Effect of dietary Spirulina (Arthrospira platensis) on the intestinal function of post-weaned piglet: An approach combining proteomics, metabolomics and histological studies.

The effect of dietary Spirulina (Arthrospira platensis) and CAZyme supplementation was assessed on the gut of weaned piglets, using an integrated NMR-metabolomics approach combined with Tandem Mass Tag labelled proteomics. Thirty weaned male piglets were assigned to one of the three following diets (n = 10): cereal and soybean meal basal diet (Control), basal diet with 10% Spirulina inclusion (SP) and SP diet supplemented with 0.01% lysozyme (SP + L). The experiment lasted 4 weeks and, upon slaughter, small intestine samples were collected for histological, metabolomic and proteomic analysis. No significant differences were found for the histology and metabolomics analysis between the three experimental groups. Lactate, glutamate, glycine and myo-inositol were the most abundant metabolites. Proteomics results showed 1502 proteins identified in the intestine tissue. A total of 23, 78, 27 differentially abundant proteins were detected respectively for the SP vs. Control, SP + L vs. Control and SP + L vs. SP comparisons. The incorporation of Spirulina and supplementation of lysozyme in the piglet's diets is associated to intestinal proteomic changes. These include increased protein synthesis and abundance of contractile apparatus proteins, related with increased nutrient availability, which has beneficial (increased glucose uptake) and detrimental (increased digesta viscosity) metabolic effects. SIGNIFICANCE: The use of conventional feedstuffs becomes increasingly prohibitive due to its environmental toll. To increase the sustainability of the livestock sector, novel feedstuffs such as microalgae need to be considered. However, its recalcitrant cell wall has antinutritional effects that can inhibit high dietary inclusion levels. The supplementation with CAZymes is a possible solution to this issue. The small intestine is a central piece in monogastric digestion and of particular importance for the weaned piglet. Studying the effect of dietary Spirulina and CAZyme supplementation on its histomorphology, metabolome and proteome allows studying relevant physiological adaptations to these diets.

Tandem Mass Tag (TMT) Proteomic Analysis of Saliva in Horses with Acute Abdominal Disease.

The aim of this study was to investigate the changes in the salivary proteome in horses with acute abdominal disease (AAD) using a tandem mass tags (TMT)-based proteomic approach. The saliva samples from eight horses with AAD were compared with six healthy horses in the proteomic study. Additionally, saliva samples from eight horses with AAD and eight controls were used to validate lactoferrin (LF) in saliva. The TMT analysis quantified 118 proteins. Of these, 17 differed significantly between horses with AAD and the healthy controls, 11 being downregulated and 6 upregulated. Our results showed the downregulation of gamma-enteric smooth muscle actin (ACTA2), latherin isoform X1, and LF. These proteins could be closely related to an impaired primary immune defense and antimicrobial capacity in the mucosa. In addition, there was an upregulation of mucin 19 (MUC19) and the serine protease inhibitor Kazal-type 5 (SPINK5) associated with a protective effect during inflammation. The proteins identified in our study could have the potential to be novel biomarkers for diagnosis or monitoring the physiopathology of the disease, especially LF, which decreased in the saliva of horses with AAD and was successfully measured using a commercially available immunoassay.

Influence of dietary Spirulina inclusion and lysozyme supplementation on the longissimus lumborum muscle proteome of newly weaned piglets.

