Unlocking the bioactivity of meat proteins: Comparison of meat and meat hydrolysate via simulated gastrointestinal digestion.

Understanding the functional attributes of meat proteins is crucial for determining their nutritional benefits. Depending on the form in which meat proteins are available, the digestive process can release peptides which are valuable for nutrition and may also possess bioactive properties, affecting physiology. Liquid chromatography - mass spectrometry (LC-MS) was used to quantitatively compare the molecular peptide features (representing non-redundant peptides), during the different stages of a simulated gastrointestinal digestion process of a minimally processed powdered meat and its enzymatically produced hydrolysate. Results from a principal component analysis (PCA) indicated that the hydrolysate did not undergo extensive additional digestion whereas the powdered meat was digested both at the gastric and in the intestinal phases. Bioactive peptide sequence prediction identified the meat hydrolysate but not the meat powder as the only source of exact and partial bioactive matches in the angiotensin-I converting enzyme and dipeptidyl peptidase IV inhibition categories. Also, a higher source of cryptides (encrypted bioactive peptides), indicated that meat hydrolysates are potentially a better substrate for the release of these enzyme inhibitory peptides. These observations thus suggest that pre-digestion of a complex food matrix such as meat, may enhance its bioavailability following oral consumption early in the digestion process. SIGNIFICANCE: This work highlights enzymatic hydrolysis of meat proteins prior to ingestion allows for potentially higher bioavailability of bioactive peptides that inhibit angiotensin-I converting enzyme and dipeptidyl peptidase IV, thus possibly aiding high blood pressure and type 2 diabetes management.

Wool fiber curvature is correlated with abundance of K38 and specific keratin-associated proteins.

Curvature in mammalian fibers, such as wool and human hair, is an important feature of the functional trait of coat structure-it affects mechanical resilience and thermo-insulation. However, to examine the relationship between fiber curvature, ultrastructure and protein composition fiber diameter variability has to be minimal. To achieve this we utilised the progeny of straight-wool domestic sheep mutant rams (crimp mutants) and wild-type ewes. Proteomic and structural results of the resulting mutant/wild-type twin pairs confirmed that straight crimp mutant wool had a normal cuticle and the same cortical protein and ultrastructural building blocks as wild-type (crimpy) fibers but differed in the layout of its cortical cells and in the relative proportions of keratin (K) and keratin-associated proteins (KAPs). In the case of the crimp mutants (straight fibers), the orthocortex was distributed in a fragmented, annular ring, with some orthocortical cells near the central medulla, a pattern similar to that of straight hairs from humans and other mammals. Crimp mutant fibers were noted for the reduced abundance of some proteins in the high glycine-tyrosine class normally associated with the orthocortex, specifically the KAP6, KAP7, and KAP8 families, while proteins from the KAP16 and KAP19 were found in increased abundance. In addition to this, the type I keratin, K38, which is also associated with the orthocortex, was also found at lower abundance in the mutant fibers. Conversely, proteins from the ultra-high sulfur class normally associated with the paracortex, specifically the KAP4 and KAP9 families, were found in higher abundance.

A detailed mapping of the readily accessible disulphide bonds in the cortex of wool fibres.

Trichocyte keratin intermediate filament proteins (keratins) and keratin associated proteins (KAPs) differ from their epithelial equivalents by having significantly more cysteine residues. Interactions between these cysteine residues within a mammalian fiber, and the putative regular organization of interactions are likely important for defining fiber mechanical properties, and thus biological functionality of hairs. Here we extend a previous study of cysteine accessibility under different levels of exposure to reducing compounds to detect a greater resolution of statistically non-random interactions between individual residues from keratins and KAPs. We found that most of the cysteines with this non-random accessibility in the KAPs were close to either the N- or C- terminal domains of these proteins. The most accessible non-random cysteines in keratins were present in the head or tail domains, indicating the likely function of cysteine residues in these regions is in readily forming intermolecular bonds with KAPs. Some of the less accessible non-random cysteines in keratins were discovered either close to or within the rod region in positions previously identified in human epithelial keratins as involved in crosslinking between the heterodimers of the tetramer. Our present study therefore provides a deeper understanding of the accessibility of disulfides in both keratins and KAPs and thus proves that there is some specificity to the disulfide bond interactions leading to these inter- and intra-molecular bonds stabilizing the fiber structure. Furthermore, these suggest potential sites of interaction between keratins and KAPs as well as keratin-keratin interactions in the trichocyte intermediate filament.

