Response of laying hens fed diet supplemented with a mixture of olive, laurel, and rosemary leaf powders: Metabolic profile, oxidative status, intestinal histomorphology, and egg quality.

This study aimed to evaluate the effects of a mixture of olive, laurel, and rosemary leaf powders, on the oxidative state, biochemical, immune, intestinal morphophysiological parameters, and egg quality of laying hens. One hundred Lohmann Brown hens (28 weeks old) were equally assigned to two groups (n. 50) corresponding to a basal control diet (CON) or the diet supplemented with 6 g/kg feed of leaf powder mixture (LPM) containing olive, laurel, and rosemary leaves (1:1:1), for 60 days. Oxidative status, biochemical indices, immune response, cecal short chain fatty acids (SCFAs), intestinal morphological characteristics, and some egg traits were evaluated at the end of the experiment. The results indicated that LPM improved (P < 0.05) the oxidative status (TOS, ROMs), the immune system (IL-6, IL-1ß, and TNF-α), the total protein and HDL cholesterol content, whereas it decreased (P < 0.05) total cholesterol and LDL cholesterol. Aspartate aminotransferase (AST), alkaline phosphatase (ALP), and alanine aminotransferase were significantly (P < 0.05) lower in the LPM than in the CON group. A significant increase (P < 0.05) in SCFA content in the caecum, as well as in villi height and crypt depth in both duodenum and ileum of LPM-treated hens, was observed. Egg quality parameters were not influenced (P > 0.05) by LPM. These findings indicate that LPM can be considered a candidate as an antioxidant ingredient for functional food in laying hens.

Label-Free Quantitative Analysis of Pig Liver Proteome after Hepatitis E Virus Infection.

Hepatitis E represents an emerging zoonotic disease caused by the Hepatitis E virus (HEV), for which the main route of transmission is foodborne. In particular, infection in humans has been associated with the consumption of contaminated undercooked meat of pig origin. The aim of this study was to apply comparative proteomics to determine if porcine liver protein profiles could be used to distinguish between pigs seropositive and seronegative for HEV. Preliminarily, an ELISA was used to evaluate the presence of anti-HEV antibodies in the blood serum of 136 animals sent to slaughter. Among the analyzed samples, a seroprevalence of 72.8% was estimated, and it was also possible to identify 10 animals, 5 positive and 5 negative, coming from the same farm. This condition created the basis for the quantitative proteomics comparison between homogeneous animals, in which only the contact with HEV should represent the discriminating factor. The analysis of the proteome in all samples of liver exudate led to the identification of 554 proteins differentially expressed between the two experimental groups, with 293 proteins having greater abundance in positive samples and 261 more represented in negative exudates. The pathway enrichment analysis allowed us to highlight the effect of the interaction between HEV and the host biological system in inducing the potential enrichment of 69 pathways. Among these, carbon metabolism stands out with the involvement of 41 proteins, which were subjected to interactomic analysis. This approach allowed us to focus our attention on three enzymes involved in glycolysis: glucose-6-phosphate isomerase (GPI), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and fructose-bisphosphate aldolase A (ALDOA). It therefore appears that infection with HEV induced a strengthening of the process, which involves the breakdown of glucose to obtain energy and carbon residues useful for the virus's survival. In conclusion, the label-free LC-MS/MS approach showed effectiveness in highlighting the main differences induced on the porcine liver proteome by the interaction with HEV, providing crucial information in identifying a viral signature on the host metabolism.

Comparative Label-Free Liquid Chromatography-Mass Spectrometry Milk Proteomic Profiles Highlight Putative Differences between the Autochthon Teramana and Saanen Goat Breeds.

Goat's milk is an excellent source of nutrients, with greater benefits compared to cow's milk. Limited information is available on autochthon goat breeds, which are important for biodiversity preservation. In this study, the aim of using label-free quantification was to investigate the milk proteome of two goat breeds, the autochthon Teramana and Saanen breeds, which are commonly used by the industry. Utilising label-free proteomic analysis, 749 and 666 proteins, respectively were identified and quantified from the Teramana and Saanen goat milk. Moreover, utilising statistical analysis, 29 proteins were able to discriminate the two goat breeds, with many of the identified proteins involved in complement and coagulation cascades. This work enhances our understanding of the goat milk proteome and shows differences between the two breeds, leading to an important contribution toward a more detailed molecular-view of this unique substrate. Additionally, charactersation of the milk proteins can help in guiding genetic improvements in the goat herds, and thus increasing its use in human nutrition.