Arthrospira platensis (Spirulina) is a microalga with a high content of crude protein. It has a recalcitrant cell wall that limits the accessibility of the animal endogenous enzymes to its intracellular nutrients. Enzymatic supplementation aiming to degrade cell walls could benefit microalgae digestibility. The objective of this study was to evaluate the impact of dietary Spirulina and lysozyme supplementation over the muscle proteome of piglets during the post-weaning stage. Thirty piglets were randomly distributed among three diets: control (no microalga), SP (10% Spirulina) and SP + L (10% Spirulina +0.01% lysozyme). After 4 weeks, they were sacrificed and samples of the longissimus lumborum muscle were taken. The muscle proteome was analysed using a Tandem Mass Tag (TMT)-based quantitative approach. A total of 832 proteins were identified. Three comparisons were computed: SP vs Ctrl, SP + L vs Ctrl and SP + L vs SP. They had ten, four and twelve differentially abundant proteins. Glycogen metabolism and nutrient reserves utilization are increased in the SP piglets. Structural muscle protein synthesis increased, causing higher energy requirements in SP + L piglets. Our results demonstrate the usefulness of proteomics to disclose the effect of dietary microalgae, whilst unveiling putative mechanisms derived from lysozyme supplementation. Data available via ProteomeXchange with identifier PXD024083. SIGNIFICANCE: Spirulina, a microalga, is an alternative to conventional crops which could enhance the environmental sustainability of animal production. Due to its recalcitrant cell wall, its use requires additional measures to prevent anti-nutritional effects on the feeding of piglets in the post-weaning period, during which they endure post-weaning stress. One of such measures could be CAZyme supplementation to help degrade the cell wall during digestion. Muscle proteomics provides insightful data on the effect of dietary microalgae and enzyme activity on piglet metabolism.

Novel biomarkers in cats with congestive heart failure due to primary cardiomyopathy.

The pathogenesis of feline cardiomyopathy and congestive heart failure (CHF) requires further understanding. In this study, we assessed serum proteome change in feline CHF, aiming to identify novel biomarker for both research and clinical use. The study comprised 15 cats in CHF, 5 cats in preclinical cardiomyopathy and 15 cats as healthy controls. Serum proteome profiles were obtained by tandem mass tag labelling followed by mass spectrometry. Protein concentrations in CHF cats were compared with healthy controls. Western blot was performed for proteomic validation. Correlations were assessed between the altered proteins in CHF and clinical variables in cats with cardiomyopathy to evaluate protein-cardiac association. Bioinformatic analysis was employed to identify pathophysiological pathways involved in feline CHF. Sixteen serum proteins were significantly different between CHF and healthy control cats (P < .05). These included serine protease inhibitors, apolipoproteins and other proteins associated with inflammation and coagulation. Clinical parameters from cats with cardiomyopathy significantly correlated with the altered proteins (P < .05). Bioinformatic analysis identified 13 most relevant functional profiles in feline CHF, which mostly associated with extracellular matrix organization and metabolism. Data are available via ProteomeXchange with identifier PXD017761. SIGNIFICANCE: Cardiomyopathies affect both cats and humans, and they can cause serious consequence such as congestive heart failure (CHF). To date, the pathophysiological mechanism of CHF is not fully understood. In this study, for the first time, we used a proteomic approach combined with bioinformatic analysis to evaluate serum protein change in cats with CHF. Results indicate systemic inflammation, coagulation protein changes, innate immunity and extracellular matrix remodeling are involved in feline CHF, which are largely comparable with findings in previous human studies. Our study provides new insights into CHF and cardiomyopathy in cats, and the identified novel biomarkers and pathophysiological pathways provide valuable information for future studies.

Extracellular vesicles in infectious diseases caused by protozoan parasites in buffaloes.

BACKGROUND: Extracellular vesicles (EVs) are small membrane-bound vesicles of growing interest in vetetinary parasitology. The aim of the present report was to provide the first isolation, quantification and protein characterization of EVs from buffalo (Bubalus bubalis) sera infected with Theileria spp. METHODS: Infected animals were identified through optical microscopy and PCR. EVs were isolated from buffalo sera by size-exclusion chromatography and characterized using western blotting analysis, nanoparticle tracking analysis and transmission electron microscopy. Subsequently, the proteins from isolated vesicles were characterized by mass spectrometry. RESULTS: EVs from buffalo sera have shown sizes in the 124-140 nm range and 306 proteins were characterized. The protein-protein interaction analysis has evidenced biological processes and molecular function associated with signal transduction, binding, regulation of metabolic processes, transport, catalytic activity and response to acute stress. Five proteins have been shown to be differentially expressed between the control group and that infected with Theileria spp., all acting in the oxidative stress pathway. CONCLUSIONS: EVs from buffaloes infected with Theileria spp. were successfully isolated and characterized. This is an advance in the knowledge of host-parasite relationship that contributes to the understanding of host immune response and theileriosis evasion mechanisms. These findings may pave the way for searching new EVs candidate-markers for a better production of safe biological products derived from buffaloes.