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.

Insights in Human Hair Curvature by Proteome Analysis of Two Distinct Hair Shapes.

Scalp hair is a universal human characteristic, and a wide range of hair shape and color variations exists. Although differences in human scalp hair shape are visually apparent, the underpinning molecular insights are yet to be fully explored. This work reports the determination of differences at the protein level between two distinct groups of hair shape: very straight samples versus very curly hair samples. An in-depth highresolution liquid-chromatography mass spectrometry proteome analysis study was performed on hair samples from 50 individuals (pooled in 10 × 5 samples) with very curly hair and 50 subjects with very straight hair (pooled in 10 × 5 samples) to decipher differences between the two experimental groups at the protein level. Our results demonstrate that a distinction between the two experimental groups (very straight vs. very curly) can be made based on their overall protein profiles in a multivariate analysis approach. Further investigation of the protein expression levels between these two groups pinpointed 13 unique proteins which were found to be significantly different between the two groups, with an adjusted p-value < 0.05 and a fold change of more than two. Although differences between the very curly and the very straight hair sample groups could be identified, linkage between population differences and curl phenotype is currently unknown and requires further investigation.

Analysis of Human Faecal Host Proteins: Responsiveness to 10-Week Dietary Intervention Modifying Dietary Protein Intake in Elderly Males.

Faecal proteomics targeting biomarkers of immunity and inflammation have demonstrated clinical application for the identification of changes in gastrointestinal function. However, there are limited comprehensive analyses of the host faecal proteome and how it may be influenced by dietary factors. To examine this, the Homo sapiens post-diet proteome of older males was analysed at the completion of a 10-week dietary intervention, either meeting the minimum dietary protein recommendations (RDA; n = 9) or twice the recommended dietary allowance (2RDA, n = 10). The host faecal proteome differed markedly between individuals, with only a small subset of proteins present in ≥ 60% of subjects (14 and 44 proteins, RDA and 2RDA, respectively, with only 7 common to both groups). No differences were observed between the diet groups on the profiles of host faecal proteins. Faecal proteins were detected from a wide range of protein classes, with high inter-individual variation and absence of obvious impact in response to diets with markedly different protein intake. This suggests that well-matched whole food diets with two-fold variation in protein intake maintained for 10 weeks have minimal impact on human faecal host proteins.

ACE inhibitory peptides in standard and fermented deer velvet: an in silico and in vitro investigation.

BACKGROUND: The use of deer velvet antler (DVA) as a potent traditional medicine ingredient goes back for over 2000 years in Asia. Increasingly, though, DVA is being included as a high protein functional food ingredient in convenient, ready to consume products in Korea and China. As such, it is a potential source of endogenous bioactive peptides and of 'cryptides', i.e. bioactive peptides enzymatically released by endogenous proteases, by processing and/or by gastrointestinal digestion. Fermentation is an example of a processing step known to release bioactive peptides from food proteins. In this study, we aimed to identify in silico bioactive peptides and cryptides in DVA, before and after fermentation, and subsequently to validate the major predicted bioactivity by in vitro analysis. METHODS: Peptides that were either free or located within proteins were identified in the DVA samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS) followed by database searching. Bioactive peptides and cryptides were identified in silico by sequence matching against a database of known bioactive peptides. Angiotensin-converting enzyme (ACE) inhibitory activity was measured by a colorimetric method. RESULTS: Three free bioactive peptides (LVVYPW, LVVYPWTQ and VVYPWTQ) were solely found in fermented DVA, the latter two of which are known ACE inhibitors. However matches to multiple ACE inhibitor cryptides were obtained within protein and peptide sequences of both unfermented and fermented DVA. In vitro analysis showed that the ACE inhibitory activity of DVA was more pronounced in the fermented sample, but both unfermented and fermented DVA had similar activity following release of cryptides by simulated gastrointestinal digestion. CONCLUSIONS: DVA contains multiple ACE inhibitory peptide sequences that may be released by fermentation or following oral consumption, and which may provide a health benefit through positive effects on the cardiovascular system. The study illustrates the power of in silico combined with in vitro methods for analysis of the effects of processing on bioactive peptides in complex functional ingredients like DVA.

