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OBJECTIVE: Human hair is regularly subjected to chemical and physical insults, such as heat, UV-irradiation and alkaline hair care products. These insults result in molecular modifications at the hair protein level that underpin mechanical and sensory property changes in the fibres. These changes can manifest itself in reduced hair quality and performance attributes observable to the consumer. In this work, changes in protein modification as a result of heat and alkaline treatments are determined. METHODS: Redox proteomic profiling using high-resolution mass spectrometry was applied to map and evaluate amino acid residue modifications in human hair exposed to a combination of thermal treatments and alkali exposure with the aim to understand the underlying chemical processes. RESULTS: Our results show that an increase in redox-related modifications is associated with exposure to higher levels of hydrothermal and alkaline insult. Post-translational modification profiling at the protein primary structural level delivered some further insights into the site-specificity of these modifications, with a clear increase in the number of cysteic acid modifications noticed in samples subjected to more extreme insults. CONCLUSION: Pinpointing modification sides within proteins and the hair shaft proteome can be used as a basis for employing mitigation or repair strategies of hair protein damage caused by environmental or hair treatment-related insults.
OBJECTIF: Les cheveux humains sont sujet à de nombreuses agressions physiques et chimiques telles que la chaleur, les radiations ultra-violettes et les produits alcalins d'entretien des cheveux. Ces agressions entrainent des modifications moléculaires dans les protéines constituant les cheveux et elles conduisent aussi à des changements mécaniques et sensoriels des fibres capillaires. Les manifestations possibles de ces transformations sont une baisse, visible pour le consommateur, de la qualité et des indicateurs de performance des cheveux. Lors de cette étude, nous mettons en évidence les changements au niveau protéique liés à la chaleur et aux traitements alcalins. MÉTHODES: Les méthodes de profilage d'oxydoréduction protéomique utilisant des spectromètres de masses à haute résolution ont été utilisées afin d'évaluer les modifications des amino-acides dans les cheveux humains après exposition à plusieurs combinaisons de traitements thermiques et alcalins dans le but de comprendre les processus chimiques impliqués. RÉSULTATS: Nos résultats montrent que l'augmentation des modifications d'oxydoréduction est associée à des niveaux élevés d'exposition aux traitements thermiques et/ou alcalins. Le profilage des modifications post-translationnelles des structures primaires des protéines ont permis de mieux comprendre les spécificités de ces modifications ; notamment une augmentation nette du nombre des modifications des acides cystéiques liée aux traitements les plus agressifs. CONCLUSION: Ce travail d'identification des modifications engendrées par les agressions liées aux traitements capillaires ou environnementales peut désormais servir de base pour évaluer et mettre en place des techniques de réduction des risques, protection et de réparation des protéines des cheveux.
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Proteínas , Proteómica , Cabello/química , Humanos , Espectrometría de Masas , Oxidación-Reducción , Proteínas/análisis , Proteómica/métodosRESUMEN
Hair curvature underpins structural diversity and function in mammalian coats, but what causes curl in keratin hair fibres? To obtain structural data to determine one aspect of this question, we used confocal microscopy to provide in situ measurements of the two cell types that make up the cortex of merino wool fibres, which was chosen as a well-characterised model system representative of narrow diameter hairs, such as underhairs. We measured orthocortical and paracortical cross-sectional areas, and cortical cell lengths, within individual fibre snippets of defined uniplanar curvature. This allowed a direct test of two long-standing theories of the mechanism of curvature in hairs. We found evidence contradicting the theory that curvature results from there being more cells on the side of the fibre closest to the outside, or convex edge, of curvature. In all cases, the orthocortical cells close to the outside of curvature were longer than paracortical cells close to the inside of the curvature, which supports the theory that curvature is underpinned by differences in cell type length. However, the latter theory also implies that, for all fibres, curvature should correlate with the proportions of orthocortical and paracortical cells, and we found no evidence for this. In merino wool, it appears that the absolute length of cells of each type and proportion of cells varies from fibre to fibre, and only the difference between the length of the two cell types is important. Implications for curvature in higher diameter hairs, such as guard hairs and those on the human scalp, are discussed.