The plasma proteome and the acute phase protein response in canine pyometra.

Canine pyometra is a common inflammatory disease of uterus in sexually mature bitches caused by secondary bacterial infection, leading to change in plasma proteins associated with the innate immune system. Proteomic investigation is increasingly being applied to canine diseases in order to identify and quantify significant changes in the plasma proteome. The aim of the study was to assess and quantify changes in plasma proteome profiles of healthy dogs and pyometra affected bitches using a TMT-based high-resolution quantitative proteomic approach. As a result, 22 proteins were significantly down-regulated including transthyretin, antithrombin, retinol-binding protein, vitamin D binding protein, paraoxonase 1, and kallikrein, while 16 were significantly up-regulated including haptoglobin light chain, alpha-1-acid glycoprotein, C-reactive protein precursor, and lipopolysaccharide-binding protein in dogs with pyometra. Pathway analysis indicated that acute inflammatory response, regulation of body fluid levels, protein activation cascade, the humoral immune response, and phagocytosis were affected in pyometra. Validation of biological relevance of the proteomic study was evident with significant increases in the concentrations of haptoglobin, C-reactive protein, alpha-1-acid glycoprotein, and ceruloplasmin by immunoassay. Pyometra in bitches was shown to stimulate an increase in host defence system proteins in response to inflammatory disease including the acute phase proteins. SIGNIFICANCE: The label-based high-resolution quantitative proteomics analysis and bioinformatic approach used in this study provide insight into the complex pathophysiology of inflammation associated with pyometra revealing proteins with biomarker potential. Early diagnosis and therapeutic intervention may prevent severe complications associated with advancing sepsis in dogs with pyometra. Therefore the identification of diagnostic biomarkers that, after clinical validation may be used in veterinary practice and protein relevant to pathways responding to disease are important findings of the study. Data are available via ProteomeXchange with identifier PXD015951.

Serum proteome profiling in canine chronic valve disease using a TMT-based quantitative proteomics approach.

Chronic valve disease (CVD) is the most common clinically significant heart disease of dogs, affecting 20 to 40% of dogs. The aim of this study was to evaluate the serum protein profile of healthy and CVD affected dogs, by means of an isobaric tandem mass tag (TMT) label-based high-resolution quantitative proteomic approach. Additionally, conventional cardiac biomarkers were measured in the serum, functional bioinformatics analysis was employed for elucidating molecular mechanisms and pathways associated with CVD, and validation of proteomic results was performed by immunoassays and Western blotting. Of 290 identified and quantified proteins, 15 proteins showed significantly different abundances (p < .05), including antithrombin-III, alpha-2-antiplasmin, tetranectin, apolipoprotein M, adiponectin, inter-alpha-trypsin inhibitor heavy chain H1, gelsolin and apolipoprotein B-100. The identified proteins with differently abundances are involved in a number of pathways, such as complement and coagulation cascades, haemostasis, regulation of actin cytoskeleton, lipid metabolism and transport. We found comparative similarities with human disease in terms of identified proteins and GO pathways, which confirmed similar pathophysiology of this disease, but also differences, mainly in lipid metabolism. SIGNIFICANCE: There have been few investigations of canine serum proteome despite the potential for biomarker discovery and comparative disease analysis. Establishing serum proteomic signatures in healthy dogs and dogs with CVD will benefit for understanding the aetiology of disease in dogs, identify putative biomarkers and provide models of comparative human disease. Circulating biomarkers are important for understanding of the mechanisms of cardiovascular disease and high incidence of CVD in dogs prioritizes the search for novel biomarkers.

