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The main goal of the work was to find biochemical protein markers specific for grapes and wine in ancient amphorae shards and fermentation pools. Grape-specific proteins are more reliable markers than tartaric acid and other small organic acids (tartaric acid natural source are not only grape but also apple, mango, and other plants). The Yavne winery (located in the Central District of Israel) is stated to be the largest known wine production complex from the Byzantine period (ca. 1500 years ago). The site has been excavated recently, and a number of wine jar have been recovered. We have applied our ethylene vinyl acetate (EVA) (EVA studded with strong cation and anion exchangers) diskettes to the inner surface of a number of jars, thus capturing residual grape proteins therein. Via mass spectrometry analyses, we have been able to identify four grape and three yeast proteins. This has been possible because the EVA films, applied to such surfaces, are able to harvest and concentrate any trace species, rendering them amenable to instrumental analysis. Our analysis makes it possible to propose an explanation for the Holy Grail phenomenon as a dish in which wine or water begins to smell pleasant. We attribute this to the slow release of terpenes, aldehydes, and ketones from the clay walls of pottery. After digital modeling, we identified that "scallop-shaped" niches in winery were used for the condensation of high percentage alcohol by passive evaporation from fermentation tanks.
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The interest of scientists in analyzing items of World Cultural Heritage has been exponentially increasing since the beginning of the new millennium. These studies have grown considerably in tandem with the development and use of sophisticated and sensitive technologies such as high-resolution mass spectrometry (MS) and the non-invasive and non-damaging technique, known under the acronym EVA (ethylene-vinyl acetate). Here, we report the results of the MS characterization of the peptides and proteins harvested by the EVA technology applied to three letters written in 1457 and 1475 by the voivode of Wallachia, Vlad III, also known as Vlad the Impaler, or Vlad Dracula. The discrimination of the "original" endogenous peptides from contaminant ones was obtained by monitoring their different levels of deamidation and of other diagenetic chemical modifications. The characterization of the ancient proteins extracted from these documents allowed us to explore the environmental conditions, in the second half of the 15th century, of the Wallachia, a region considered as a meeting point for soldiers, migrants, and travelers that probably carried not only trade goods and cultural traditions but also diseases and epidemics. In addition, the identification of many human peptides and proteins harvested from the letters allowed us to uncover more about Vlad Dracula the Impaler. Particularly, the experimental data show that he probably suffered from inflammatory processes of the respiratory tract and/or of the skin. In addition, proteomics data, although not exhaustive, suggest that, according to some stories, he might also have suffered from a pathological condition called hemolacria, that is, he could shed tears admixed with blood. It is worth noting that more medieval people may have touched these documents, which cannot be denied, but it is also presumable that the most prominent ancient proteins should be related to Prince Vlad the Impaler, who wrote and signed these letters. The data have been deposited to the ProteomeXchange with the identifier ⟨PXD041350⟩.
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Proteómica , Masculino , Humanos , Espectrometría de MasasRESUMEN
The discovery of low- and very low-abundance proteins in medical applications is considered a key success factor in various important domains. To reach this category of proteins, it is essential to adopt procedures consisting of the selective enrichment of species that are present at extremely low concentrations. In the past few years pathways towards this objective have been proposed. In this review, a general landscape of the enrichment technology situation is made first with the presentation and the use of combinatorial peptide libraries. Then, a description of this peculiar technology for the identification of early-stage biomarkers for well-known pathologies with concrete examples is given. In another field of medical applications, the determination of host cell protein traces potentially present in recombinant therapeutic proteins, such as antibodies, is discussed along with their potentially deleterious effects on the health of patients on the one hand, and on the stability of these biodrugs on the other hand. Various additional applications of medical interest are disclosed for biological fluids investigations where the target proteins are present at very low concentrations (e.g., protein allergens).
