Stefan Clerens

Proteomic evaluation and location of UVB-induced photo-oxidation in wool

Photo-oxidation of proteinaceous fibres correlates directly to lowered appearance retention and performance, with particular commercial significance for wool and human hair. We here outline the first detailed proteomic evaluation of differential photo-oxidation occurring in the cuticle and cortex of wool fibres. After exposure of whole wool to UVB irradiation, physical disruption techniques designed to minimise further oxidative modification were utilised to prepare enriched cuticle and cortex fractions. This was followed by comprehensive redox proteomic analyses of photo-oxidation via the location within the fibre components of modifications to aromatic residues. An oxidative classification system was developed and applied to provide further insight into differential photo-oxidation. These results were compared with coloration changes observed within the cuticular and cortical components of the fibre. In this study, although the cuticle was observed to have a higher level of baseline oxidation, the cortex exhibited significantly higher levels of photo-oxidation under UVB irradiation. These proteomic results were supported by the observation of significantly higher photoyellowing within the cortex than within the cuticle. It has been assumed that fibre photo-oxidation was predominantly confined to the wool cuticle, and that changes within the cuticle had the greatest effect on appearance retention. These results provide new insight into the contribution of the cortex to photo-induced discoloration of proteinaceous animal fibres.

Direct profiling and identification of peptide expression differences in the pancreas of control and ob/ob mice by imaging mass spectrometry

Imaging mass spectrometry (IMS) technology utilizes MALDI MS to map molecules of interest in thin tissue sections. In this study, we have evaluated the potential of MALDI IMS to study peptide expression patterns in the mouse pancreas under normal and pathological conditions, and to in situ identify peptides of interest using MS/MS. Different regions of the pancreas of both control and ob/ob mice were imaged, resulting in peptide‐specific profiles. The distribution of ions of m/zu20053120 and 3439 displayed a striking resemblance with Langerhans islets histology and, following MS/MS fragmentation and database searching were identified as C‐peptide of insulin and glicentin‐related polypeptide, respectively. In addition, a significant increase of the 3120 peak intensity in the obese mice was observed. This study underscores the potential of MALDI IMS to study the contribution of peptides to pancreas pathology.

Anti–α-enolase Antibodies in Patients with Inflammatory Bowel Disease

BACKGROUNDnPatients with inflammatory bowel disease (IBD) carry autoantibodies such as perinuclear antineutrophil cytoplasmic antibodies (pANCA). alpha-Enolase has been proposed as a target antigen in IBD. We evaluated the prevalence and diagnostic value of anti-alpha-enolase antibodies in IBD and related disorders.nnnMETHODSnWe used a classic proteomic approach with extracts from granulocytes and pANCA-positive ulcerative colitis (UC) sera to confirm alpha-enolase as a target antigen. By means of Western blot analysis, we screened a cohort of 525 subjects for the presence of anti-alpha-enolase antibodies. We performed GeneArray experiments on RNA extracted from colonic mucosal biopsies from 35 IBD and 6 control patients.nnnRESULTSnWe detected anti-alpha-enolase antibodies 49.0% of patients with UC, 50.0% of patients with Crohns disease, 30.5% of patients with primary sclerosing cholangitis, 37.8% of patients with autoimmune hepatitis, 34.0% of patients with ANCA-positive vasculitis, 31.0% of non-IBD gastrointestinal controls, and 8.5% of healthy controls. Gene array experiments showed a significant upregulation of alpha-enolase mRNA in colonic mucosal biopsies from patients with IBD, but not from controls. There was no association between the presence of pANCA and anti-alpha-enolase antibodies. Preabsorption with alpha-enolase did not eliminate the pANCA pattern on indirect immunofluorescence.nnnCONCLUSIONSnAnti-alpha-enolase antibodies are present in a substantial proportion of patients with IBD, patients with various inflammatory/autoimmune disorders, and non-IBD gastrointestinal controls. Therefore, anti-alpha-enolase antibodies are of limited diagnostic value for the diagnosis of IBD.

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.

Effect of Cooking on Meat Proteins: Mapping Hydrothermal Protein Modification as a Potential Indicator of Bioavailability

Thermal treatment of meat proteins induces a range of observable and molecular-level changes. In order to understand and track these heat-induced modifications at the amino acid level, various analytical techniques were used. Changes were observed both in the soluble and in the insoluble fractions after hydrothermal treatment of minced beef samples. Redox proteomics clearly indicated increasing oxidative modification of proteins with increased heat exposure. Collagens in the soluble fraction and myosin in the insoluble fraction were found to be highly susceptible to such modifications. Maillard reaction products in the insoluble and pyrrolidone formation in the soluble fraction steadily increased with increased heat exposure. Fluorescence studies indicated a rapid increase in fluorescence with heat, suggesting the formation of advanced glycation end products. Overall these results provide a deeper understanding of the effect of cooking on meat proteins and the possible relationship to processing conditions in meat-derived food.