Plasma proteomic profiling and pathway analysis of normal and overconditioned dairy cows during the transition from late pregnancy to early lactation.

This study applied a quantitative proteomics approach along with bioinformatics analyses to investigate changes in the plasma proteome of normal and overconditioned dairy cows during the transition period. Fifteen weeks before their anticipated calving date, 38 multiparous Holstein cows were selected based on their current and previous body condition scores (BCS) and allocated to either a high or a normal BCS group (19 cows each). They received different diets until dry-off to reach targeted differences in BCS and back fat thickness (BFT) until dry-off. At dry-off, normal BCS cows had a BCS <3.5 (minimum, 2.75) and BFT <1.2 cm (minimum, 0.58), and the high BCS cows had a BCS >3.75 (maximum, 4.50) and BFT >1.4 cm (maximum, 2.90). The proteomics study used a subset of 5 animals from each group. These cows were selected based on their circulating concentrations of fatty acids (FA) on d 14 postpartum and ß-hydroxybutyrate (BHB) on d 21 postpartum, representing the greater or the lower extreme values within their BCS group, respectively. The high BCS subset (HE-HBCS) had 4.50 < BCS > 3.75, FA = 1.17 ± 0.46 mmol/L, and BHB = 2.15 ± 0.42 mmol/L (means ± SD), and the low BCS subset (LE-NBCS) had 3.50 < BCS > 2.75, FA = 0.51 ± 0.28 mmol/L, and BHB = 0.84 ± 0.17 mmol/L. Plasma samples from d -49, +7, and +21 relative to parturition were used for proteome profiling by applying the quantitative tandem mass tags (TMT) approach. Nondepleted plasma samples were subjected to reduction and digestion and then labeled with TMT 10plex reagents. High-resolution liquid chromatography-tandem mass spectrometry analysis of TMT-labeled peptides was carried out, and the acquired spectra were analyzed for protein identification and quantification. In total, 254 quantifiable proteins (criteria: 2 unique peptides and 5% false discovery rate) were identified in the plasma samples. From these, 24 differentially abundant proteins (14 more abundant, 10 less abundant) were observed in the LE-NBCS cows compared with the HE-HBCS cows during the transition period. Plasma α-2-macroglobulins were more abundant in HE-HBCS versus LE-NBCS cows at d +7 and +21. Gene Ontology enrichment analyses of differentially abundant proteins revealed that the acute inflammatory response, regulation of complement activation, protein activation cascade, and regulation of humoral immune response were the most enriched terms in the LE-NBCS group compared with the HE-HBCS group. In addition, we identified 24 differentially abundant proteins (16 in the LE-NBCS group, and 8 in the HE-HBCS group) during the transition period. The complement components C1q and C5 were less abundant, while C3 and C3d were more abundant in LE-NBCS compared with HE-HBCS cows. Overall, overconditioning around calving was associated with alterations in protein pathways related to acute inflammatory response and regulation of complement and coagulation cascades in transition cows.

Metabolome and proteome changes in skeletal muscle and blood of pre-weaning calves fed leucine and threonine supplemented diets.