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Queratinas/química , Fibra de Lana/análisis , Lana/química , Animales , Recuento de Células , Oveja DomésticaRESUMEN
Oxidation of keratin results in a range of deleterious effects, including discolouration and compromised physical and mechanical properties. Keratin oxidative degradation is driven by molecular-level events, with accumulation of modifications at the protein primary level resulting directly in changes to secondary, tertiary and quaternary structure, as well as eventually changes in the observable physical and chemical properties. Advances in proteomic analysis techniques provide an increasingly clearer insight into the cascade of molecular modification underpinning keratin oxidation and how this translates through to higher order changes in properties. This chapter summarises the effects of oxidation on keratin-based materials, the types of molecular modification associated with this, and advances in techniques and approaches for characterising this modification.
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Queratinas/química , Estrés Oxidativo , Proteómica , Animales , Humanos , Conformación ProteicaRESUMEN
INTRODUCTION: In several biomedical research fields, the cross-linking of peptides and proteins has an important impact on health and wellbeing. It is therefore of crucial importance to study this class of post-translational modifications in detail. The huge potential of mass spectrometric technologies in the mapping of these protein-protein cross-links is however overshadowed by the challenges that the field has to overcome. Areas covered: In this review, we summarize the different pitfalls and challenges that the protein-protein cross-linking field is confronted with when using mass spectrometry approaches. We additionally focus on native disulfide bridges as an example and provide some examples of cross-links that are important in the biomedical field. Expert commentary: The current flow of methodological improvements, mainly from the chemical cross-linking field, has delivered a significant contribution to deciphering native and insult-induced cross-links. Although an automated data analysis of proteome-wide peptide cross-linking is currently only possible in chemical cross-linking experiments, the field is well on the way towards a more automated analysis of native and insult-induced cross-links in raw mass spectrometry data that will boost its potential in biomedical applications.
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Disulfuros/química , Espectrometría de Masas/métodos , Procesamiento Proteico-Postraduccional , Proteoma/química , Cisteína/química , Cisteína/metabolismo , Disulfuros/metabolismo , Humanos , Proteoma/metabolismoRESUMEN
Food ingredients commonly undergo heat treatment. Meat, in particular, is typically consumed after some form of heating, such as boiling or roasting. Heating of meat can introduce a wide range of structural changes in its proteinaceous components. At the 3-dimensional structural level, meat proteins may denature and form aggregates upon heating. At the molecular level, primary structure (amino acid residue) alterations reported in cooked meat include protein carbonylation, modification of aromatic residues, and the formation of Maillard reaction products. Identification of these modifications is essential for determining the mechanism of thermal processing of meat and allowing better control of the nutritional and functional properties of products. This article reviews and summarizes the current state of understanding of protein modifications at the molecular level in commonly consumed mammalian food. In addition, relevant case studies relating to characterization of heat-induced amino acid residue-level modifications in other biological materials such as milk and wool are discussed to provide complementary insights.
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Lactoferrin and ß-lactoglobulin are important protein components of mammalian milk. Maillard reactions, as well as redox chemistry, are of particular interest for dairy products because they are known to occur during common processing steps, notably heating procedures such as pasteurization. Using a redox proteomics approach, we characterized AA residue side-chain modification across a range of heating times and with or without the specific addition of lactose, to both map the key modification sites within these proteins and evaluate their sensitivity to process-induced modification. Heating in the presence of lactose resulted in significant Maillard modification (both lactosylation and carboxymethylation) to both bovine lactoferrin and ß-lactoglobulin. Notably, Lys47, a key residue in the bioactive peptide lactoferricin, was particularly susceptible to modification. Lactoferrin appeared to be fairly robust to hydrothermal treatment, with relatively low levels of oxidative modification observed. In contrast, ß-lactoglobulin was susceptible to significant oxidative modification under hydrothermal treatment, with the range and type of modifications observed suggesting compromised nutritional value. These results have important implications for processing applications in dairy foods where retention of biological function and optimal protein quality is desired.