Comparative Proteomics Analysis of Pig Muscle Exudate through Label-Free Liquid Chromatography-Mass Spectrometry.

Capital-driven animal husbandry systems undertaken in the last century led to the abandoning of many pig breeds that were not profitable. These local pig breeds and their respective production systems have great potential as they are able to respond to the high criteria and needs of modern society concerning some environmental aspects, animal-welfare, healthiness, etc. This is the case of the black pigs of Italy. The Apulo-Calabrese is a breed of black pig, known by many other names such as Nero d'Abruzzo. In order to further understand the biological differences between different types of porcine genetics (Nero d'Abruzzo and commercial-hybrid) we used a label-free LC-MS strategy and Western-blot to characterize the proteomes of muscle-exudate collected from these pigs. This proteomics approach identified 1669 proteins of which 100 changed significantly in abundance between breeds. Bioinformatics functional analysis indicated that differentially expressed proteins were involved in several biological processes related to energy-metabolism and response to oxidative stress, suggesting that these functions might distinguish between these pigs. Fatty-acid synthase, catalase and glutathione-peroxidase, involved in enzymatic activity were found to be more represented in samples obtained from the Nero d'Abruzzo. This biological information can potentially provide new biological factors that could determine the different production performances of these pigs, distinguished by their different genetic backgrounds.

DIGE Analysis of Animal Tissues.

Two-dimensional difference gel electrophoresis (2D-DIGE) is an acrylamide gel electrophoresis-based technique for protein separation and quantification in complex mixtures. The technique addresses some of the drawbacks of conventional 2D polyacrylamide gel electrophoresis (2D-PAGE), offering improved sensitivity, more limited experimental variation, and accurate within-gel matching. 2D-DIGE is based on direct labeling of proteins with isobaric fluorescent dyes (known as CyDyes: Cy2, Cy3, and Cy5) prior to isoelectric focusing (IEF). Here, up to two samples and a reference pool (internal standard) can be mixed and loaded onto IEF for first dimension prior to SDS (sodium dodecyl sulfate)-PAGE separation in the second dimension. After the electrophoretic run, the gel is imaged at the specific excitation wavelength for each dye, in sequence, and gel scans are recorded separately. For each individual protein spot, intensities recorded at the different wavelengths are integrated and the ratio between volumes normalized to that of the internal standard. This provides an immediate appreciation of protein amount variations under the different conditions tested. In addition, proteins of interest can still be excised and identified with conventional mass spectrometric techniques and further analyzed by other biochemical methods. In this chapter, we describe application of this methodology to separation and quantitation of protein mixtures from porcine muscle exudate, collected following centrifugation of muscle specimens (centrifugal drip) for the characterization of quality parameters of importance in meat industry.

A Label-Free Quantitative Analysis for the Search of Proteomic Differences between Goat Breeds.

The intensification and standardization of livestock farming are causing a decline in the number of animal breeds in many species, such as the goat. The availability of more studies on the potentiality of goat breeds could raise awareness of their importance, conservation and productive possibilities. Label-free quantitative analysis was applied in this study to investigate the proteomic differences between the autochthon Teramana and Saanen goats that could be useful for defining peculiar features of these breeds. A total of 2093 proteins were characterized in the muscle exudate proteome of the Teramana and Saanen breeds. A total of 41 proteins clearly separated the two breeds. Eukaryotic initiation factor proteins and aldehyde-dehydrogenase 7 family-member A1 were up-regulated in the autochthon breed and associated with its resilience, whereas catalase was down-regulated and associated with lower muscular mass. This study is the most detailed report of goat muscle proteome. Several differentially regulated proteins between the two breeds were identified, providing insights into functional pathways that define this organism and its biology.