In pre-weaning calves, both leucine and threonine play important roles in growth and muscle metabolism. In this study, metabolomics, proteomics and clinical chemistry were used to assess the effects of leucine and threonine supplementation added to milk replacer on 14 newborn Holstein male calves: 7 were fed a control diet (Ctrl) and 7 were fed the Ctrl diet supplemented with 0.3% leucine and 0.3% threonine (LT) from 5.6 days of age to 53.6 days. At this time, blood and semitendinosus muscle biopsies were collected for analysis. Integrated metabolomics and proteomics showed that branched-chain amino acids (BCAA) degradation and mitochondrial oxidative metabolism (citrate cycle and respiratory chain) were the main activated pathways in muscle because of the supplementation. BCAA derivatives and metabolites related to lipid mobilization showed the major changes. The deleterious effects of activated oxidative phosphorylation were balanced by the upregulation of antioxidant proteins. An increase in protein synthesis was indicated by elevated aminoacyl-tRNA biosynthesis and increased S6 ribosomal protein phosphorylation in skeletal muscle. In conclusion, LT group showed greater BCAA availability and mitochondrial oxidative activity; as the muscle cells undergo greater aerobic metabolism, antioxidant defenses were activated to compensate for possible cell damage. Data are available via ProteomeXchange (PXD016098). SIGNIFICANCE: Leucine and threonine are essential amino acids for the pre-weaning calf, being of high importance for growth. In this study, we found that leucine and threonine supplementation of milk replacer to feed pre-weaning calves led to differences in the proteome, metabolome and clinical chemistry analytes in skeletal muscle and plasma, albeit no differences in productive performance were recorded. This study extends our understanding on the metabolism in dairy calves and helps optimizing their nutritional status.

Differences on salivary proteome at rest and in response to an acute exercise in men and women: A pilot study.

The aim of the present study was to evaluate the differences in salivary proteome at rest and in response to an acute exercise in men and women. For this, unstimulated whole saliva samples in rest and after a bout resistance exercise leading to failure (ELF) of both men and women (n = 5 for each sex) were subjected to isobaric Tandem Mass Tags (TMT) labelling followed by LC-MS/MS. A total of 274 proteins were identified and met the inclusion criteria. 16 proteins were modulated for the interaction sex*exercise, 6 were modulated because of the exercise, and 65 were differentially expressed between men and women at rest. In conclusion, these results indicate sex-related differences in the salivary proteome at rest and after an acute exercise, pointing out possible candidate biomarkers for sports performance and allowing further knowledge of the physiological processes occurring during ELF. SIGNIFICANCE: The present study describes for the first time the changes that occur in salivary proteome detected by TMT-based proteomics in response to an acute exercise and the differences in these changes depending on sex. Of the 274 protein identified, the 87 differentially expressed proteins and their related pathways were discussed, focusing on the sex- and exercise-related differences in the salivary proteome.

Extracellular vesicles in infectious diseases caused by protozoan parasites in buffaloes

Extracellular vesicles (EVs) are small membrane-bound vesicles of growing interest in vetetinary parasitology. The aim of the present report was to provide the first isolation, quantification and protein characterization of EVs from buffalo (Bubalus bubalis) sera infected with Theileria spp. Methods: Infected animals were identified through optical microscopy and PCR. EVs were isolated from buffalo sera by size-exclusion chromatography and characterized using western blotting analysis, nanoparticle tracking analysis and transmission electron microscopy. Subsequently, the proteins from isolated vesicles were characterized by mass spectrometry. Results: EVs from buffalo sera have shown sizes in the 124-140 nm range and 306 proteins were characterized. The protein-protein interaction analysis has evidenced biological processes and molecular function associated with signal transduction, binding, regulation of metabolic processes, transport, catalytic activity and response to acute stress. Five proteins have been shown to be differentially expressed between the control group and that infected with Theileria spp., all acting in the oxidative stress pathway. Conclusions: EVs from buffaloes infected with Theileria spp. were successfully isolated and characterized. This is an advance in the knowledge of host-parasite relationship that contributes to the understanding of host immune response and theileriosis evasion mechanisms. These findings may pave the way for searching new EVs candidate-markers for a better production of safe biological products derived from buffaloes.(AU)

Changes in saliva of dogs with canine leishmaniosis: A proteomic approach.

In the present study, a quantitative proteomic approach to study changes in saliva proteins associated with canine leishmaniosis (CanL) was performed. For this, canine salivary proteins were analysed and compared between dogs before (T0) and after (T1) experimental infection with Leishmania infantum by high-throughput label-based quantitative LC-MS/MS proteomic approach and bioinformatic analysis of the in silico inferred interactome protein network was created from the initial list of differential proteins. More than 2000 proteins were identified, and of the 90 differentially expressed proteins between T0 and T1, 12 were down-regulated with log2 fold change lower than -0.5849, and 19 were up-regulated with log2 fold change greater than 0.5849. This study provides evidence of changes in salivary proteome that can occur in canine leishmaniosis and revealed biological pathways in saliva modulated in canine leishmaniosis with potential for further targeted research.