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Lactoferrina/metabolismo , Lactoglobulinas/química , Animales , Bovinos , Proteínas de la Leche/metabolismo , Oxidación-Reducción , ProteómicaRESUMEN
The disulfide bond network within the cortex of mammalian hair has a critical influence on the physical and mechanical characteristics of the fiber. The location, pattern, and accessibility of free and crosslinked cysteines underpin the properties of this network, but have been very difficult to map and understand, because traditional protein extraction techniques require the disruption of these disulfide bonds. Cysteine accessibility in both trichocyte keratins and keratin associated proteins (KAPs) of wool was investigated using staged labeling, where reductants and chaotropic agents were used to expose cysteines in a stepwise fashion according to their accessibility. Cysteines thus exposed were labeled with distinguishable alkylation agents. Proteomic profiling was used to map peptide modifications and thereby explore the role of KAPs in crosslinking keratins. Labeled cysteines from KAPs were detected when wool was extracted with reductant only. Among them were sequences from the end domains of KAPs, indicating that those cysteines were easily accessible in the fiber and could be involved in forming interdisulfide linkages with keratins or with other KAPs. Some of the identified peptides were from the rod domains of Types I and II keratins, with their cysteines positioned on the exposed surface of the α-helix. Peptides were also identified from keratin head and tail domains, demonstrating that they are not buried within the filament structure and, hence, have a possible role in forming disulfide linkages. From this study, a deeper understanding of the accessibility and potential reactivity of cysteine residues in the wool fiber cortex was obtained.
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Cistina/química , Queratinas Específicas del Pelo/química , Lana/química , Secuencia de Aminoácidos , Animales , Datos de Secuencia Molecular , Mapeo Peptídico , Estructura Secundaria de Proteína , Oveja DomésticaRESUMEN
Fungal endophytes belonging to the genus Epichloë form associations with temperate grasses belonging to the sub-family Poöideae that range from mutualistic through to pathogenic. We previously identified a novel endophyte gene (designated gigA for grass induced gene) that is one of the most abundantly expressed fungal transcripts in endophyte-infected grasses and which is distributed and highly expressed in a wide range of Epichloë grass associations. Molecular and biochemical analyses indicate that gigA encodes a small secreted protein containing an imperfect 27 amino acid repeat that includes a kexin protease cleavage site. Kexin processing of GigA liberates within the plant multiple related products, named here as epichloëcyclins, which we have demonstrated by MS/MS to be cyclic peptidic in nature. Gene deletion of gigA leads to the elimination of all epichloëcyclins with no conspicuous phenotypic impact on the host grass, suggesting a possible bioactive role. This is a further example of a ribosomal peptide synthetic (RiPS) pathway operating within the Ascomycetes, and is the first description of such a pathway from a mutualistic symbiotic fungus from this Phylum.
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Endófitos/genética , Epichloe/genética , Proteínas Fúngicas/genética , Poaceae/microbiología , Endófitos/fisiología , Epichloe/fisiología , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Oligopéptidos/genética , Oligopéptidos/metabolismo , Péptidos Cíclicos/genética , Péptidos Cíclicos/metabolismo , Biosíntesis de Proteínas , Proteínas Ribosómicas/química , Proteínas Ribosómicas/metabolismo , Simbiosis , Espectrometría de Masas en TándemRESUMEN
The astonishing growth rate of deer antlers offers a valuable model for the discovery of novel factors and regulatory systems controlling rapid tissue growth. Numerous molecules have been identified in growing antlers using a variety of techniques. However, little is known about the spatial distribution of these molecules in situ. A technique that has the potential to help in this regard is direct proteomic analysis of tissue sections by matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS). The present study applied this technique to spatially map molecules in antler tissue sections. Two protonated molecular ions were selected: m/z 6679 and m/z 6200 corresponding to VEGF and thymosin beta-10, respectively. Superimposition of the respective ion images on to histologically stained samples showed distinct spatial distribution across the antler tissue sections which were consistent with the previous reports using in situ hybridization. Two other molecular ions specifically m/z 8100 and m/z 11,800 were also selected, corresponding to reported masses of urocortin precursor and thioredoxin, respectively. As the spatial distribution of these proteins is not specifically known, MALDI-IMS was used as a potential technique to obtain information on their distribution on antler tips. The presence of all these molecules in deer antlers were further confirmed using LC-MS/MS data. The present study also demonstrated that MALDI-IMS could be further used to image antler sections with an extended ion mass range of up to m/z 45,000, thus potentially increasing the ability to discover the distribution of a larger set of molecules that may play an important role in antler growth. We have thus demonstrated that MALDI-IMS is a promising technique for generating molecular maps with high spatial resolution which can aid in evaluating the function of novel molecules during antler growth.
