Characterisation of white and black merino wools: a proteomics study.

Wool is an important agricultural commodity with merino wool being rated alongside the finest quality fibres, which include the goat fibres Mohair and Cashmere. Although pigmented wool merinos have become extremely rare, the market for this wool is increasing. In Portugal, there are two merino breeds: white and black, descendants of animals originally bred on the Iberian Peninsula. These breeds have the potential to assist in our understanding of how protein expression relates to wool traits of importance to the textile industry. Herein, we study the characteristics and protein expression profiles of wool from ewes of the Portuguese black and white merino (n=15). Both breeds had very similar results for fibre diameter (25 µm) and curvature (105 to 111°/mm). Significant between-breed differences were found in the two types of keratin-associated proteins (KAPs): high-sulphur proteins (HSPs) and high-glycine-tyrosine proteins (HGTPs). The expression of HSPs, KAP2-3 and KAP2-4, decreased expression in the pigmented animals, whereas KAP13-1 was found in higher amounts. Likewise, the expression of the ultra-high-sulphur proteins, KAP4-3 and KAP4-7-like, was reduced in black sheep to half the levels of the white wools, whereas the HGTPs, KAP6, KAP6-1, KAP6-2 and KAP16-2, were more abundant in black sheep. These results suggest structural differences between the black and white merino wool, because of differences among some KAPs. These differences have important implications for the textile industry.

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

OBJECTIVE: To understand the structural and chemical effects of cosmetic peroxide bleaching on human hair. METHODS: Human hair was progressively bleached using alkaline peroxide-persulphate treatment. Proteins lost through leaching were examined using amino acid analysis and mass spectrometric sequencing. Fibre damage was assessed using transmission electron microscopy, amino acid analysis and redox proteomics. RESULTS: Protein loss through leaching increased with bleaching severity. Leached proteins were not limited to the cuticle, but also included cortical intermediate filaments and matrix keratin-associated proteins. The leached proteins were progressively oxidized as bleaching severity increased. Bleached fibres demonstrated substantial damage to the cuticle layers and to the cortex. Extensive melanin granule degradation was present after the mildest bleach treatment. Protein oxidation in bleached fibres was principally in cortical intermediate filaments - the most abundant hair proteins - and targeted the sulphur-containing amino acids, particularly the conversion of cystine disulphide bonds to cysteic acid. CONCLUSION: Peroxide chemical treatments quickly access the cortex, causing untargeted oxidative damage across the fibre in addition to the desired loss of melanin. Peroxide ingress is likely facilitated by the considerable structural degradation caused to the cuticle layers of hair fibres. The consequences of the peroxide action within the cuticle and cortex are oxidation of the proteins, and subsequent protein loss from the fibre that correlates to bleaching severity.

Comparative analysis of human milk and infant formula derived peptides following in vitro digestion.

It has long been recognised that there are differences between human milk and infant formulas which lead to differences in health and nutrition for the neonate. In this study we examine and compare the peptide profile of human milk and an exemplar infant formula. The study identifies both similarities and differences in the endogenous and postdigestion peptide profiles of human milk and infant formula. This includes differences in the protein source of these peptides but also with the region within the protein producing the dominant proteins. Clustering of similar peptides around regions of high sequence identity and known bioactivity was also observed. Together the data may explain some of the functional differences between human milk and infant formula, while identifying some aspects of conserved function between bovine and human milks which contribute to the effectiveness of modern infant formula as a substitute for human milk.

Redox proteomic evaluation of oxidative modification and recovery in a 3D reconstituted human skin tissue model exposed to UVB.