Label-free quantitative proteomics and stress responses in pigs-The case of short or long road transportation.

Ethical livestock production is currently a major concern for consumers. In parallel, research has shown that transport duration is an important factor affecting animal welfare and has a negative impact on the final product quality and on the production cost. This study applied proteomics methods to the animal stress/welfare problem in pigs muscle-exudate with the aim to identify proteins indicative of molecular processes underpinning transport stress and to better characterise this species as a biomedical model. A broader perspective of the problem was obtained by applying label-free LC-MS to characterise the proteome response to transport stress (short or long road transportation) in pigs within the same genetic line. A total of 1,464 proteins were identified, following statistical analysis 66 proteins clearly separating pigs subject to short road transportation and pigs subject long road transportation. These proteins were mainly involved in cellular and metabolic processes. Catalase and stress-induced phosphoprotein-1 were further confirmed by Western blot as being involved in the process of self-protection of the cells in response to stress. This study provide an insight into the molecular processes that are involved in pig adaptability to transport stress and are a step-forward for the development of an objective evaluation method of stress in order to improve animal care and management in farm animals.

Qualitative attributes of meat from Teramana goat kids, an Italian native breed of the Abruzzo region.

OBJECTIVE: The aim of this work was the characterization of the qualitative aspects of meat obtained from Teramana goats, an Italian indigenous breed of the Abruzzo region. Specifically, the study included a comparison with meat samples deriving from Saanen goat kids reared in the same environment and applying the same feeding protocol. METHODS: Upon reaching about 7 months of age the animals were slaughtered and samples of muscle tissue were collected to be analyzed. Specifically, meat samples were subjected to evaluations of the physical parameters, including color and the meat ability to retain water, in addition to chemical evaluations that were focused to the determination of the total lipids amount, fatty acids composition, lipid oxidation, and volatile profile. RESULTS: The meat samples obtained from the indigenous breed showed a less intense reddish color and no significant variations for the muscle tissue tendency to retain water, both regarding fresh and cooked meat. Several differences were instead observed in the fatty acid profile. The Teramana samples were richer in saturated fatty acids (p<0.01) and interestingly showed higher concentrations of rumenic acid (p<0.05), a conjugate of linoleic acid that has been associated with important health benefits for the consumers. Another important finding for these meat samples was the marked resistance to oxidative events, as evidenced by the thiobarbituric acid reactive substances-test (p<0.05) and by the characterization of the volatile profile that highlighted a strong reduction in the relative percentage of hexanal (p<0.05), commonly associated to lipid oxidation and the development of unpleasant aromatic notes. CONCLUSION: The collected data, therefore appeared useful for the valorization of the food product derived from the Teramana goat, although no sensory information has been collected to define the degree of acceptability by the consumers.

Influence of Grape Pomace Intake on Nutritional Value, Lipid Oxidation and Volatile Profile of Poultry Meat.

Grape pomace (GP) represents the main solid by-product deriving from grape processing. The aim of this study was to evaluate the effect of dietary GP intake on nutritional quality, lipid oxidation and volatile profile of chicken meat. A total of 112 Ross 508 broilers were randomly divided into 4 groups and fed for 21 days with a standard diet. For the remaining 28 days of the trial, the control group (CG) continued to receive a standard diet, while the experimental groups (EGs) were fed with diets containing different GP concentrations: 2.5% (EG1), 5% (EG2) and 7% (EG3). Following the slaughtering, samples of breast meat were collected from each group. No significant differences were observed for pH, cooking loss and meat brightness, whereas the GP intake showed effectiveness in inducing variations in drip loss, meat yellowness and redness. The experimental feeding strategy also induced changes in the fatty acid profile, with an overall increase in polyunsaturated fatty acids (PUFA), mainly due to the increase in concentration of linoleic acid. The dietary supplementation also induced a decrease in lipid oxidation in meat, a finding also confirmed by the reduction in volatile aldehydes in 7 days stored raw meat. The feeding strategy based on the use of GP did not induce detrimental effects on the quality of broiler meat and showed the potential to lengthen the shelf-life as a direct consequence of the improvement in the oxidative stability. Overall, the present study showed a viable way for the recovery and the valorization of an agro-industrial by-product, with potential benefits also from an environmental point of view.

