Characterizing lysinoalanine crosslinks in food systems: Discovery of a diagnostic ion in model peptides using MALDI mass spectrometry.

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.

Insight into the self-assembly and gel formation of a bioactive peptide derived from bovine casein.

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.

The protein dynamics of bovine and caprine ß-lactoglobulin differ as a function of pH.

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.

Redox proteomics analysis of hair shaft proteins upon hydrothermal and alkaline insult.

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.

The effects of blanching on composition and modification of proteins in navy beans (Phaseolus vulgaris).

Blanching is an important process in the preparation of navy beans (Phaseolus vulgaris L.) for canning. We here explore the effect of blanching which can profoundly affect protein composition and introduce protein-primary-level modifications. Amino acid analysis showed significantly decreased protein abundance (58.5%) in blanched beans compared to raw beans. Proteomic analyses revealed a decrease in high molecular weight isoforms of the major storage globulin proteins phaseolin (mean fold-change -3.7) and legumin (mean fold-change -2.5) and concomitant increase in their low molecular weight isoforms (mean fold-change 6.4 and 8.3, respectively). Blanched beans also had decreased abundance of lipoxygenase (mean fold-change -13.1), an enzyme responsible for product spoilage during storage. Increased lysinoalanine (up to 47%) and highly modified protein fragments were found in the processing waters, indicating heat- induced modifications. Correlating these molecular level changes thus provides a basis for evaluating how processing parameters can be modified to increase protein food quality.

Oral delivery of self-assembling bioactive peptides to target gastrointestinal tract disease.

Peptides are known for their diverse bioactivities including antioxidant, antimicrobial, and anticancer activity, all three of which are potentially useful in treating colon-associated diseases. Beside their capability to stimulate positive health effects once released in the body, peptides are able to form useful nanostructures such as hydrogels. Combining peptide bioactivity and peptide gel-forming potentials can create interesting systems that can be used for oral delivery. This combination, acting as a two-in-one system, has the potential to avoid the need for delicate entrapment of a drug or natural bioactive compound. We here review the context and research progress, to date, in this area.

Sub-Ångstrom structure of collagen model peptide (GPO)10 shows a hydrated triple helix with pitch variation and two proline ring conformations.

Collagens are large structural proteins that are prevalent in mammalian connective tissue. Peptides designed to include a glycine-proline-hydroxyproline (GPO) amino acid triad are biomimetic analogs of the collagen triple helix, a fold that is a hallmark of collagen-like sequences. To inform the rational engineering of collagen-like peptides and proteins for food systems, we report the crystal structure of the (GPO)10 peptide at 0.89-Å resolution, solved using direct methods. We determined that a single chain in the asymmetric unit forms a pseudo-hexagonal network of triple helices that have a pitch variation consistent with the model 7/2 helix (3.5 residues per turn). The proline rings occupied one of two states, while the helix was found to have a well-defined hydration shell involved in the stabilization of the inter-helix crystal network. This structure offers a new high-resolution basis for understanding the hierarchical assembly of native collagens, which will aid the food industry in engineering new sustainable food systems.

Changes in Milk Protein Interactions and Associated Molecular Modification Resulting from Thermal Treatments and Storage.

We investigated protein modifications that occur during short- and long-term storage of raw, pasteurized, and ultra-high-temperature processed (UHT) milks using RE-HPLC and redox proteomics. The RE-HPLC results show that casein dissociation and whey protein/κ-casein association occurred in both pasteurized and UHT milk. The extent of protein interactions was more pronounced in UHT milk after storage. The redox proteomics analyses show that primary structural level protein modifications were not correlated to processing type on the of day processing but did occur and increase during storage. Methionine oxidation was the most significant type of oxidative modification in all samples, particularly in the caseins. Methionine oxidation increased in the UHT-treated milk samples with longer storage times, especially in the micelle-phase proteins, likely due to the increasing exposure of these proteins as they migrated to the serum phase. Glycated and lactosylated early-stage Maillard reaction products were also found after heat treatment, particularly in UHT-treated milk, with the levels of these products maintained and generally increased with increasing storage time. PRACTICAL APPLICATION: Understanding changes in protein modification during heat processing and storage of liquid milk products may help develop a model to predict the quality and shelf-life stability of heat treated milk products.

The effects of a wool hydrolysate on short-chain fatty acid production and fecal microbial composition in the domestic cat (Felis catus).