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Biblioteca de Péptidos , Proteómica , Humanos , Proteómica/métodos , Proteínas Recombinantes , Anticuerpos , Técnicas Químicas CombinatoriasRESUMEN
The recent paleoproteomic studies, including paleo-metaproteomic analyses, improved our understanding of the dietary of ancient populations, the characterization of past human diseases, the reconstruction of the habitat of ancient species, but also provided new insights into the phylogenetic relationships between extant and extinct species. In this respect, the present work reports the results of the metaproteomic analysis performed on the middle part of a trunk, and on the portion of a trunk tip tissue of two different woolly mammoths some 30,000 years old. In particular, proteins were extracted by applying EVA (Ethylene-Vinyl Acetate studded with hydrophilic and hydrophobic resins) films to the surface of these tissues belonging to two Mammuthus primigenus specimens, discovered in two regions located in the Russian Far East, and then investigated via a shotgun MS-based approach. This approach allowed to obtain two interesting results: (i) an indirect description of the habitat of these two mammoths, and (ii) an improved characterization of the collagen type I, alpha-1 and alpha-2 chains (col1a1 and col1a2). Sequence characterization of the col1a1 and col1a2 highlighted some differences between M. primigenius and other Proboscidea together with the identification of three (two for col1a1, and one for col1a2) potentially diagnostic amino acidic mutations that could be used to reliably distinguish the Mammuthus primigenius with respect to the other two genera of elephantids (i.e., Elephas and Loxodonta), and the extinct American mastodon (i.e., Mammut americanum). The results were validated through the level of deamidation and other diagenetic chemical modifications of the sample peptides, which were used to discriminate the "original" endogenous peptides from contaminant ones. The data have been deposited to the ProteomeXchange with identifier < PXD029558 > .
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Mamuts , Animales , Humanos , Recién Nacido , Colágeno Tipo I/genética , Ecosistema , Fósiles , Mamuts/genética , Espectrometría de Masas , Filogenia , Proteómica/métodos , TecnologíaRESUMEN
After a decade of experimental applications, it is the objective of this review to make a point on combinatorial peptide ligand libraries dedicated to low-abundance proteins from animals to plants and to microorganism proteomics. It is, thus, at the light of the recent technical developments and applications that we will examine the state of the art, its usage within the scientific community, and its openness to unexplored fields. The improvements of the methodology and its implementation in connection with analytical determinations of combinatorial peptide ligand library (CPLL)-treated samples are extensively reviewed and commented upon. Relevant examples covering few critical aspects describe the performance of the technology. Finally, a reflection on the technological future is attempted in particular by involving new concepts adapted to the limited availability of certain biological samples.
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Biblioteca de Péptidos , Proteínas de Plantas , Animales , Técnicas Químicas Combinatorias/métodos , Ligandos , Proteínas de Plantas/química , Plantas/metabolismo , Proteómica/métodosRESUMEN
It is well established that the ink pigment used for writing the Dead Sea Scrolls (DSS) is mainly composed of carbon soot. The ink's binder however has yet to be securely identified. By applying EVA (ethylene vinyl acetate containing strong anion and cation exchangers admixed with C8 and C18) diskettes on one fragment and analyzing the captured material, the following study was able to determine the composition of the binder. Proteins admixed of plant proteins (ribulose biphosphate carboxylase, rhamnogalacturonate lyase, α-galactosidase A, calmodulin, among those identified) as well as of a few glycoproteins with different combinations of pentosyl and hexosyl units with plant acids (stearic, palmitic, oleic, linoleic and linolenic acids) and terpenes (triacontanol, catechin, lupeol) are mixed attributes of acacia trees which suggests the use of gum Arabic as the ink's binder. SIGNIFICANCE: Whereas a huge body of reports has explored any possible aspect of the Dead Sea Scrolls, including the dating and the animal origin of the parchment, one aspect had not been investigated so far, namely which kind of ligand had been adopted to ensure a firm binding of the ink (in reality carbon soot) to the supporting parchment. In the present investigation it has been demonstrated that this "glue" is a mixture of plant proteins, as well as a few glycoproteins, together with plant acids and terpenes. These proteins and metabolites belong to two species of trees, Vachellia nilotica and Acacia Albida, widespread in this Middle East region. The EVA methodology here adopted has shown that it is possible to explore any item pertaining to the world Cultural Heritage in the absence of damage or contamination thus permititng to analyze any possible precious document stored in museum, public libraries and private collections.