Dietary supplementation of Saanen goats with dried licorice root modifies chemical and textural properties of dairy products.

The aim of the present study was to evaluate the effect of dietary integration with dried licorice root on the chemical-nutritional qualities of goat milk and cheeses. The study was conducted for 60 d, during which 30 Saanen goats were divided into 2 groups: a control group (CG) that received a standard diet and an experimental group (LG+) whose diet was supplemented with licorice. At the end of the study, milk samples were collected to determine chemical-nutritional compositions and fatty acid (FA) profiles. Cheeses produced with CG and LG+ bulk milk were analyzed for chemical-physical parameters after 3 (T3) and 30 (T30) d of ripening. A different FA profile and a significant increase in protein and casein were observed in LG+ milk samples compared with CG milk. Regarding cheeses, an increase of proteins and fat was found in LG+ cheeses, which also were harder, more elastic, and more gummy than the CG samples after both 3 and 30 d of ripening. A different protein profile was detected in the 2 groups without significant variations in casein fractions (αS2-casein and ß-casein) during ripening. Moreover, greater oxidative stability was found in LG+ cheeses at both T3 and T30. Different families of volatile compounds were detected in T30 cheeses obtained from both groups. A significant reduction of octanoic acid and an increase in nonanal and ketones were found in LG+ T3 cheeses, whereas the LG+ T30 cheeses were characterized by a significant decrease of hexanoic acid an increase of 3-methyl-1-butanol and acetoin. We concluded that it is possible to assert that dietary integration with dried licorice root modified chemical and technological properties of goat cheeses, reducing lipid oxidation during ripening and inducing changes in texture that could improve consumer acceptability, although further studies are needed from this point of view.

A comparative analysis of label-free liquid chromatography-mass spectrometry liver proteomic profiles highlights metabolic differences between pig breeds.

The liver is a complex organ governing several physiological processes that define biological mechanisms affecting growth, feed efficiency and performance traits in all livestock species, including pig. Proteomics may contribute to a better understanding of the relationship between liver functions and complex production traits in pigs and to characterize this species as biomedical model. This study applied, for the first time, a label-free liquid chromatography-mass spectrometry (LC-MS) proteomic approach to compare the liver proteome profiles of two important heavy pig breeds, Italian Duroc and Italian Large White. Liver specimens were collected (after slaughtering) from performance tested pigs of these two breeds, raised in standard conditions. The label-free LC-MS method captured a total of 501 proteins of which 200 were subsequently considered in the between breeds comparison. A statistical pipeline based on the sparse Partial Least Squares Discriminant Analysis (sPLS-DA), coupled with stability and significance tests, was applied for the identification of up or down regulated proteins between breeds. This analysis revealed a total of 25 proteins clearly separating Italian Duroc and Italian Large White pigs. Among the top proteins differentiating the two breeds, 3-ketoacyl-CoA thiolase, mitochondrial (ACAA2) and histone H2B type 2-F (HIST2H2BF) were up-regulated in Italian Duroc pigs and carboxylesterase 3 (CES3) and ketohexokinase (KHK) were up-regulated in Italian Large White pigs. Fatty acid synthase (FASN), involved in fatty acid metabolism and encoded by a gene located in a QTL region for fatty acid composition, was up-regulated in Italian Large White pigs. The in silico protein interaction analysis showed that 16 of these proteins were connected in one big module. Bioinformatic functional analysis indicated that differentially expressed proteins were involved in several biological processes related to the metabolism of lipids, amino-acids, carbohydrates, cofactors and antibiotics/drugs, suggesting that these functions might distinguish Italian Duroc and Italian Large White pigs. This pilot comparative proteomic analysis of the porcine liver highlighted several biological factors that could determine the peculiar production potentials of these two heavy pig breeds, derived by their different genetic backgrounds.

DIGE Analysis of Animal Tissues.