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Cuernos de Venado/crecimiento & desarrollo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Tiorredoxinas/metabolismo , Timosina/metabolismo , Urocortinas/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Cuernos de Venado/citología , Ciervos , Masculino , Proteómica/métodosRESUMEN
Human scalp hairs are comprised of a central cortex enveloped by plate-like cuticle cells. The elongate cortex cells of mature fibres are composed primarily of macrofibrils-bundles of hard-keratin intermediate filaments (IFs) chemically cross-linked within a globular protein matrix. In wool, three cell types (ortho-, meso- and paracortex) contain macrofibrils with distinctly different filament arrangements and matrix fractions, but in human hair macrofibril-cell type relationships are less clear. Here we show that hair macrofibrils all have a similar matrix fraction (â¼0.4) and are typically composed of a double-twist architecture in which a central IF is surrounded by concentric rings of tangentially-angled IFs. The defining parameter is the incremental angle increase (IF-increment) between IFs of successive rings. Unlike the wool orthocortex, hair double-twist macrofibrils have considerable inter-macrofibril variation in IF increment (0.05-0.35°/nm), and macrofibril size and IF increment are negatively correlated. Correspondingly, angular difference between central and outer-most IFs is up to 40° in small macrofibrils, but only 5-10° in large macrofibrils. Single cells were observed containing mixtures of macrofibrils with different diameters. These new observations advance our understanding of the nano-level and cell-level organisation of human hair, with implications for interpretation of structure with respect the potential roles of cortex cell types in defining the mechanical properties of hair.
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Cabello/química , Cuero Cabelludo/química , Tomografía con Microscopio Electrónico , Humanos , Filamentos Intermedios/química , Filamentos Intermedios/ultraestructuraRESUMEN
Deamidation of glutamine (Q) and asparagine (N) has been recognized as a marker of degradation and aging in ancient proteins. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to study deamidation in wool textiles, we identified eight peptides from α-keratin proteins in sheep wool that could potentially be used to assess the level of degradation. For each chosen peptide, the extent of deamidation was determined by comparing the calculated theoretical distribution with the measured distribution using a genetic algorithm that gives the best fit to the measured distribution. Variations in the levels of deamidation were observed between peptides and in modern wool samples buried for up to 8 years in which deamidation levels were relatively low under short-term burial. In contrast, deamidation was higher in archeological textile fragments from medieval sites ranging from the 9th to 13th century in York (United Kingdom) and Newcastle (United Kingdom) and from the 13th to 16th century in Reykholt (Iceland). Major differences were observed between the British and the Icelandic samples, showing a negative correlation between age of samples and levels of deamidation, but highlighting the effect of local environment. In addition, nanoscale liquid chromatography-electrospray ionization tandem mass spectrometry (nanoLC-ESI-MS/MS) data indicated that deamidation in wool's α-keratin was influenced by primary and higher-order structures. Predominance of deamidation on glutamine rather than asparagine in the archeological samples was attributed to a higher abundance of Q in the α-helical core domain of keratins, neighboring residues and steric hindrance preventing deamidation of N.