OBJECTIVE: Exposure to UV in humans resulting in sunburn triggers a complex series of events that are a mix of immediate and delayed damage mediation and healing. While studies on the effects of UV exposure on DNA damage and repair have been reported, changes in the oxidative modification of skin proteins are poorly understood at the molecular level, despite the important role played by structural proteins in skin tissue, and the effect of the integrity of these proteins on skin appearance and health. Proteomic molecular mapping of oxidation was here applied to try to enhance understanding of skin damage and recovery from oxidative damage and UVB exposure. METHODS: A redox proteomic-based approach was applied to evaluating skin protein modification when exposed to varying doses of UVB after initial oxidative stress, via tracking changes in protein oxidation during the healing process in vitro using a full-thickness reconstituted human skin tissue model. Bioassays and structural evaluation confirmed that our cultured skin tissues underwent a normal physiological response to UVB exposure. RESULTS: A set of potential skin marker peptides was generated, for use in tracking skin protein oxidative modification. Exposure to UVB after thermal oxidative stress was found to result in higher levels of skin protein oxidation than a non-irradiated control for up to seven days after exposure. Recovery of the skin proteins from oxidative stress, as assessed by the overall protein oxidation levels, was found to be impaired by UVB exposure. Oxidative modification was largely observed in skin structural proteins. CONCLUSION: Exposure of skin proteins to UVB exacerbates oxidative damage to structural skin proteins, with higher exposure levels leading to increasingly impaired recovery from this damage. This has potential implications for the functional performance of the proteins and inter-related skin health and cosmetic appearance.

Effect of pre-slaughter handling, exercise and the presence of a dog on lamb welfare and meat quality.

Before slaughter, lambs may experience several stressors such as feed and water deprivation, handling and transport that have the potential to negatively impact welfare and meat quality. The objective of this study was to evaluate the effect of pre-slaughter handling, exercise and the presence of a dog on the behaviour and physiology of lambs and meat quality at slaughter. At 6 months of age, 60 lambs (n=20 lambs/replicate; three replicates) were allocated to one of the two treatment groups (n=30 lambs/treatment): low (LOW) intensive handling or high (HIGH) intensive handling. LOW lambs were moved short distances, quietly and without the use of a dog before transport. HIGH lambs were moved quickly, long distances and with a dog present before transport. Lamb behaviour (standing, lying, rumination and panting) was recorded for 1 h before (post-treatment) and after transport (post-transport), and for 30 min before slaughter (pre-slaughter). Blood samples were collected before (baseline), after transport (post-transport) and at exsanguination (at slaughter) to assess cortisol, lactate and non-esterified fatty acid (NEFA) concentrations. At slaughter, lamb carcases (M. longissimus lumborum) were evaluated for pH levels, drip and cook loss, and tenderness. HIGH lambs spent more time standing (P<0.001) and panting (P<0.001) and less time lying (P<0.001) and ruminating (P<0.001) post-treatment than LOW lambs, but more (P<0.001) time ruminating post-transport. All lambs spent more time standing (P<0.001) and less time lying (P<0.001) and panting (P<0.001) post-transport and pre-slaughter than post-treatment. Cortisol concentrations were greater (P<0.001) in lambs post-transport and at slaughter compared with baseline values. Lactate concentrations were lower (P=0.002) in HIGH than LOW lambs. In addition, NEFA concentrations were higher (P<0.001) post-transport and at slaughter in HIGH compared with LOW lambs. Ultimate pH was higher (P<0.001) in HIGH than LOW lambs and pH declined quicker (P=0.012) in LOW than HIGH lambs. Cook loss, drip loss and shear force were lower (P⩽0.05) in HIGH than LOW lambs. The HIGH intensive pre-slaughter handling regime used in the present study caused stress in lambs and increased ultimate pH that could potentially negatively impact welfare, product quality and consistency.

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.

Efferent intestinal lymph protein responses in nematode-resistant, -resilient and -susceptible lambs under challenge with Trichostrongylus colubriformis.