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.

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.

In silico modeling of protein hydrolysis by endoproteases: a case study on pepsin digestion of bovine lactoferrin.

This paper presents a novel model of protein hydrolysis and release of peptides by endoproteases. It requires the amino-acid sequence of the protein substrate to run, and makes use of simple Monte-Carlo in silico simulations to qualitatively and quantitatively predict the peptides that are likely to be produced during the course of the proteolytic reaction. In the present study, the model is applied to the case of pepsin, the gastric protease. Unlike pancreatic proteases, pepsin has a low substrate specificity and therefore displays a stochastic behavior that is particularly challenging to model and predict. Two versions of the model are studied and compared with peptidomic data obtained during pepsin hydrolysis of bovine lactoferrin. The first version of the model takes into account cleavage probabilities according to the amino acids in position P1-P1' only, whereas the second version also accounts for the influence of neighbor amino acids (P4, P3, P2, P2', P3', P4') and peptide terminal ends. The second version of the model was able to reproduce many real-world features of the reported behavior of pepsin, such as the peptide size distribution, or the quantity of free amino-acids. More remarkably, 50% of the experimentally monitored peptides (44/87) lay within the 120 most abundant simulated peptides. The presented methodology has the advantage of being applicable not only to different proteins, but to different enzymes as well, as long as cleavage frequency data are available.

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 tracking of hydrothermal Maillard and redox modification in lactoferrin and ß-lactoglobulin: Location of lactosylation, carboxymethylation, and oxidation sites.

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.

Oxidative Modification in Human Hair: The Effect of the Levels of Cu (II) Ions, UV Exposure and Hair Pigmentation.

Protein oxidative degradation is implicated in a wide range of deleterious effects. For human hair, this oxidative damage can lead to significant observable changes in fiber physical and visual properties. A redox proteomic approach was applied to map molecular modification in human hair proteins and correlate this modification with the abundance of copper (II) ions, the levels of UV exposure and the general level of hair pigmentation. An increase in oxidative modification was observed with increasing copper (II) ion levels, regardless of the pigmentation level. Significantly, increased protein oxidative modification was also observed to occur in both lightly and darkly pigmented hair tresses even in the absence of irradiation, albeit at lower relative levels. Modification levels increased with increased copper (II) ion concentration. This new finding indicates that the level of copper (II) ions in human hair plays a key role in mediating protein oxidation, with or without exposure to UV light. Overall, these results strongly suggest that minimization of the level of copper (II) ions in human hair will mitigate and/or slow protein oxidative modification and therefore lower overall hair damage.

Digestion proteomics: tracking lactoferrin truncation and peptide release during simulated gastric digestion.

The extent to which nutritional and functional benefit is derived from proteins in food is related to its breakdown and digestion in the body after consumption. Further, detailed information about food protein truncation during digestion is critical to understanding and optimising the availability of bioactives, in controlling and limiting allergen release, and in minimising or monitoring the effects of processing and food preparation. However, tracking the complex array of products formed during the digestion of proteins is not easily accomplished using classical proteomics. We here present and develop a novel proteomic approach using isobaric labelling to mapping and tracking protein truncation and peptide release during simulated gastric digestion, using bovine lactoferrin as a model food protein. The relative abundance of related peptides was tracked throughout a digestion time course, and the effect of pasteurisation on peptide release assessed. The new approach to food digestion proteomics developed here therefore appears to be highly suitable not only for tracking the truncation and relative abundance of released peptides during gastric digestion, but also for determining the effects of protein modification on digestibility and potential bioavailability.