Novel animal-derived fibers are of interest for the pet food industry. We here introduce a method for extracting wool proteins using controlled hydrolysis of wool. This results in an appropriate form and we demonstrate its application in pet food using the domestic cat. The effect of the wool hydrolysate on biomarkers of digestive health (e.g., fecal short-chain fatty acids and fecal microbial composition, apparent amino acid (AA) and protein digestibility), are also described. In a feeding study, a cohort of cats (n = 8 per treatment) were fed a basal diet (Control), or the basal diet supplemented with 2% wool hydrolysate, 2% inulin (Synergy1; as is) or 2% cellulose (Novagel; as is). The concentration of butyric acid was not significant (P = 0.102) between treatment groups. The concentration of fecal lactic acid was greatest (P = 0.007) in cats on the Novagel diet. Valeric acid was increased (P = 0.001) in cats fed Synergy1. Supplementation of cat diet with a wool hydrolysate showed similarities to Novagel supplementation in terms of its effects on fecal short-chain fatty acid concentrations and fecal microbiota composition. Wool hydrolysate increased apparent cysteine digestibility compared to Synergy 1 or Novogel. In terms of fecal health, intake, and palatability, the diet supplemented with wool hydrolysate was not detrimental, being similar to currently used dietary fiber supplements. These findings indicate that wool hydrolysates offer promise as an animal-derived supplement source for pet diets.

Oxidative Modification of Trichocyte Keratins.

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.

Intrinsic curvature in wool fibres is determined by the relative length of orthocortical and paracortical cells.

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.

Improved Detection and Fragmentation of Disulphide-Linked Peptides.

Characterisation of peptides containing intact disulphide bonds (DSBs) via mass spectrometry is challenging. Our study demonstrates that the addition of aniline to alpha-cyano-4-hydroxycinnamic acid improves detection and fragmentation of complex DSB peptides by matrix-assisted laser desorption/ionization, tandem time-of-flight mass spectrometry (MALDI-TOF-TOF MS). This improved assignment will be a significant new tool when a simple screening to confirm the DSB existence is required.

Protein-protein cross-linking and human health: the challenge of elucidating with mass spectrometry.

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.

Molecular markers in keratins from Mysticeti whales for species identification of baleen in museum and archaeological collections.

Baleen has been harvested by indigenous people for thousands of years, as well as collected by whalers as an additional product of commercial whaling in modern times. Baleen refers to the food-filtering system of Mysticeti whales; a full baleen rack consists of dozens of plates of a tough and flexible keratinous material that terminate in bristles. Due to its properties, baleen was a valuable raw material used in a wide range of artefacts, from implements to clothing. Baleen is not widely used today, however, analyses of this biomolecular tissue have the potential to contribute to conservation efforts, studies of genetic diversity and a better understanding of the exploitation and use of Mysticeti whales in past and recent times. Fortunately, baleen is present in abundance in museum natural history collections. However, it is often difficult or impossible to make a species identification of manufactured or old baleen. Here, we propose a new tool for biomolecular identification of baleen based on its main structural component alpha-keratin (the same protein that makes up hair and fingernails). With the exception of minke whales, alpha-keratin sequences are not yet known for baleen whales. We therefore used peptide mass fingerprinting to determine peptidic profiles in well documented baleen and evaluated the possibility of using this technique to differentiate species in baleen samples that are not adequately identified or are unidentified. We examined baleen from ten different species of whales and determined molecular markers for each species, including species-specific markers. In the case of the Bryde's whales, differences between specimens suggest distinct species or sub-species, consistent with the complex phylogeny of the species. Finally, the methodology was applied to 29 fragments of baleen excavated from archaeological sites in Labrador, Canada (representing 1500 years of whale use by prehistoric people), demonstrating a dominance of bowhead whale (Balaena mysticetus) in the archaeological assemblage and the successful application of the peptide mass fingerprinting technique to identify the species of whale in unidentified and partially degraded samples.

Cooking-Induced Protein Modifications in Meat.

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.

Proteomic and peptidomic differences and similarities between four muscle types from New Zealand raised Angus steers.

Four muscles from New Zealand-raised Angus steers were evaluated (musculus semitendinosus, m. longissimus thoracis et lumborum, m. psoas major and m. infraspinatus) to test their differences and common features in protein and peptide abundances. The ultimate goal of such a comparison is to match muscle types to products with targeted properties. Protein profiling based on two-dimensional electrophoresis showed that the overall profiles were similar, but, between muscle types, significant (p<0.05) intensity differences were observed in twenty four protein spots. Profiling of endogenous peptides allowed characterisation of 346 peptides. Quantitative analysis showed a clear distinction between the muscle types. Forty-four peptides were identified that showed a statistically significant (p<0.05) and substantial (>2-fold change) difference between at least two muscle types. These analyses demonstrate substantial similarities between these four muscle types, but also clear distinctions in their profiles; specifically a 25% difference between at least two muscles at the peptidomic level, and a 14% difference at the proteomic level.