The mechanisms underlying resistance to challenge by gastrointestinal nematode parasites in sheep are complex. Using DIGE, we profiled ovine lymph proteins in lambs with host resistance (R), resilience (Ri) or susceptibility (S) to a daily trickle challenge with the nematode Trichostrongylus colubriformis. Efferent intestinal lymph was collected prior to infection (day 1) and on days 5 and 10 post-infection. Eight proteins identified by LC-MS/MS, showed differences relating to host genotype. Of these, Serpin A3-3 and Serpin A3-7 have not been reported previously in the lymph proteome. Three acute phase proteins showed significant differences relating to interactions between breeding line and parasite challenge, including complement C3ß, C3α and haptoglobin (Hp) ß. In the R lambs C3α was significantly up regulated (P<0.05) on day 10, while in the Ri lambs Hp ß was significantly down regulated (P<0.05). In the S lambs, levels of C3ß were up regulated and levels of Hp ß down regulated (both P<0.05) on day 10. Hence we demonstrate that acute phase inflammation proteins contribute to differences in the innate immune response of sheep to challenge by T. colubriformis. The findings may lead to the development of new approaches to combat nematode infestations in sheep production systems. BIOLOGICAL SIGNIFICANCE: Breeding lines of sheep with resistance (R), resilience (Ri) or susceptibility (S) to nematode infections provide an experimental model to examine the biological mechanisms underlying the ability of some sheep to expel worms and remain healthy without the use of an anthelmintic. Using proteomics we identified differences in the expression of acute phase lymph proteins in the R, Ri and S lambs. The results will assist the development of alternative control strategies to manage nematode infections in livestock.

Redox proteomic evaluation of bleaching and alkali damage in human hair.

OBJECTIVE: 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. METHODS: Redox proteomic profiling of virgin, bleached and relaxed hair tresses was performed, with comprehensive qualitative characterization of modification and semi-quantitative evaluation of damage through adaptation of a new damage scoring system. Modifications were mapped to specific locations in the hair proteome and a range of potential damage marker peptides identified. RESULTS: Virgin hair contained a baseline level of modification, consistent with environmental oxidative insult during hair growth. Hydrogen peroxide bleaching resulted in significantly increased levels of oxidative damage observable at the molecular level. This treatment also resulted in enhanced levels of dehydroalanine and dehydration products; modifications typically associated with alkali or thermal treatment and not previously been reported as a product of hair bleaching. Relaxation treatment with sodium hydroxide increased the formation of dehydroalanine and dehydration products and moderately enhanced the levels of oxidation. Cysteine was the predominant modification site for both bleaching and alkali damage. CONCLUSION: This study validates the utility and power of redox proteomic-based approaches to characterizing hair modification. This offers potential application to a wide range of damage types, as well as evaluation of new damage mitigation and repair technologies.

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.

Neurotoxicity of polychlorinated biphenyls (PCBs) by disturbance of thyroid hormone-regulated genes.

PCBs are known as neurotoxic compounds. Part of this neurotoxicity could be due to an alteration of the expression of TH-regulated genes in brain. To identify such genes, brain protein extracts of hypo- and hyperthyroid as well as PCB-treated embryos were compared by fluorescent 2D-DIGE. In total, we observed 109 differentially expressed proteins, of which 17 differed with both PCB and hypo- or hyperthyroid treatment. It was found that the interaction of PCBs with the expression of TH-regulated genes is congener-specific and that both hyperthyroidism- and hypothyroidism-related effects occur.

Immunohistochemical localization of chromogranin A in gonadotrophs and somatotrophs of the turkey and chicken pituitary.