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Tinta , Proteómica , Animales , MetabolómicaRESUMEN
During the last decade, paleoproteomics allowed us to open a direct window into the biological past, improving our understanding of the phylogenetic relationships of extant and extinct species, past human diseases, and reconstruction of the human diet. In particular, meta-proteomic studies, mainly carried out on ancient human dental calculus, provided insights into past oral microbial communities and ancient diets. On the contrary, very few investigations regard the analysis of ancient gut microbiota, which may enable a greater understanding of how microorganisms and their hosts have co-evolved and spread under the influence of changing diet practices and habitat. In this respect, this paper reports the results of the first-ever meta-proteomic analysis carried out on a gut tissue sample some 40,000 years old. Proteins were extracted by applying EVA (ethylene-vinyl acetate) films to the surface of the gut sample of a woolly mammoth (Mammuthus primigenus), discovered in 1972 close to the Shandrin River (Yakutia, Russia), and then investigated via a shotgun MS-based approach. Proteomic and peptidomic analysis allowed in-depth exploration of its meta-proteome composition. The results were validated through the level of deamidation and other diagenetic chemical modifications of the sample peptides, which were used to discriminate the "original" endogenous peptides from contaminant ones. Overall, the results of the meta-proteomic analysis here reported agreeing with the previous paleobotanical studies and with the reconstructed habitat of the Shandrin mammoth and provided insight into its diet. The data have been deposited to the ProteomeXchange with identifier < PXD025518 > .
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Microbioma Gastrointestinal , Mamuts/microbiología , Espectrometría de Masas/métodos , Proteómica/métodos , Animales , Mamuts/genéticaRESUMEN
We describe here a modern tool for exploring documents pertaining to the world Cultural Heritage while avoiding their contamination or damage. Known under the acronym EVA, it consists of a plastic foil of Ethylene Vinyl Acetate studded with strong cation and anion resins admixed with C8 and C18 hydrophobic beads. When applied to any surface such foils can harvest any type of surface material, which is then eluted and analyzed via standard means, such as GS/MS (typically for metabolites), MS/MS (for peptide and protein analysis), X-ray (for elemental analysis). We briefly review here a number of past data, such as screening of original documents by Bulgakov, Chekov, Casanova, Kepler, while dealing in extenso with very recent data, pertaining to Orwell and Stalin and analysis of the skin of an Egyptian mummy. The technique was also successfully applied to paintings, such as the Donna Nuda at the Hermitage in St. Petersburg, attributed to Leonardo and his school. This novel methodology represents a formidable tool for exploring the past life of famous authors, scientist and literates in that it can detect traces of their pathologies and even drug consumption left by saliva and sweat traces on their original hand-written documents.
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Pinturas , Espectrometría de Masas en Tándem , Animales , Femenino , Caballos , Resinas de Plantas , SalivaRESUMEN
Undoubtedly, the two leaders who were under enormous pressure during World War II (WWII) were Winston Churchill and Joseph Stalin' since their respective countries had to sustain most of the war weight, at least in Europe. Lord Moran recounted in his memoir Winston Churchill: The Struggle for Survival that he had diagnosed a middle-aged Churchill with bipolar disorder. Churchill himself often referred to his periods of intense and prolonged depression as his "black dog." On the contrary, not much is known about Stalin's mental conditions, although in 1927 the neurologist V. M. Bekhterev, the day prior to his sudden death, upon a long examination of the leader's mental status, declared that he had found him affected by paranoia. No chemical evidence via clinical chemistry analyses was provided for the two leaders, though. We have had access to the collection of books (stored in the Russian Government Archive of Social and Political History, RGASPI, of the former Institute of Marxism and Leninism under the Central Committee of the USSR Communist Party) that Stalin was reading during WWII, with pages containing personal annotations on the margins. Upon harvesting surface material via EVA disks (ethylene-vinyl acetate studded with strong cation and anion exchangers and C8-C18 resins) and instrumental analysis via X-ray photoelectron spectroscopy, we detected lithium levels (~ 100 ± 8 ng/cm2) compatible with those present in the sweat and/or saliva of patients treated with lithium salts for curing bipolarity and paranoia or probably gout. These data are the first clear indication that Stalin was under cure for this pathology.Graphical abstract.