Two-dimensional difference gel electrophoresis (2D-DIGE) is an acrylamide gel electrophoresis-based technique for protein separation and quantification in complex mixtures. The technique addresses some of the drawbacks of conventional 2D polyacrylamide gel electrophoresis (2D-PAGE), offering improved sensitivity, more limited experimental variation and accurate within-gel matching. DIGE is based on direct labeling of proteins with isobaric fluorescent dyes (known as CyDyes: Cy2, Cy3, and Cy5) prior to isoelectric focusing (IEF). Here, up to two samples and a reference pool (internal standard) can be mixed and loaded onto IEF for first dimension prior to SDS-PAGE separation in the second dimension. After the electrophoretic run, the gel is imaged at the specific excitation wavelength for each dye, in sequence, and gel scans are recorded separately. For each individual protein spot, intensities recorded at the different wavelengths are integrated and the ratio between volumes normalized to that of the internal standard. This provides an immediate appreciation of protein amount variations under the different conditions tested. In addition, proteins of interest can still be excised and identified with conventional mass spectrometry techniques and further analyzed by other biochemical methods. In this chapter, we describe the application of this methodology to separation and quantitation of proteins mixtures from porcine muscle exudate, collected following centrifugation of muscle specimens (centrifugal drip) for the characterization of quality parameters of importance in the meat industry.

Identification of the metabolic alterations associated with the multidrug resistant phenotype in cancer and their intercellular transfer mediated by extracellular vesicles.

Multidrug resistance (MDR) is a serious obstacle to efficient cancer treatment. Overexpression of P-glycoprotein (P-gp) plays a significant role in MDR. Recent studies proved that targeting cellular metabolism could sensitize MDR cells. In addition, metabolic alterations could affect the extracellular vesicles (EVs) cargo and release. This study aimed to: i) identify metabolic alterations in P-gp overexpressing cells that could be involved in the development of MDR and, ii) identify a potential role for the EVs in the acquisition of the MDR. Two different pairs of MDR and their drug-sensitive counterpart cancer cell lines were used. Our results showed that MDR (P-gp overexpressing) cells have a different metabolic profile from their drug-sensitive counterparts, demonstrating decreases in the pentose phosphate pathway and oxidative phosphorylation rate; increases in glutathione metabolism and glycolysis; and alterations in the methionine/S-adenosylmethionine pathway. Remarkably, EVs from MDR cells were capable of stimulating a metabolic switch in the drug-sensitive cancer cells, towards a MDR phenotype. In conclusion, obtained results contribute to the growing knowledge about metabolic alterations in MDR cells and the role of EVs in the intercellular transfer of MDR. The specific metabolic alterations identified in this study may be further developed as targets for overcoming MDR.

Data supporting the shedding of larger extracellular vesicles by multidrug resistant tumour cells.

To date, there are no simple and minimally invasive methods to diagnose MDR. Extracellular vesicles (EVs) are shed by all cells, carry a specific cargo from the donor cells and are present in several body fluids, which means that they can potentially be easily collected from cancer patients and become the source of biomarkers to diagnose cancer. This data article contains a full list of the proteins identified in the EVs shed by an isogenic pair of chronic myeloid leukaemia cells (MDR cells and their drug-sensitive counterparts) by LC/MS/MS analysis, together with their GeneOntology analysis. In addition, it also contains data from protein content analysis and Dynamic light scattering count-rate events of the referred EVs as well as of the EVs shed from an isogenic pair of non-small cell lung cancer cells (MDR cells and their drug-sensitive counterparts). The interpretation of the data presented in this article and further extensive insights can be found in "Multidrug resistant tumour cells shed more microvesicles-like EVs and less exosomes than their drug-sensitive counterpart cells" [1].

Comparative Proteomic Profiling of Divergent Phenotypes for Water Holding Capacity across the Post Mortem Ageing Period in Porcine Muscle Exudate.