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Amidas/análisis , Queratinas/análisis , Fragmentos de Péptidos/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Textiles/análisis , Anciano , Secuencia de Aminoácidos , Animales , Historia Medieval , Humanos , Queratinas/genética , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , OvinosRESUMEN
There is increasing awareness of the importance of reductive and oxidative (redox) protein damage in protein-based materials including, hair, wool, nails, and skin. Light-induced damage to protein-based materials is of particular concern because of its impact on age-related degradation and product life spans. Consequently, cosmetic applications frequently target hair and skin restoration, where the integrity of the constituent filamentous proteins is essential to a healthy appearance. The keratins constitute an important subset of the structural proteins within skin, hair, and wool. We will introduce a means to assess damage to this important group of proteins at the molecular level, utilizing proteomic techniques to track the formation or degradation of sensitive peptides within intermediate filament proteins. The degradation of three molecular markers of redox damage, the peptides SFGYR, LASDDFR, and DVEEWYIR, along with the formation of their oxidized products, is demonstrated after exposure to ultraviolet A, ultraviolet B, and blue light. The method is shown to be suitable for evaluating the protective effect of treatments, as lower levels of oxidative markers were observed after the application of a protective fiber treatment. Molecular-level redox tracking will allow more targeted design and evaluation of protection and repair treatments for protein systems.
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Queratinas/química , Luz , Proteínas/química , Secuencia de Aminoácidos , Oxidación-Reducción , ProteolisisRESUMEN
RATIONALE: In ancient and/or damaged artefacts containing keratinous materials, the species of origin of the materials can be difficult to identify through visual examination; therefore, a minimally destructive methodology for species identification is required. While hair fibres from some species have seen substantial characterisation, others such as horn or baleen have received little or no attention, or lack protein sequences allowing formal identification using proteomics techniques. METHODS: We used the PMF method (Peptide Mass Fingerprinting with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS)) to catalogue and identify diagnostic peptide markers up to the genus level. Sequences were checked using nanoflow liquid chromatography/electrospray ionisation tandem mass spectrometry (nanoLC/ESI-MS/MS) and unidentified peptides were searched against a theoretical database generated by substituting amino acids in keratin sequences. RESULTS: Specific peptides were identified by m/z and sequences characterised whenever possible for a range of species belonging to Bovidae and Camelidae, and for tissues such as baleen and horn. The theoretical database allowed an increase in the number of peptides of up to 10% in species with little genetic information. CONCLUSIONS: A proteomics approach can successfully identify specific markers for the identification of materials to the genus level, and should be considered when identification by other means is not possible. Identification by PMF is fast, reliable and inexpensive.
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Cabello/química , Queratinas/química , Espectrometría de Masas en Tándem/métodos , Lana/química , Secuencia de Aminoácidos , Animales , Camélidos del Nuevo Mundo , Camelus , Bovinos , Bases de Datos de Proteínas , Perros , Cabras , Cuernos/química , Datos de Secuencia Molecular , Proteómica , Conejos , OvinosRESUMEN
The effect of reactive oxidation species (ROS) on tryptophan or tyrosine was investigated by qualitatively determining the major detectable oxidation products generated by hydroxyl radicals, produced by the Fenton process, or singlet oxygen, generated by exposure to green light in the presence of Rose Bengal, on these photosensitive amino acids in synthetic pentapeptides. Based on mass spectrometric analysis it would appear that the hydroxyl radical favours a pathway leading to the formation of tryptophandione-based products from tryptophan. In contrast singlet oxygen attack appears to favour the formation of kynurenine-type products from tryptophan. Specific oxidative products observed proteomically are therefore potentially able to discriminate between predominant ROS-mediated pathways. To validate these findings, a keratin-enriched extract was exposed to UVB light under aqueous conditions. The observation of the conversion of tryptophan to hydroxytryptophan in marker peptides, and the absence of singlet-oxygen specific modifications, suggested that under these conditions oxidative degradation occurred primarily via hydroxyl radical attack. These observations provide the first direct proteomic evidence of the dominant photodegradation pathways in wet wool.