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Trastorno Bipolar/diagnóstico , Personajes , Trastornos Paranoides/diagnóstico , Anciano , Historia del Siglo XX , Humanos , Masculino , Persona de Mediana EdadRESUMEN
The margins of several pages of the Aleppo codex have been found to be corroded and contaminated by diffuse maculae. In order to understand the origin of this decay these margins have been analysed by applying EVA (ethylene vinyl acetate plastic embedded with strong cation and anion exchangers and mixed with C8 and C18 hydrophobic resins) diskettes for harvesting surface material. The captured compounds have been eluted, digested with trypsin and analysed by nano-HPLC-MS. Three major strains of Aspergillus have been identified, namely Aspergillus fumigatus, Aspergillus pseudoglaucus, Aspergillus amstelodami, together with a lactobacillus strain and human keratins. The novelty of this investigation is that for the first time the EVA technology has been applied to ancient parchments in the absence of mechanical deformation or distortion that could be induced if there had been water exchange between the EVA diskettes and the parchment. These findings should help curators to find suitable restoration protocols for these precious documents belonging to the world Cultural Heritage.
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Aspergillus/aislamiento & purificación , Queratinas/aislamiento & purificación , Lactobacillus/aislamiento & purificación , Archivos/historia , Historia Antigua , Polivinilos/químicaRESUMEN
A novel way for exploring the world's cultural heritage in the absence of damage or contamination (such as removing pigments in paintings or chipping away pieces of bones) of the items under investigation is here reported, called the EVA technique. It is based on films of ethylene vinyl acetate (EVA) impregnated with strong anion and cation exchangers, admixed with hydrophobic resins, C8 and C18. When in contact with any surface these films can harvest nanomoles of macromolecules (proteins and DNA) as well as metabolites, which can then be identified by standard instrumentation. Some important applications are reported, such as the findings of the renal pathology and assumption of morphine in the original manuscript of Master I Margarita by Bulgakov, the presence of TBC bacterium in Chekhov's shirt and in a letter by Orwell, the Y. pestis and anthrax bacteria in the death registries of Milan's lazaretto in the 1630 plague bout, as well as ample traces of five metals in Kepler's manuscripts, suggesting his potential practice of alchemy. Also, in the pages of the Memoirs of Casanova, although the gonorrhea bacterium could not be found, spots of HgS could be measured, suggesting its use for curing the disease. A family of EVA films is described, enlarging its use to dedicated applications, such as the capture of drugs of abuse in the pages of famous writers and even in the paintings of fauvists. It is hoped that the present methodology could open the doors of museums, state archives, and private collections for detecting biological traces left by artists, literates, and men of culture in their masterpieces.
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Proteómica , Compuestos de Vinilo , Etilenos , Humanos , Masculino , TecnologíaRESUMEN
The detection and identification of low-abundance proteins from plant tissues is still a major challenge. Among the reasons are the low protein content, the presence of few very high-abundance proteins, and the presence of massive amounts of other biochemical compounds. In the last decade numerous technologies have been devised to resolve the situation, in particular with methods based on solid-phase combinatorial peptide ligand libraries. This methodology, allowing for an enhancement of low-abundance proteins, has been extensively applied with the advantage of deciphering the proteome composition of various plant organs. This general methodology is here described extensively along with a number of possible variations. Specific guidelines are suggested to cover peculiar situations or to comply with other associated analytical methods.
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Técnicas Químicas Combinatorias/métodos , Biblioteca de Péptidos , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Proteoma/análisis , Proteoma/metabolismoRESUMEN
George Orwell, fighter for the Republican Army during the Spanish Civil War, was shot through the throat by a sniper on 20th May 1937 and nearly killed. After receiving only a summary external treatment, on the 29th, he was cured in a Barcelona hospital where he was infected by the Koch bacillus. After fleeing from Spain on 23rd June 1937, he repaired to his cottage in Wallington, Hertfordshire, wherefrom he wrote a letter to Sergey Dynamov, Editor of Soviet journal "Foreign Literature." This typewritten letter was analyzed by application of five EVA strips (ethylene vinyl acetate studded with strong cation and anion and with C8 and C18 resins; four on the corners and one over his signature), searching for biological traces. Upon elution of the captured biologicals, trypsin digestion and Orbitrap Fusion trihybrid mass spectrometer analyses, three of the five strips yielded clear traces of six unique proteins (via proteotypic peptides) of the tuberculosis bacterium. Additionally, MALDI TOF analysis of saliva of a tuberculosis patient and the EVA strip eluates gave a spectrum of 14 peptide bands (Mr 2700 to 6700 Da range) coincident between the two samples, thus, fully confirming Orwell's pathology. These results are attributed to saliva traces on Orwell's fingertips and to the fact that the letter was written on 2nd July 1937, when Orwell's pathology was at its peak.