Two dimensional Difference Gel Electrophoresis (2-D DIGE) and mass spectrometry were applied to investigate the changes in metabolic proteins that occur over a seven day (day 1, 3 and 7) post mortem ageing period in porcine centrifugal exudate from divergent meat quality phenotypes. The objectives of the research were to enhance our understanding of the phenotype (water holding capacity) and search for biomarkers of this economically significant pork quality attribute. Major changes in protein abundance across nine phenotype-by-time conditions were observed. Proteomic patterns were dominated by post mortem ageing timepoint. Using a machine learning algorithm (l1-regularized logistic regression), a model was derived with the ability to discriminate between high drip and low drip phenotypes using a subset of 25 proteins with an accuracy of 63%. Models discriminating between divergent phenotypes with accuracy of 72% and 73% were also derived comparing respectively, high drip plus intermediate phenotype (considered as one phenotype) versus low drip and comparing low drip plus intermediate phenotype (considered as one phenotype) versus high drip. In all comparisons, the general classes of discriminatory proteins identified include metabolic enzymes, stress response, transport and structural proteins. In this research we have enhanced our understanding of the protein related processes underpinning this phenotype and provided strong data to work toward development of protein biomarkers for water holding capacity.

Multidrug resistant tumour cells shed more microvesicle-like EVs and less exosomes than their drug-sensitive counterpart cells.

BACKGROUND: Multidrug resistance (MDR) is a serious impediment to cancer treatment, with overexpression of drug efflux pumps such as P-glycoprotein (P-gp) playing a significant role. In spite of being a major clinical challenge, to date there is no simple, minimally invasive and clinically validated method for diagnosis of the MDR phenotype using non-tumour biological samples. Recently, P-gp has been found in extracellular vesicles (EVs) shed by MDR cancer cells. This study aimed to compare the EVs shed by MDR cells and their drug-sensitive cellular counterparts, in order to identify biomarkers of MDR. METHODS: Two pairs of MDR and drug-sensitive counterpart tumour cell lines were studied as models. EVs were characterized in terms of size and molecular markers and their protein content was investigated by proteomic analysis and Western blot. RESULTS: We found that MDR cells produced more microvesicle-like EVs and less exosomes than their drug-sensitive counterpart. EVs from MDR cells contained P-gp and presented a different content of proteins known to be involved in the biogenesis of EVs, particularly in the biogenesis of exosomes. CONCLUSIONS: The determination of the size and of this particular protein content of EVs shed by tumour cells may allow the development of a minimally-invasive simple method of detecting and predicting MDR. GENERAL SIGNIFICANCE: This work describes for the first time that cancer multidrug resistant cells shed more microvesicle-like EVs and less exosomes than their drug-sensitive counterpart cells, carrying a specific content of proteins involved in EV biogenesis that could be further studied as biomarkers of MDR.

Label-free LC-MS analysis of HER2+ breast cancer cell line response to HER2 inhibitor treatment.

BACKGROUND: Human epidermal growth-factor receptor (HER)-2 is overexpressed in 25 % of breast-cancers and is associated with an aggressive form of the disease with significantly shortened disease free and overall survival. In recent years, the use of HER2-targeted therapies, monoclonal-antibodies and small molecule tyrosine-kinase inhibitors has significantly improved the clinical outcome for HER2-positive breast-cancer patients. However, only a fraction of HER2-amplified patients will respond to therapy and the use of these treatments is often limited by tumour drug insensitivity or resistance and drug toxicities. Currently there is no way to identify likely responders or rational combinations with the potential to improve HER2-focussed treatment outcome. METHODS: In order to further understand the molecular mechanisms of treatment-response with HER2-inhibitors, we used a highly-optimised and reproducible quantitative label-free LC-MS strategy to characterize the proteomes of HER2-overexpressing breast-cancer cell-lines (SKBR3, BT474 and HCC1954) in response to drug-treatment with HER2-inhibitors (lapatinib, neratinib or afatinib). RESULTS: Following 12 ours treatment with different HER2-inhibitors in the BT474 cell-line; compared to the untreated cells, 16 proteins changed significantly in abundance following lapatinib treatment (1 µM), 21 proteins changed significantly following neratinib treatment (150 nM) and 38 proteins changed significantly following afatinib treatment (150 nM). Whereas following 24 hours treatment with neratinib (200 nM) 46 proteins changed significantly in abundance in the HCC1954 cell-line and 23 proteins in the SKBR3 cell-line compared to the untreated cells. Analysing the data we found that, proteins like trifunctional-enzyme subunit-alpha, mitochondrial; heterogeneous nuclear ribonucleoprotein-R and lamina-associated polypeptide 2, isoform alpha were up-regulated whereas heat shock cognate 71 kDa protein was down-regulated in 3 or more comparisons. CONCLUSION: This proteomic study highlights several proteins that are closely associated with early HER2-inhibitor response and will provide a valuable resource for further investigation of ways to improve efficacy of breast-cancer treatment.