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Radical Hidroxilo/metabolismo , Oligopéptidos/química , Oligopéptidos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Oxígeno Singlete/metabolismo , Triptófano/metabolismo , Tirosina/metabolismo , Radical Hidroxilo/química , Oxidación-Reducción , Oxígeno Singlete/química , Triptófano/química , Tirosina/química , Rayos UltravioletaRESUMEN
Formation of lysinoalanine protein-protein crosslinks during food processing adversely impacts nutritional value. However, mapping lysinoalanine directly in food is challenging. We characterized the fragmentation pattern of lysinoalanine crosslinks in synthetic peptide models over a range of pH and time treatments using mass spectrometry. A putative diagnostic ion resulting from the cleavage of the α-carbon and ß-carbon of lysinoalanine is identified in MALDI MS/MS spectra. This represents the first step in mapping lysinoalanine in real food samples with higher precision than currently identifiable through standard or customized software. We then determined a correlated trend in the reduction of disulfide bonds and formation of lysinoalanine with increasing pH and time. Mapping lysinoalanine formation is critical to enhance our understanding of molecular processes impacting the nutritional value of foods, including notably in the development of protein alternatives that use alkaline treatment to extract protein isolates.
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Abstract: The self-assembling and gelation properties of a bioactive peptide derived from bovine casein (FFVAPFPEVFGK) were studied in the peptide's natural form (uncapped, uncapFFV) and capped with protecting groups added to both termini (capped, capFFV). Although the natural peptide (uncapFFV) did not demonstrate self-assembly, the capped peptide (capFFV) spontaneously self-assembled and formed a self-supporting gel. Variations in peptide concentration and incubation time influenced the gel's mechanical properties, suggesting the peptide's properties could be tuned and exploited for different applications. These results suggest that food-derived bioactive peptides have good potential for self-assembly and therefore utilisation as gels in functional foods and nutraceuticals. Background: Self-assembly is a natural phenomenon that occurs in many fundamental biological processes. Some peptides can self-assemble and form gels with tunable properties under given conditions. These properties, along with peptide bioactivity, can be combined to make unique biomaterials. Instead of synthesising the self-assembling bioactive peptides, we aim to extract them from natural sources. In order to use these peptides for different applications, it is essential to understand how we can trigger self-assembly and optimise the assembly conditions of these peptide gels. Scope: The self-assembling and gelation properties of a bioactive peptide derived from bovine casein (FFVAPFPEVFGK) were studied in the peptide's natural form (uncapped, uncapFFV) and capped with protecting groups added to both termini (capped, capFFV). Major conclusions: Although the natural peptide (uncapFFV) did not demonstrate self-assembly, the capped peptide (capFFV) spontaneously self-assembled and formed a self-supporting gel. Variations in peptide concentration and incubation time influenced the gel's mechanical properties, suggesting the peptide's properties could be tuned and exploited for different applications. General significance: These results suggest that food-derived bioactive peptides have good potential for self-assembly and therefore utilisation as gels in functional foods and nutraceuticals.
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The properties of milk proteins differ between mammalian species. ß-Lactoglobulin (ßlg) proteins from caprine and bovine milk are sequentially and structurally highly similar, yet their physicochemical properties differ, particularly in response to pH. To resolve this conundrum, we compared the dynamics of both the monomeric and dimeric states for each homologue at pH 6.9 and 7.5 using hydrogen/deuterium exchange experiments. At pH 7.5, the rate of exchange is similar across both homologues, but at pH 6.9 the dimeric states of the bovine ßlg B variant homologue have significantly more conformational flexibility compared with caprine ßlg. Molecular dynamics simulations provide a mechanistic rationale for the experimental observations, revealing that variant-specific substitutions encode different conformational ensembles with different dynamic properties consistent with the hydrogen/deuterium exchange experiments. Understanding the dynamic differences across ßlg homologues is essential to understand the different responses of these milks to processing, human digestion, and differences in immunogenicity.