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Proteínas Bacterianas , Correspondencia como Asunto/historia , Mycobacterium tuberculosis/química , Proteómica/métodos , Tuberculosis , Conflictos Armados/historia , Proteínas Bacterianas/análisis , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Historia del Siglo XX , Humanos , Literatura , Masculino , Saliva/microbiología , Extracción en Fase Sólida , España , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tuberculosis/diagnóstico , Tuberculosis/historia , Tuberculosis/microbiologíaRESUMEN
In plant tissues proteins are present in low amounts but in a very large number. To this peculiar situation many complex foreign components render protein extraction and purification very difficult. In the last several years interesting technologies have been described to improve the technical situation to the point that some methodologies allow reaching very low-abundance proteins and minor allergens. Among enrichment methods the one documented in this report is based on combinatorial peptide ligand libraries (CPLLs) that emerged in the last decade by contributing to largely improve the knowledge in plant proteomics. It is the aim of this review to describe how this technology allows detecting low-abundance proteins from various plant tissues and to report the dynamics of the proteome components in response to environmental changes and biotic attacks. Typical documented examples with the description of their scientific interest are reported. The described technical approach and selected applications are considered as one of the most advanced approaches for plant proteomics investigations with possibilities not only to enlarge the knowledge of plant proteomes but also to discover novel allergens as well as plant biomarkers subsequent to stressful situations.
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Biblioteca de Péptidos , Proteínas de Plantas/genética , Plantas/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Proteínas de Plantas/metabolismo , Plantas/genéticaRESUMEN
The application of analytical chemistry to the exploration of the World Cultural Heritage represents a major challenge in that most protocols and strategies are invasive and require micro-sampling. We report a novel methodology for harvesting material deposited on the surface of ancient documents while avoiding their damage or contamination. The technology here described relates to the capture of metals on these specimens. It is based on the use of plastic films (ethylene vinyl acetate, EVA) impregnated with different metal chelators (sodium 2,3-dimercapto-1-propanesulfonate, DMPS, meso-2,3-dimercaptosuccinic acid, DMSA and ethylene diamino tetra acetic acid, EDTA, as calcium salt), for harvesting from surfaces of different supports potential traces of metals therein deposited. The EVA film technology has been used to explore the pages of a manuscript written by Kepler concerning the movements of the moon and catalogued under the title "Hipparchus" at the Archives of the Russian Academy of Sciences (St. Petersburg branch). The EVA-based chelating diskettes were able to capture very significant amounts of different metals, namely: Au, Ag, Hg, As, Pb, suggesting that Kepler, well known as astronomer, astrologist, mathematician and Lutheran theologian, might have started practicing alchemy, a pseudo-chemical science he had learned from his colleague Tycho Brahe in Prague.
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The painting "Donna Nuda" by Leonardo was acquired by Catherine II (the Great) from the R. Walpole collection, Houghton Hall, England, in 1779 for the Hermitage in St. Petersburg. By exploiting the EVA film technology (ethylene vinyl acetate polymer embedded with strong cation and anion exchangers and with C8 and/or C18 resins) we have explored the surface of the painting in order to ascertain the techniques used in its drawing. Five EVA films were affixed on the body and on the landscape for 60â¯min. Upon elution from the recovered films, the harvested material was analyzed by gas-chromatography/mass spectrometry as well as by liquid chromatography/mass spectrometry. "Tempera grassa" (consisting of linseed oil admixed with egg yolk) was used in the entire painting. The surface was then protected by a layer of conifer resin. It is hypothesized that access to the layer underneath the protective layer was obtained via micro-cracks on the conifer resin itself. Rosemary oil was used as diluent to slow down the drying process and so to perform the glazing technique, thus obtaining the "aerial perspective" in correspondence of the landscape. To our reckoning, this is the first time in which a Leonardo painting is analyzed in depth but also in which his artistic technique is deciphered via modern techniques for exploring Cultural Heritage. The EVA film technology might be used for ascertaining the authenticity of paintings and uncover frauds. SIGNIFICANCE STATEMENT: Leonardo da Vinci was the most famous Italian polymath of the Renaissance and one of the most important innovators of his time. He was the author of several important artworks such as "La Gioconda", but he also painted the "Donna Nuda" conserved at the Hermitage Museum. Although some attempts permitted the identification of part of the materials used by Leonardo, to date no analytical investigations were able to fully characterize and decipher the recipes. We explored the surface of the "Donna Nuda" painting through a non-invasive approach that uses a functionalized film to adsorb nano-scopic amounts of materials that were then analyzed by mass spectrometry. This method has the potential to revolutionize the approaches used to analyze cultural heritage.