Changes in saliva proteins in two conditions of compromised welfare in pigs: An experimental induced stress by nose snaring and lameness.

The aim of this study was to identify biological pathways and proteins differentially expressed in saliva of pigs in two conditions of compromised welfare: an acute stress consisting of restraint with a nose snare and in pigs with lameness which is a highly frequent problem in the swine industry. For this purpose, high-resolution quantitative proteomics based on Tandem Mass Tags labelling was used. Four proteins showed significant differences in the conditions of compromised welfare, namely cornulin, the heat shock protein 27 and lactate dehydrogenase (LDH), that showed significant increases, whereas immunoglobulin J chain showed a significant decrease. LDH, which was the protein that showed the highest differences, was selected for validation and clinical evaluation as a diagnostic biomarker. Significant changes in this protein were observed between pigs restrained with a nose snare and pigs with lameness compared with healthy pigs when measured with available commercial assays in a larger population of pigs. In conclusion, this study reports that in situations of compromised welfare on farm, such as acute stress and lameness in pigs, there are changes in proteins and metabolic pathways in saliva, and describes a series of proteins that could potentially be used as biomarkers for both short term acute stress and longer term chronic stress of lameness. These biomarkers would have the advantage of being measured in saliva by a noninvasive and not stressful collection sampling procedure.

Identification of possible new salivary biomarkers of stress in sheep using a high-resolution quantitative proteomic technique.

The aim of this study was to identify biological pathways and proteins differentially expressed in the saliva proteome of sheep after the application of a model of stress, using high-resolution quantitative proteomics. In addition, one of the proteins differently expressed was verified and evaluated as a possible biomarker of stress in this species. Saliva paired samples from eight sheep before and after the application of a model of stress based on shearing were analysed using tandem mass tags (TMT). The TMT analysis allowed for the identification of new stress-related metabolic pathways and revealed 13 proteins, never described in saliva of sheep, that were differentially expressed between before and after the stress. Six of these proteins pertain to four major metabolic pathways affected, namely: canonical glycolysis, oxygen transport, neural nucleus development, and regulation of actin cytoskeleton reorganization. The rest of proteins were unmapped original proteins such as acyl-coenzyme-A-binding protein; complement C3; alpha-2-macroglobulin isoform-X1; type-II small proline-rich protein; lactoferrin; secretoglobin family-1D-member; and keratin, type-II cytoskeletal 6. Of these proteins, based on its biological significance and specific immunoassay availability, lactoferrin was selected for further validation. The immunoassay intra- and inter-assay coefficients of variation were lower than 13%. The method showed good linearity under dilution and recovery, and the detection limit was low enough to detect salivary lactoferrin levels. A significant decrease (P < 0.01) in salivary lactoferrin concentration in the sheep following the application of the model of stress was observed, suggesting that this protein could be a potential salivary biomarker of stress situations in sheep.

Quantitative proteomics using tandem mass tags in relation to the acute phase protein response in chicken challenged with Escherichia coli lipopolysaccharide endotoxin.