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Cabras , Lactoglobulinas , Humanos , Animales , Lactoglobulinas/genética , Lactoglobulinas/química , Deuterio , Cabras/genética , Hidrógeno , Concentración de Iones de HidrógenoRESUMEN
Keratin-associated proteins (KAPs) are a structural component of the wool fibre and form the matrix between the keratin intermediate filaments (KIFs). The gene encoding high sulphur-protein KAP11-1 has been identified in human, cattle and mouse, but not yet in sheep, despite the economic importance of wool. In this study, PCR using primers based on the cattle KAP11-1 gene sequence produced an amplicon of the expected size with sheep DNA. Upon using PCR-Single Stranded Conformational Polymorphism (PCR-SSCP) analysis in 260 sheep, six different PCR-SSCP patterns were detected. Either one or a combination of two banding patterns was observed for each sheep, suggesting they were either homozygous or heterozygous for this gene. Sequencing of the amplicons confirmed the occurrence of six DNA sequences. All of these were unique, and the greatest homology was with KRTAP11-1 sequences from cattle, human and mouse, suggesting that they were derived from the ovine KAP11-1 gene and were allelic variants. The ovine KAP11-1 gene had an open reading frame of 477 nucleotides encoding 159 amino acids. The putative protein was rich in serine, cysteine, and threonine which account for 18.2-18.9, 12.6 and 12.0 mol%, respectively. Of these, approximately 20 of the serine and threonine residues might be phosphorylated. Five nucleotide substitutions were identified, and one was non-synonymous and would result in an amino acid change at a potential phosphorylation site. The genetic variation found in KRTAP11-1 may influence its expression, protein structure, and/or post-translational modifications, and consequently affect wool fibre structure and wool traits.
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Variación Genética , Queratinas/genética , Sistemas de Lectura Abierta/genética , Oveja Doméstica/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Secuencia de Bases , Cruzamiento , Frecuencia de los Genes/genética , Humanos , Queratinas/química , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple/genética , Alineación de SecuenciaRESUMEN
A protocol for improved extraction of peptides from in-gel protein digests, using a combination of the acid labile surfactant, sodium deoxycholate (SDC) and C18 Empore™ membranes, is presented. This approach results in better mass spectrum quality, higher numbers of identified peptide peaks and improved identification scores compared to standard tryptic digestion protocols, or protocols using only SDC or only C18 Empore™ disks. The advantages of the new protocol are demonstrated for two different types of samples: Merino wool intermediate filament proteins and Elaeis guineensis (oil palm) mesocarp proteins.
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Electroforesis en Gel Bidimensional/métodos , Mapeo Peptídico/métodos , Proteínas de Plantas/química , Lana/química , Secuencia de Aminoácidos , Animales , Arecaceae/química , Ácido Desoxicólico/química , Detergentes/química , Electroforesis en Gel Bidimensional/instrumentación , Datos de Secuencia Molecular , Mapeo Peptídico/instrumentación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
BACKGROUND: Peptide and protein damage contributes to the loss of quality and value in protein-based food and textile products as well as to the degeneration of biological tissues such as hair and skin. The effects of elevated temperature on such substrates at the molecular level are, however, relatively unknown. This paper examines the response of peptides and proteins to hydrothermal damage using mass spectrometry and reports the location of molecular markers of hydrothermal damage within wool proteins. RESULTS: The hydrothermal exposure of model peptides containing the oxidatively sensitive residues tryptophan and tyrosine revealed the formation of a number of products such as hydroxytryptophan and dihydrophenylalanine. A variety of degradation products were also observed in intermediate filament proteins, including products arising from deamidation and from oxidation of histidine, tyrosine and tryptophan residues. CONCLUSION: The products observed to form during hydrothermal exposure indicated the involvement of reactive oxygen species. Molecular markers were identified within a proteinaceous system to allow the evaluation of damage type or severity. These findings have important implications for the thermal processing of foods and textiles.