Effect of beef ultimate pH and large structural protein changes with aging on meat tenderness

This study investigated the effect of ultimate pH (pHu) in beef on the degradation of large structural proteins during refrigerated storage using SDS-PAGE. M. longissimus dorsi from bull carcasses were selected and classified into three groups: low pHu (≤5.79), intermediate pHu (5.80-6.19) and high pHu (≥6.2) muscles. Samples were then stored at -1.5°C for 1, 2, 7, 14, 21 and 28days. Meat tenderness was measured at each aging time. Depending on meat pHu, different protein patterns and degradation rates of structural proteins were found. Rapid changes of large structural proteins took place within 48h post mortem. Besides titin and nebulin, degradation of filamin was clearly revealed. Two more large protein bands corresponding to myosin family members also exhibited fast decline with storage time. It suggested that the fast degradation of these proteins is a key factor in the improvement of meat tenderness.

The Proteome of the Wool Cuticle

The cuticle is responsible for important wool fiber characteristics such as handle and abrasion resistance, which impact on the fibers performance in both interior and apparel textiles. The cuticle proteome, however, is not well understood due to the difficulty in isolating pure wool cuticle and its significant resistance to protein extraction, which is attributed to the presence of extensive disulfide and isopeptide cross-linking. We investigated the proteome of highly pure Merino wool cuticle using a combined strategy of chemical and enzymatic digestion and identified 108 proteins, including proteins responsible for a variety of cellular processes. The majority of identified proteins belonged to keratin and nonkeratin protein families known to play an important role in molecular assembly and cellular structure. Keratin-associated, intermediate filament and cytoskeletal keratin proteins were identified as the most prominent keratinous cuticular constituents, while histones, tubulins, and desmosomes were the key nonkeratin structural proteins. We conclude that a variety of proteins contribute to cuticle structure and fiber characteristics, and that the keratinous protein families of IFPs and KAPs represent the most important cuticular constituents.

Influence of feed restriction on the wool proteome: A combined iTRAQ and fiber structural study

UNLABELLEDnSeasonal weight loss is the main limitation to animal production worldwide, significantly affecting the productivity of milk, meat and wool farms, particularly in drought-prone areas of the world where most of the large-scale wool production farms are located. Although the effect of nutritional status on wool quality parameters has been extensively studied, little is known on how it affects wool protein composition. Here, a proteomic approach has been applied to study changes in fiber structure and protein composition in wool from merino sheep subjected to experimentally induced weight loss. Results indicate that there is a significant reduction in the fiber diameter of wool from the animals on a restricted diet over a 42-day period. At the same time, significant increases in the expression of the high sulfur protein KAP13.1 and proteins from the high glycine-tyrosine protein KAP6 family in the wools from the animals on the restricted diet were also detected. Such findings have strong implications for the wool industry that favors finer wool.nnnBIOLOGICAL SIGNIFICANCEnSeasonal weight loss caused by poor pasture availability has strong effects on wool productivity parameters and quality traits. In this work we determine that experimentally induced weight loss causes a decrease in fiber diameter associated with an increase in the level of high sulfur protein KAP13.1 and proteins from the high glycine-tyrosine protein KAP6 family. The implication of this is that decreasing the fiber diameter of the wool by this process could result in a fiber reduced prickle but with reduced wearability and appearance retention.

Modeling deamidation in sheep α-keratin peptides and application to archeological wool textiles.

Deamidation of glutamine (Q) and asparagine (N) has been recognized as a marker of degradation and aging in ancient proteins. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to study deamidation in wool textiles, we identified eight peptides from α-keratin proteins in sheep wool that could potentially be used to assess the level of degradation. For each chosen peptide, the extent of deamidation was determined by comparing the calculated theoretical distribution with the measured distribution using a genetic algorithm that gives the best fit to the measured distribution. Variations in the levels of deamidation were observed between peptides and in modern wool samples buried for up to 8 years in which deamidation levels were relatively low under short-term burial. In contrast, deamidation was higher in archeological textile fragments from medieval sites ranging from the 9th to 13th century in York (United Kingdom) and Newcastle (United Kingdom) and from the 13th to 16th century in Reykholt (Iceland). Major differences were observed between the British and the Icelandic samples, showing a negative correlation between age of samples and levels of deamidation, but highlighting the effect of local environment. In addition, nanoscale liquid chromatography-electrospray ionization tandem mass spectrometry (nanoLC-ESI-MS/MS) data indicated that deamidation in wools α-keratin was influenced by primary and higher-order structures. Predominance of deamidation on glutamine rather than asparagine in the archeological samples was attributed to a higher abundance of Q in the α-helical core domain of keratins, neighboring residues and steric hindrance preventing deamidation of N.

Characterization of the exocuticle a-layer proteins of wool.

Abstract:u2002 The outermost protein layer of wool cuticle cells is known as the exocuticle a‐layer. This layer is a resistant barrier to the degradation of the fibre and, as a result, little is known of its proteinaceous composition. Merino wool fibres were subjected to both proteolytic and chemical digestion and the resulting material was found by transmission electron microscopy to be highly enriched in a‐layer. Amino acid analysis revealed a high cysteine and glycine content, with a close, but not exact, match to the Allwörden membrane. Subsequent digestion of the a‐layer preparation by 2‐nitro‐5‐thiocyano‐benzoic acid produced a large number of short peptides, and analysis by mass spectrometry revealed peptides with strong homologies to cuticular ultra‐high sulphur proteins of sheep wool and cuticular ultra‐high and high‐sulphur proteins of human hair, thus supporting other evidence for the presence of these sulphur‐rich proteins in the a‐layer.