Charisa D. Cornellison

Developing the wool proteome

The wool proteome has been largely uncharted due to a lack of database coverage, poor protein extractability and dynamic range issues. Yet, investigating correlations between wool physical properties and protein content, or characterising UV-, heat- or processing-induced protein damage requires the availability of an identifiable and identified proteome. In this study we have achieved unprecedented wool proteome identification through a strategy of comprehensive data acquisition, iterative protein identification/validation and concurrent augmentation of the sequence database. Data acquisition comprised a range of different hyphenated MS techniques including LC-MS/MS, LC-MALDI, 2D-LC-MS/MS and SDS-PAGE LC-MS. Using iterative searching of databases and search result combination using ProteinScape, a systematic expansion of identifiable proteins in the sequence database was achieved. This was followed by extensive validation and rationalisation of the protein identifications. In total, 72 complete and 30 partial ovine-specific protein sequences were added to the database, and 113 wool proteins were identified. Enhanced access to ovine-specific protein identification and characterisation will facilitate all wool fibre protein chemistry and proteomics research.

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.

Photoproducts Formed in the Photoyellowing of Collagen in the Presence of a Fluorescent Whitening Agent

Fibrous proteins discolor on exposure to the UV component of sunlight. This effect is exacerbated in the presence of fluorescent whitening agents (FWAs), which are often applied to textiles to improve product brightness. Tryptophan photoproducts have been identified as significant contributors to protein photoyellowing; however, the role of non–tryptophan‐derived chromophores is less clear. In this study bovine collagen, containing no tryptophan residues, was irradiated in the presence and absence of the stilbene‐derived FWA, 4,4′‐bis(2‐sulfostyryl)biphenyl (DSBP) and photoproducts were identified using mass spectrometry. Photoyellowing was found to be dependent on the presence of the FWA, attributed to amplified generation of reactive oxygen species (ROS), particularly hydroxyl radicals and peroxynitrite. Four key proteinaceous photomodifications contributing directly to photoyellowing were located in irradiated collagen pretreated with DSBP, namely dopa, nitrophenylalanine, nitrotyrosine and nitrohistidine. This represents the first direct characterization of the three nitrated residues in the photoyellowing of an isolated fibrous protein, and implicates the ROS, peroxynitrite, as a key contributor to protein photoyellowing. Direct oxidative modification of the FWA itself was also observed. This study demonstrates that, even in the absence of tryptophan residues, significant photomodification of protein residues leading to chromophore formation occurs in the presence of an FWA.

The Photoyellowing of Stilbene‐derived Fluorescent Whitening Agents—Mass Spectrometric Characterization of Yellow Photoproducts

The application of fluorescent whitening agents (FWAs) significantly accelerates the photoyellowing of wool and silk under exposure to the ultraviolet and visible components of sunlight <500 nm. The photochemistry involved in this process is poorly understood, particularly the role of photoproducts derived directly from the FWA itself. Hydroxylation was identified as the key initial mechanism of photodegradation leading to coloration of the solution in the irradiation of the stilbene‐derived FWA 4,4′‐bis(2‐sulfostyryl)biphenyl (DSBP) in the presence of hydrogen peroxide (H2O2). Polyhydroxylated DSBP derivatives were implicated as critical intermediates in the formation of yellow photoproducts under these conditions. The formation of trace quantities of DSBP quinone derivatives subsequent to hydroxylation was identified as the key cause of DSBP photoyellowing. These results are the first successful characterization of yellow photoproducts resulting directly from irradiation of a stilbene‐based FWA. Formation of these yellow stilbene‐based FWA‐derived photoproducts may occur on the surface of FWA‐treated wool exposed to simulated sunlight, as previous work has shown that H2O2 is photogenerated when wet FWA‐treated wool is exposed to light. These results therefore suggest that yellow FWA‐derived photoproducts contribute to the accelerated photoyellowing of FWA‐treated wool.

Redox proteomic evaluation of bleaching and alkali damage in human hair

Protein modification and damage in human hair, resulting from environmental, cosmetic and grooming stresses, create changes to visual and tactile characteristics and correlates with consumer perception of quality. This study outlines molecular‐level evaluation of modification resulting from peroxide (bleaching) and alkaline straightening (relaxing) treatments.

Unravelling the proteome of wool: towards markers of wool quality traits.

With ongoing efforts to make wool more competitive alongside other fibres, notably synthetics, there is a need to obtain a better understanding of the relationship between protein composition and characteristic wool properties to assist sheep breeding programmes. Before this can be achieved, the wool proteome needs to be mapped, by gel and non-gel techniques, and methods developed to reliably quantitate protein expression. Nevertheless, in setting out to achieve this, there are numerous challenges to be faced in the application of proteomics to wool, including the relative lack of wool protein sequence information in the publically accessible databases, the wide variety of proteins in the wool fibre, the high homology within the Type I and Type II keratins, the high degree of homology and polymorphism within individual keratin associated protein families, the dominance of the keratin proteins over others in wool and the peculiar chemistries found in keratins and their associated proteins. This review will discuss the various strategies that have been developed to both identify these proteins in the wool protein map and quantify them with the view to their application to the identification of markers for wool quality traits.

Like Follicle, like Fibre? Diameter and not Follicle Type Correlates with Fibre Ultrastructure

The hair follicles of most mammals are of two types, primary and secondary. Primary follicles develop earlier and have a prominent arrectorpili muscle. Secondary follicles have less prominent muscles and are often clumped, sharing a common opening from which fibres emerge. It is not entirely clear what types of follicles occur in human scalps. Partly this is because human hairs have a uniform appearance, unlike many mammals in which robust primary hairs differ markedly from narrow secondary fibres. Some sheep breeds are an exception because like humans, wool fibres have a similar macro-scale appearance irrespective of follicle type. How deep does this similarity go Using electron microscopy, we examined wool primary fibres from different breeds and contrasted them to secondary fibres. For fibres of similar diameter, there was no significant difference in the ultrastructure or proportion and distribution of cortex cell types in primary and secondary fibres. We conclude that fibre diameter is the most important fibre parameter with respect to structural differences between fibres, not whether the fibres originate from primary or secondary follicles.

MALDI-MS Redox Lipidomics Applied to Human Hair: A First Look.

Lipids are an amazingly diverse group of biomolecules with an array of biological functions including protecting and maintaining key properties and structure. Oxidative insult in the form of UV, hydrothermal, or other damage leads to compromised lipid function and can be linked to a wide range of consumer complaints. This proof-of-principle study applied and evaluated redox lipidomic approaches for the characterization and profiling of selected lipids and their oxidation products in human hair. It was observed that cholesterol and cholesterol derivatives regions appeared to be the most susceptible to oxidative damage and this leads to further experiments, including the systematic characterization of oxidative products, and correlation of modifications with damage protocol.