In the course of producing monoclonal antibodies to turkey prolactin, three monoclonal antibodies to turkey chromogranin A (CgA) were also produced, apparently arising from minor contamination of the turkey prolactin immunogen with peptide fragments of CgA. The identity of the antigen recognized by these antibodies was established by tandem mass spectrometry de novo sequencing of seven tryptic peptides from a turkey pituitary protein purified by immunoaffinity chromatography. These peptides showed high homology with distinctly separate regions of mammalian and ostrich CgA, and in silico cloned chicken CgA sequences. Chromogranin A immunostaining patterns on Western blots and pituitary tissue sections differed from those of prolactin, growth hormone, or luteinizing hormone (LH). Dual-label fluorescent immunohistochemistry revealed that CgA was co-localized with LH in most avian gonadotrophs in young chickens and turkeys, but not in adult, laying birds. Conversely, CgA was found in a majority of somatotrophs in laying birds but was absent from somatotrophs in young, growing chickens and turkeys. Lactotrophs contained no detectable CgA immunoreactivity in the tissues studied. These results suggest that CgA may modulate hormone secretion by gonadotrophs and somatotrophs in a manner that differs between cell type with age or reproductive state.

Peptide profiling of a single Locusta migratoria corpus cardiacum by nano-LC tandem mass spectrometry.

The pars intercerebralis-corpora cardiaca complex in insects is the functional equivalent of the vertebrate brain-pituitary axis. During the past few decades more than 40 neuropeptides have been isolated from the locust brain-corpus cardiacum complex. Tedious and time-consuming successive purification rounds of large tissue extracts were necessary to achieve the purification and sequencing of most of these signal molecules. Nowadays, the combination of nanoscale liquid chromatography and the very sensitive tandem mass spectrometry allows us to identify and sequence peptides in very low concentration directly from tissue extracts. In this manuscript, we review previous data on the peptidome analysis of the locust corpora cardiaca, with emphasis on AKH processing. In addition, we report the peptide profiling of a single corpus cardiacum from Locusta migratoria. 23 peptides were isolated and sequenced in a single nano-LC-MS/MS experiment, demonstrating the sensitivity and effectiveness of mass spectrometry in peptide research.

Defoliation induces fructan 1-exohydrolase II in Witloof chicory roots. Cloning and purification of two isoforms, fructan 1-exohydrolase IIa and fructan 1-exohydrolase IIb. Mass fingerprint of the fructan 1-exohydrolase II enzymes.

The cloning of two highly homologous chicory (Cichorium intybus var. foliosum cv Flash) fructan 1-exohydrolase cDNAs (1-FEH IIa and 1-FEH IIb) is described. Both isoenzymes could be purified from forced chicory roots as well as from the etiolated "Belgian endive" leaves where the 1-FEH IIa isoform is present in higher concentrations. Full-length cDNAs were obtained by a combination of reverse transcriptase-polymerase chain reaction (PCR), PCR and 5'- and 3'-rapid amplification of cDNA ends using primers based on N-terminal and conserved amino acid sequences. 1-FEH IIa and 1-FEH IIb cDNA-derived amino acid sequences are most homologous to a new group of plant glycosyl hydrolases harboring cell wall-type enzymes with acid isoelectric points. Unlike the observed expression profiles of chicory 1-FEH I, northern analysis revealed that 1-FEH II is expressed when young chicory plants are defoliated, suggesting that this enzyme can be induced at any developmental stage when large energy supplies are necessary (regrowth after defoliation).

Identification and characterization of novel chromogranin B-derived peptides from porcine chromaffin granules by liquid chromatography/electrospray tandem MS.

Chromogranin B (CgB) is a regulated secretory protein that is stored in endocrine and neuroendocrine cells. It can be processed proteolytically to small peptide fragments. In the present study three proteolytic products of porcine CgB were obtained after size-exclusion, immunoaffinity, and reversed-phase chromatography, and then identified by electrospray tandem MS. One novel peptide was identified as S586-R602 (SR-17) and is phosphorylated at one or two serine residues. Another novel peptide H603-Q636 (HQ-34), with molecular mass 3815.56 Da, was found to be oxidized at the methionine residue. In addition, a secretolytin-like peptide fragment (KR-11), which is two amino acids shorter than the bovine secretolytin, was found. This is the first report that the C-terminal region of CgB, the homologue of human CCB, is proteolytically processed further into three small peptide fragments.