Proteomic investigation of protein profile changes and amino acid residue-level modification in cooked lamb longissimus thoracis et lumborum: The effect of roasting.

Protein modifications of meat cooked by typical dry-heat methods (e.g., roasting) are currently not well understood. The present study utilised a shotgun proteomic approach to examine the molecular-level effect of roasting on thin lamb longissimus thoracis et lumborum patties, in terms of changes to both the protein profile and amino acid residue side-chain modifications. Cooking caused aggregation of actin, myosin heavy chains and sarcoplasmic proteins. Longer roasting time resulted in significantly reduced protein extractability as well as protein truncation involving particularly a number of myofibrillar and sarcoplasmic proteins, e.g., 6-phosphofructokinase, beta-enolase, l-lactate dehydrogenase A chain, alpha-actinin-3, actin and possibly myosin heavy chains. Modifications that have potential influence on nutritional properties, including carboxyethyllysine and a potentially glucose-derived N-terminal Amadori compound, were observed in actin and myoglobin after roasting. This study provided new insights into molecular changes resulting from the dry-heat treatment of meat, such as commonly used in food preparation.

Wool Keratin-Associated Protein Genes in Sheep-A Review.

The importance of sheep's wool in making textiles has inspired extensive research into its structure and the underlying genetics since the 1960s. Wool keratin-associated proteins (KAPs) are a key structural component of the wool fibre. The characterisation of the genes encoding these proteins has progressed rapidly with advances in the nucleotide and protein sequencing. This review describes our knowledge of ovine KAPs, their categorisation into families, polymorphism in the proteins and genes, the clustering and chromosomal location of the genes, some characteristics of gene expression and some potential effects of the KAPs on wool traits. The extent and nature of genetic variation in wool KAP genes and its association with fibre characteristics, provides an opportunity for the development of gene-markers for selective breeding of sheep to produce better wool with properties highly matched to specific end-uses.

Identification of four new gene members of the KAP6 gene family in sheep.

KAP6 is a high glycine-tyrosine keratin-associated protein (HGT-KAP) family. This family is thought to contain multiple genes. In this study, we used a KRTAP6 coding sequence to search the Ovine Genome (v3.1) and identified five homologous regions (R1-R5). All these regions contained an open reading frame, and they were either identical to, or highly similar to, sheep skin Expressed Sequence Tags (ESTs). Phylogenetic analysis revealed that R1-R5 were clustered with KAP6 sequences from different species and formed a group distinct to other HGT-KAPs. R1 was very similar to the characterised KRTAP6-1 sequence, but the remaining genes appeared to be new. PCR primers were designed to amplify and confirm the presence of these new genes. Amplicons were obtained for all of the 96 sheep investigated. Six, five, three and six PCR-SSCP patterns representing six, five, three and six DNA sequences were observed for KRTAP6-2 to KRTAP6-5 respectively. KRTAP6-2 and KRTAP6-4 had five and three SNPs respectively. Three SNPs and a 45-bp insertion/deletion were detected for KRTAP6-3, and five SNPs and an 18-bp insertion/deletion were identified for KRTAP6-5. Allele frequencies for these KAP6 genes differed between Merino and Romney sheep.

Application of a Mass Spectrometric Approach to Detect the Presence of Fatty Acid Biosynthetic Phosphopeptides.

The details of plant lipid metabolism are relatively well known but the regulation of fatty acid production at the protein level is still not understood. Hence this study explores the importance of phosphorylation as a mechanism to control the activity of fatty acid biosynthetic enzymes using low and high oleic acid mesocarps of oil palm fruit (Elaeis guineensis variety of Tenera). Adaptation of neutral loss-triggered tandem mass spectrometry and selected reaction monitoring to detect the neutral loss of phosphoric acid successfully found several phosphoamino acid-containing peptides. These peptides corresponded to the peptides from acetyl-CoA carboxylase and 3-enoyl-acyl carrier protein reductase as identified by their precursor ion masses. These findings suggest that these enzymes were phosphorylated at 20th week after anthesis. Phosphorylation could have reduce their activities towards the end of fatty acid biosynthesis at ripening stage. Implication of phosphorylation in the regulation of fatty acid biosynthesis at protein level has never been reported.