70 microT) magnetic field for 60 min at 16+/-0.4 degrees C resulted in a significant enhancement of the hemi channel permeability from DeltaA/min=0.0007+/-0.0002 to DeltaA/min=0.0010+/-0.0001 (P=0.030). This enhancement was not found for magnetic field exposures of liposomes kept at the higher temperatures tested. Temperature appears to influence lipid bilayer arrangement in such a way as being capable to mask possible effects induced by the magnetic field. Although the observed effect was very low, it seems to confirm the applicability of our model previously proposed for the interaction of low frequency electromagnetic fields with lipid membrane.     

Spermatogenesis and blood-testis barrier in rats after long-term Vitamin A deprivation

It has been established that experimental avitaminosis A in rats results in a 'Sertoli cell-only situation' after about 10 weeks, and that replacing the vitamin immediately reinitiates spermatogenesis. The present study deals with testicular recovery after prolonged deprivation (up to 19 weeks). The Sertoli cell-only situation reached under this condition was thought to be refractory to Vitamin A replacement. However, spermatogenesis did reinitiate about 11 weeks after vitamin restoration, although in an atypical manner. The blood-testis barrier, normally assembled when spermatocytes reaches the zygotene stage, remained under this condition permeable to the lanthanum intercellular tracer. Concomitantly, primary spermatocytes normally found in the adluminal compartment isolated by the barrier (zygotene onward) became massively apoptotic. All the tubules containing early spermatocytes (preleptotene or leptotene cells), normally found in the basal compartment, exhibited normal features with no signs of degeneration. Based on these results, a possible relationship between blood-testis barrier assembly and spermatocyte differentiation is proposed.     

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.     

Ten Years of Global Evolution of the Human Respiratory Syncytial Virus BA Genotype with a 60-Nucleotide Duplication in the G Protein Gene

The emergence of natural isolates of human respiratory syncytial virus group B (HRSV-B) with a 60-nucleotide (nt) duplication in the G protein gene in Buenos Aires, Argentina, in 1999 (A. Trento et al., J. Gen. Virol. 84:3115-3120, 2003) and their dissemination worldwide allowed us to use the duplicated segment as a natural tag to examine in detail the evolution of HRSV during propagation in its natural host. Viruses with the duplicated segment were all clustered in a new genotype, named BA (A. Trento et al., J. Virol. 80:975-984, 2006). To obtain information about the prevalence of these viruses in Spain, we tested for the presence of the duplicated segment in positive HRSV-B clinical samples collected at the Severo Ochoa Hospital (Madrid) during 12 consecutive epidemics (1996-1997 to 2007-2008). Viruses with the 60-nt duplication were found in 61 samples, with a high prevalence relative to the rest of B genotypes in the most recent seasons. Global phylogenetic and demographic analysis of all G sequences containing the duplication, collected across five continents up until April 2009, revealed that the prevalence of the BA genotype increased gradually until 2004-2005, despite its rapid dissemination worldwide. After that date and coinciding with a bottleneck effect on the population size, a relatively new BA lineage (BA-IV) replaced all other group B viruses, suggesting further adaptation of the BA genotype to its natural host.Human respiratory syncytial virus (HRSV), a member of the Pneumovirus genus within the Paramyxoviridae family, is recognized as the leading agent responsible for severe respiratory infections in the pediatric population (31, 34, 35) and a pathogen of considerable importance in vulnerable adults (23, 24). The global respiratory syncytial virus (RSV) disease burden is estimated at 64 million cases and 160,000 deaths every year (70). This virus causes regular seasonal epidemics which take place during the winter months in temperate countries or during the rainy season in tropical areas (12). A peculiar aspect of HRSV is that the immune response produced by infection does not confer long-lasting protection, which is why reinfections are common throughout life (30).Neutralization tests performed with hyperimmune serum (16) and reactivity with specific monoclonal antibodies (4, 45) were used to classify HRSV isolates into two antigenic groups, A and B, which correlated with genetically distinct viruses (18). The main differences between these two groups are located in the major attachment G protein. This protein is a type II glycoprotein that shares neither sequence nor structural features with the attachment proteins (HN or H) of other paramyxoviruses (69), and it represents one of the targets of the immune response (27, 43). The full-length membrane-bound G protein (Gm) of 292 to 319 amino acids (depending on the viral strain) is also expressed in a secreted version (Gs) that lacks the transmembrane domain due to alternative initiation of translation at a second in-frame AUG codon in the G open reading frame (M48) (52). The G protein is the viral gene product with the highest degree of antigenic and genetic diversity among viral isolates (4, 18, 28, 45). Most changes are concentrated in two hypervariable regions that flank a highly conserved central region of the G protein ectodomain, which includes a cluster of four cysteines and the putative receptor binding site (43). It has been suggested that antigenic differences within this protein could facilitate repeated HRSV infections (37, 59). In addition, positive selection of amino acid changes was observed in the two hypervariable regions of the G protein ectodomain (7, 43, 71, 73, 74). One of the hypervariable regions, located in the C-terminal one-third of the G molecule, contains multiple epitopes recognized by monoclonal antibodies (43), suggesting that immune selection of new variants by antibodies may contribute to generation of HRSV diversity.Phylogenetic studies based on sequence analysis of the G protein have identified numerous genotypes in the antigenic groups A and B that show complex circulation patterns, since multiple genotypes of both antigenic groups may circulate within the same season and community, with one or two dominant genotypes being replaced in successive years (13, 14, 26, 27, 32, 49, 50). Each community shows a seasonal circulation pattern of genotypes, probably determined by local factors, such as the level of herd immunity to certain strains (3, 14, 49).The capacity of the G protein to accommodate drastic sequence changes was illustrated best by three antigenic group B viruses isolated in Buenos Aires, Argentina, in 1999 that contained a duplication of 60 nucleotides (nt) in the C-terminal third of the G protein gene (63). The global dissemination of these viruses allowed us to use the duplicated segment as a natural tag to reexamine the evolution of HRSV during propagation in its natural host. Phylogenetic analysis of G sequences revealed that all viruses with the duplicated segment clustered in a new genotype, named BA, and this finding supported the idea of a common ancestor for all viruses with the 60-nt duplication, dated about 1998 (64). The limited information about the molecular epidemiology of HRSV in Spain, together with an increase in G sequences with the duplicated segment reported worldwide, prompted us to conduct both a local search in Madrid for these viruses and a global phylogenetic analysis of HRSV with the 60-nt duplication from the time that these viruses were first detected, taking into account the geographic and temporal distribution of each isolate.     