Monitoring post mortem changes in porcine muscle through 2-D DIGE proteome analysis of Longissimus muscle exudate.

BACKGROUND: Meat quality is a complex trait influenced by a range of factors with post mortem biochemical processes highly influential in defining ultimate quality. High resolution two-dimensional DIfference Gel Electrophoresis (2-D DIGE) and Western blot were applied to study the influence of post mortem meat ageing on the proteome of pork muscle. Exudate collected from the muscle following centrifugation was analysed at three timepoints representing a seven day meat ageing period. RESULTS: The intensity of 136 spots varied significantly (p < 0.05) across this post mortem period and 40 spots were identified using mass spectrometry. The main functional categories represented were metabolic proteins, stress-related proteins, transport and structural proteins. Metabolic and structural proteins were generally observed to increase in abundance post mortem and many likely represent the accumulation of the degradation products of proteolytic enzyme activity. In contrast, stress-related proteins broadly decreased in abundance across the ageing period. Stress response proteins have protective roles in maintaining cellular integrity and a decline in their abundance over time may correlate with a reduction in cellular integrity and the onset of meat ageing. Since cellular conditions alter with muscle ageing, changes in solubility may also contribute to observed abundance profiles. CONCLUSIONS: Muscle exudate provided valuable information about the pathways and processes underlying the post mortem ageing period, highlighting the importance of post mortem modification of proteins and their interaction for the development of meat quality traits.

2D DIGE proteomic analysis of early post mortem muscle exudate highlights the importance of the stress response for improved water-holding capacity of fresh pork meat.

Variation in water-holding capacity (WHC), which presents a major economic burden to the swine industry, is considered to be underpinned by variation at a molecular and biochemical level. High-resolution 2D DIGE followed by MS analysis and Western blot were used to unravel the proteome of muscle exudate, collected following centrifugation, in the pH 4-7 range. A first 2DE-based protein map of this substrate was produced where 89 spots were successfully characterised. Two phenotypes divergent for WHC plus one intermediate were compared with a view to deciphering the biochemical processes impacting on variation in WHC. Twenty spots were observed to be altered across the phenotypes. Of these, 14 represented sixteen proteins including metabolic enzymes, stress response proteins and structural proteins. Triosephosphate isomerase and transferrin showed a major difference between the two extreme phenotypes, and may have potential as biological markers for WHC prediction. Several members of the HSPs family were highlighted. This proteomic study makes an important contribution towards a more detailed molecular view of the processes behind WHC and will provide a valuable resource for future investigations.

Centrifugal drip is an accessible source for protein indicators of pork ageing and water-holding capacity.

Achieving an improvement in water-holding capacity (WHC) of pork and a reduction in the incidence of pale, soft and exudative (PSE)- and dark, firm and dry (DFD)-like meat is a major challenge for the swine industry. Using proteomics, we sought to identify proteins associated with WHC and to monitor postmortem protein degradation. Twenty longissimus samples were categorised into WHC phenotypes. The centrifugal drip was subjected to SDS-PAGE and mass-spectrometry. Forty-four proteins were identified in the centrifugal drip proteome. Changes occurred in volume of five bands across the ageing period, with most significant changes representing increases between day 3 and day 7. Seven proteins were identified in these bands, most with functions in glycolysis. One band significantly differed in abundance across WHC phenotypes. Peptide signatures of the heat shock protein family were identified in this band.