Investigating amino acid residue-level damage using novel proteomic approaches, with application to wool proteins

Abstract

Damage to wool is derived from the modification of its constituent proteins, as the dry matter of wool is principally made up of protein. A significant component of protein damage consists of modifications to the amino acid side chains. In wool, these modifications can lead to lowered quality in the form of reduced strength and elasticity (phototendering), and to undesirable colour changes (photobleaching and photoyellowing). These limitations hinder the competitiveness of natural wool fibres against synthetic counterparts in carpet and apparel manufacture and consumer appeal. Better approaches to resolving these limitations will arise from an increased understanding of the process of protein damage in wool at the molecular level. Wool damage occurs during on-farm production, processing, and over the product lifespan with the consumer. During processing, elevated temperatures and chemical treatments are applied to the fibre, while the primary degradative influences experienced during subsequent product life are abrasion and light exposure. This study used novel proteomic techniques to investigate the heat- and light-induced degradation of synthetic model peptides and of intermediate filaments derived from wool. Electrospray ionisation (ESI) and matrix-assisted laser desorption ionisation (MALDI) mass spectrometric techniques were used to comprehensively characterise the degradation of model peptides containing tryptophan and tyrosine. A total of sixteen residue side-chain degradation products were detected and confirmed using tandem mass spectrometry and detailed de novo sequencing. The relative abundance of parent peptides and their degradation products was determined using two mass spectrometric approaches: a label-free approach, based on observed parent ion abundance, and an isobaric-labelling approach, based on the abundance of the reporter fragment ions of a commercially available isobaric tag (iTRAQ) in tandem mass spectra. The influence of reactive oxygen species was apparent by the UVA-, UVB-, and hydrothermallyinduced modifications observed in the model peptides. The deduced oxidative degradation pathways shed light into the mechanisms behind protein damage under these conditions. In the UVA and UVB-irradiated proteins, the modifications observed were consistent with the hydroxyl radical playing a key role, as well as the involvement of peroxynitrite. The observation of reactive oxygen species-generated modifications in the hydrothermally-damaged peptides was particularly noteworthy, as the heat-induced generation of reactive oxygen species is not well recognised or reported. When wool-derived intermediate filament proteins were exposed to hydrothermal insult, the formation of residue-level modifications was also observed after reverse phase high performance liquid chromatography (HPLC) and MALDI mass spectrometric analysis. The presence of oxidative modifications confirmed the involvement of reactive oxygen species. Using quantitative isobaric iTRAQ labels, the degradation of a number of marker peptides and the formation of selected modified products was monitored. Such molecular-level markers of damage provided sensitive and specific evaluation of the type and extent of protein damage experienced. It is anticipated that utilisation of these markers will provide a sensitive and effective tool for determining and tracking protein damage at the molecular level, as well as facilitating validation and optimisation of protection or repair strategies for wool and other protein fibres. Although this research focussed on understanding protein degradation in wool, the understandings gleaned from the work on model peptides and the molecular damage marker approach may be extended to a variety of other substrates in which protein degradation is a concern. These include a number of living tissues, in which protein damage plays a role in the development of disease states, and other protein-based substrates such as skin, hair, leather, and protein-based foods.