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Cromatografía Liquida , Yema de Huevo/química , Aceites Volátiles/análisis , Pintura/análisis , Pinturas , Resinas de Plantas/análisis , Espectrometría de MasasRESUMEN
Introduction: Capture of proteins and metabolites from Cultural Heritage (paintings, manuscripts, parchment etc.) has been done in the past via surface scraping and erasing, a method discouraged. The EVA (ethylene vinyl acetate) method consists of a plastic polymer in which strong cation and anion resins, admixed with C8 and/or C18, are embedded. Areas covered: We review here the findings on different items stored in public libraries and archives: (a) the original manuscript of the novel Master y Margarita by Bulgakov; (b) the death registries of the lazaretto in the 1630 Milano plague; (c) the shirt worn by A. Chekhov in his death bed; (d) Kepler's script on Hipparchus (in St. Petersburg National Archives); (e) the Memoirs of G. Casanova. Expert opinion: The technique here surveyed appears to be a unique tool enabling exploration of any document stored in public archives, museum and private collections without damaging or contaminating the items under analysis. The amounts harvested from any surface are very minute, yet sufficient for analysis via advanced mass spectrometry instrumentation, thus permitting the identification of all captured material. It is hoped that the present review will stimulate the scientific community to adopt it for projects pertaining to Cultural Heritage.
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Ensayo de Materiales/métodos , Polivinilos/química , HumanosRESUMEN
The original manuscript of Casanova's Memoirs is stored at the Bibliothèque Nationale de France in Paris. We have gained access to it and explored the surfaces of chapters one and two (via the ethylene vinyl acetate [EVA] film technology, i.e., of diskettes of ethylene vinyl acetate with embedded strong cation and anion exchangers and C8 resins) in search of potential diseases of the author, especially of the gonorrhea bacterium, since Casanova reported that he had several bouts of this pathology along his adventurous life. Although the bacterium was not found, we have detected high levels of HgS as red spots along the lines of the manuscript, suggesting that Casanova was using this chemical as a cure for his venereal disease. Additionally, among the several bacteria identified on the surface via mass spectrometry, we could detect traces of Streptococcus uberis, a typical animal infection, found also in humans, together with a few strains of Lactobacilli, probably present in his saliva. The EVA film technology appears to open new horizons for investigating the world Cultural Heritage.
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Libros/historia , Tipificación Molecular/métodos , Escritura/historia , Proteínas Bacterianas/análisis , Proteínas Bacterianas/química , Proteínas Bacterianas/clasificación , Francia , Historia del Siglo XVIII , Humanos , Lactobacillus/química , Espectrometría de Masas/métodos , Compuestos de Mercurio/análisis , Compuestos de Mercurio/química , Polivinilos , Streptococcus/químicaRESUMEN
The present review covers proteomics discoveries in farm animals using combinatorial peptide ligand libraries (CPLLs). These libraries enhance the identification of low-abundance proteins by compressing the dynamic range of the protein concentration. This technology can be applied in multiple biological fluids, such as plasma or serum, follicular and cerebrospinal fluids, urine, saliva, tears seminal fluid, milk whey and even lymph. The discovery of low-abundance proteins contributes to the continuous monitoring for animal pathologies, which allows early treatment and disease prevention. Additionally, evidencing rare proteins in animal products used for human consumption can help in assessing their nutritional properties and their positive or negative effects on human health. SIGNIFICANCE: Many investigations are made on the identification of proteins of farm animals. Protein concentration changes under abnormal stressful conditions as a sign of a first physiological reaction. Many of these protein changes are of low-abundance and their detection is dependent on enrichment technologies. These changes are potentially correlated with the type of stressor and may determine actions to preserve the animal welfare.