Horam nonam exclamavit: sitio. The trace proteome of your daily vinegar

The trace proteome of white-wine vinegar has been identified via capture with home-made combinatorial peptide ligand libraries under conditions mimicking reverse-phase capture, i.e. at pH 2.2 in presence of 0.1% trifluoroacetic acid. A total of 27 unique gene products have been identified, of which 10 specific of the database Vitis vinifera, 13 found in the general database Uniprot_viridiplantae and 4 in Swiss Prot_all entries. The most abundant species detected, on the basis of spectral counts, appears to be the whole genome shotgun sequence of line PN40024, scaffold_22 (a protein of the glycosyl hydrolase family). Curiously, up to the present, no information had been available on vinegar proteome.     

Assessment of the floral origin of honey via proteomic tools

The honey from chestnut, acacia, sunflower, eucalyptus and orange was analysed for its proteome content, in order to see if any plant proteins present would allow the proteo-typing of these different varieties. Since the total protein content turned out to be minute, 200g of each honey type were diluted to 1L and then added with ProteoMiner to enhance the visibility of the proteinaceous material. All bands visible in the SDS-PAGE profile of each type of honey were eluted, digested and identified by mass spectrometry in a LTQ-XL instrument. It turned out that all proteins identified (except one, the enzyme glyceraldehyde-3-phosphate dehydrogenase from Mesembryanthemum crystallinum) were not of plant origin but belonged to the Apis mellifera proteome. Among the total proteins identified (eight, but only seven as basic constituents of all types of honey) five belonged to the family of major royal jelly proteins 1-5, and were also the most abundant ones in any type of honey, together with α-glucosidase and defensin-1. It thus appears that honey has a proteome resembling the royal jelly proteome (but with considerably fewer species), except that its protein concentration is lower by three to four orders of magnitude as compared to royal jelly. Attempts at identifying additional plant (pollen, nectar) proteins via peptidome analysis were unsuccessful.