Molecular marker approaches for tracking redox damage and protection in keratins.

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.

Protein oxidation: identification and utilisation of molecular markers to differentiate singlet oxygen and hydroxyl radical-mediated oxidative pathways.

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.

Proteomic characterisation of hydrothermal redox damage.

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.

Isobaric labeling approach to the tracking and relative quantitation of peptide damage at the primary structural level.

Protein oxidative damage lies behind skin and hair degradation and the deterioration of protein-based products, such as wool and meat, in addition to a range of serious health problems. Effective strategies to ameliorate degenerative processes require detailed fundamental knowledge of the chemistry at the molecular level, including specific residue-level products and their relative abundance. This paper presents a new means of tracking damage-induced side-chain modification in peptides using a novel application for isobaric label quantification. Following exposure to heat and UVA and UVB irradiation, tryptophan and tyrosine damage products in synthetic peptides were characterized and tracked using ESI-MS/MS and iTRAQ labeling-based relative quantification. An in-depth degradation profile of these peptides was generated, enabling the formation of even low-abundance single-residue-level modifications to be sensitively monitored. The development of this novel approach to profiling and tracking residue-level protein damage offers significant potential for application in the development and validation of protein protection treatments.

Characterisation of low abundance wool proteins through novel differential extraction techniques.

Fibres from human hair and wool are characterised by two main types of proteins: intermediate filament proteins (IFPs) and keratin associated proteins (KAPs). The IFPs, comprising over 50% of the fibre, tend to dominate 2-D electrophoretic maps, hindering identification of the less-abundant KAPs. This has been compounded in wool fibres by the relatively limited amount of sequence information available, with approximately 35 distinct protein sequences from ten KAP families being available, in contrast to human hair, where the sequences from well over 80 proteins from 26 KAP families are known. Additional complications include the high degree of homology within these families, ranging from 70 to 95%, and the dominance of cysteine residues in a number of KAP families with their high propensity to form cross-links. The lack of sequence information for wool KAPs has been partly overcome through the recent acquisition of new sequences. Fractionation of the proteins on the basis of their solubility with pH, urea and DTT concentration has resulted in protein extracts in which the IFP concentration has been considerably reduced. These improvements have enabled the identification of low-abundance proteins in 2-D electrophoretic maps and represent a significant advance in our knowledge of the wool proteome.

Profiling of residue-level photo-oxidative damage in peptides.

Protein and peptide oxidation is a key feature in the progression of a variety of disease states and in the poor performance of protein-based products. The present work demonstrates a mass spectrometry-based approach to profiling degradation at the amino acid residue level. Synthetic peptides containing the photosensitive residues, tryptophan and tyrosine, were used as models for protein-bound residue photodegradation. Electrospray ionisation tandem mass spectrometry (ESI-MS/MS) was utilised to characterise and provide relative quantitative information on the formation of photoproducts localised to specific residues, including the characterisation of low abundance photomodifications not previously reported, including W + 4O modification, hydroxy-bis-tryptophandione and topaquinone. Other photoproducts observed were consistent with the formation of tyrosine-derived dihydroxyphenylalanine (dopa), trihydroxyphenylalanine, dopa-quinone and nitrotyrosine, and tryptophan-derived hydroxytryptophan, dihydroxytryptophan/N-formylkynurenine, kynurenine, hydroxyformylkynurenine, tryptophandiones, tetrahydro-beta-carboline and nitrotryptophan. This approach combined product identification and abundance tracking to generate a photodegradation profile of the model system. The profile of products formed yields information on formative mechanisms. Profiling of product formation offers new routes to identify damage markers for use in tracking and controlling oxidative damage to polypeptides.