Anita J. Grosvenor

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.

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.

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

Lactoferrin and β-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 β-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, β-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.

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.

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.

Protein Fibre Surface Modification

Many natural fibres, including wool, cashmere and silk, are protein-based materials; the dry weight of wool is almost entirely derived from proteins (Maclaren & Milligan 1981). As such, they possess an inherent structural and chemical heterogeneity not found in synthetic polymers. Although typically less heterogeneous than biological fibres, the rapidly emerging range of commercially available protein-based biomaterials also contain a wide range of functionality derived from their constituent primary and secondary protein structure. The response of fibres to processes such as dyeing and finishing treatments correlates directly to their structural and chemical properties, and this is particularly true for surface treatments. Due to its barrier function in the fibre, modification of the surface has a profound impact on processing and performance. Keratinous fibres such as wool and cashmere have an outer lipid layer which results in a hydrophobic surface. Recently a range of innovative and novel fibre surface technologies has been developed, many of which involve altering surface properties by the removal of the lipid layer, which exposes a proteinaceous surface with a variety of reactive chemical moieties. Treatments that can be covalently bound to fibre surface components, rather than simply physically applied to the surface, offer the potential for superior durability. The surface modification of proteinaceous fibres has a long history. These include plasma applications, which expose and generate functional groups on the protein surface, etching into the surface of the cuticle scales, to improve properties such as surface wettability, dyeability, shrink-resistance and felting-resistance. Chemical approaches utilised include ozone treatments, which cause oxidation of the surface and altered ionic balance, leading to a more plastic and reactive fibre surface and shrinkage control; chlorination, which improves sorption characteristics and reduces shrinkage; hydrogen peroxide treatment; and acid anhydride acetylation of silk and wool, which improves the textile response to dyeing, shrink resist, and setting treatments. Enzymatic treatments have been utilised to decuticulate the surface and improve properties such as shrink resistance. More recent developments include reaction of functional chemical agents or branched molecules to exposed reactive groups on the fibre surface, enabling the attachment of covalently-bound smart treatments, or the amplification of reactive groups for increased functionality. This chapter outlines developments in the area of targeted surface modification of proteinbased fibres and textiles, including summarising applications and future directions. It is not

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

ABSTRACT 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. Highlighted Article: Curvature in mammalian hairs is underpinned by the relative difference in length between orthocortical and paracortical cells rather than their proportion or number along each side of the fibre.

The physical and chemical disruption of human hair after bleaching - studies by transmission electron microscopy and redox proteomics

To understand the structural and chemical effects of cosmetic peroxide bleaching on human hair.

Feather meal-based thermoplastics: Methyl vinyl ether/maleic anhydride copolymer improves material properties

The poultry meat processing industry produces large amounts of feather meal, which is traditionally used as lowvalue plant fertilizer or fish nutrient. A higher value application for feather meal is described in this paper - a thermal blending and compression molding method to create compostable composites out of environmentally friendly materials: feather meal, glycerol, and a biodegradable copolymer of methyl vinyl ether and maleic anhydride (MVEMA). The composite’s mechanical, microstructural and chemical characteristics are described. Feather meal plasticized only with glycerol is mechanically fragile, with average tensile strength of 1.7 MPa, Young’s modulus of 296 MPa and strain-at-failure of 0.6 %. With the addition of MVEMA copolymer, feather meal is transformed into a ductile plastic composite, with tensile modulus reduced 2- to 5-fold and strain-at-failure increased 4- to 25-fold. These properties are ideal for creating feather mealbased compostable bioplastics for agricultural and industrial applications.

Novel Approaches to Tracking the Breakdown and Modification of Food Proteins through Digestion

Abstract Understanding how foods break down during consumption and digestion is a key goal of food science. The cascade of protein breakdown and modification, in particular, is a critical concern in optimizing the release and uptake of biologically active peptide sequences, as well as in monitoring potential allergen uptake. Additionally, the effect of processing, storage, and food preparation protocols on protein breakdown is highly relevant with respect to human nutrition, health, and well-being. However, in spite of the need to understand these processes, robust technologies for the profiling and tracking of protein breakdown through digestion have remained elusive, largely due to the complexity of the digestion products and their relative abundances. The emerging proteomic subdiscipline of redox proteomics provides new powerful tools to track protein breakdown and modification through digestion, and evaluate the effect of processing and preparation protocols on this digestive process. These technologies offer the potential for accurate profiling and tracking of protein truncation modification and cascades within complex food systems. The approaches exemplified here have application to the evaluation of the digestive behavior of any food protein, determining and controlling the impact of food processing on nutrition and functionality, and developing new food delivery systems that can deliver specific active proteins and peptides through digestion with minimum degradation and modification.