The inflammatory response in chickens (Gallus Gallus domesticus) is an integral part of the bird's response to infection. Detailing proteomic changes occurring during infection would be beneficial to the poultry industry, offering opportunities for comparative pathophysiological analysis. The objective of this study was to quantify the changes in the plasma proteome in chickens challenged with lipopolysaccharide (LPS), a bacterial endotoxin known to stimulate the host innate immune system. Plasma from chicken (N = 6) challenged with Escherichia coli (LPS) (2 mg/kg body weight) was collected pre (0 h) and at 12, 24, 48, and 72 h post-injection along with plasma from a control group (N = 6) challenged with sterile saline. Samples were analysed by a quantitative Tandem Mass Tags approach using a Q-Exactive-Plus mass-spectrometer. Identification and relative quantification were performed using Proteome Discoverer, and data were analysed using R. Gene Ontology terms were analysed by Cytoscape based on the Gallus gallus database. Finally, 87 significantly regulated proteins were found, including serum-amyloid-A, ovotransferrin and alpha-1-acid-glycoprotein, showing a significant effect of time post-injection in the LPS-treated group. Different pathways related with protein activation cascade and heterotopic cell-cell adhesion were affected by LPS-challenge. LPS-challenged chickens demonstrate significant changes to the plasma proteome with both increases and decreases of individual proteins within 12 h of challenge. SIGNIFICANCE: The injection of chicken with bacterial lipopolysaccharide followed by sequential plasma and clinical analysis of the bird, is a long established and a widely used model for inflammation and infection studies. This study, utilising and combining proteomic and immunoassay analysis with bioinformatic analysis, revealed that several biological pathways are modulated during this early period of inflammation. In addition, proteins with biomarker potential were identified and successfully validated. This experimental model also demonstrated potential for pathophysiological mechanism investigation and as an inflammatory model for biomedical research. There is, despite plasma being an easily accessible biological matrix which is representative of the health status of the bird, scarce data on the chicken plasma proteome. This research makes a positive contribution to the current field, generating significant data for continuing comparative analysis.

Changes in salivary analytes in canine parvovirus: A high-resolution quantitative proteomic study.

The present study evaluated the changes in salivary proteome in parvoviral enteritis (PVE) in dogs through a high-throughput quantitative proteomic analysis. Saliva samples from healthy dogs and dogs with severe parvovirosis that survived or perished due to the disease were analysed and compared by Tandem Mass Tags (TMT) analysis. Proteomic analysis quantified 1516 peptides, and 287 (corresponding to 190 proteins) showed significantly different abundances between studied groups. Ten proteins were observed to change significantly between dogs that survived or perished due to PVE. Bioinformatics' analysis revealed that saliva reflects the involvement of different pathways in PVE such as catalytic activity and binding, and indicates antimicrobial humoral response as a pathway with a major role in the development of the disease. These results indicate that saliva proteins reflect physiopathological changes that occur in PVE and could be a potential source of biomarkers for this disease.

Proteomics in Veterinary Medicine and Animal Science: Neglected Scientific Opportunities with Immediate Impact.

Animal/veterinary proteomics is an evolving field which holds a great promise not only for fundamental and applied discoveries regarding biology and pathology of domestic species, but can also be implemented in comparative applications of human diseases research. Experimental proteomics in domestic animals have advantages over use of rodents, such as multiple sampling in time series and availability of biological samples in sufficient volume for multiple analyses, such that both experimental and natural disease processes can be investigated. While there are certain technical limitations in the expansion of the field, they can currently be circumvented and in the future mastered with a greater participation of proteomic experts, which will in turn drive the accessibility of species-specific reagents, data volume expansion in bioinformatic databases, and increased funding. This Viewpoint highlights some comparative proteomics studies addressing important issues and encourages readers to expand their horizons of domestic animal proteomics research. It will hopefully inspire new fruitful collaborations between veterinary and animal scientists and proteomic specialists for research in these areas that can have immediate and direct impact on health, society, and the economy.

Serum proteome profiling in canine idiopathic dilated cardiomyopathy using TMT-based quantitative proteomics approach.

Idiopathic dilated cardiomyopathy (iDCM) is a primary myocardial disorder with an unknown aetiology, characterized by reduced contractility and ventricular dilation of the left or both ventricles. Naturally occurring canine iDCM was used herein to identify serum proteomic signature of the disease compared to the healthy state, providing an insight into underlying mechanisms and revealing proteins with biomarker potential. To achieve this, we used high-throughput label-based quantitative LC-MS/MS proteomics approach and bioinformatics analysis of the in silico inferred interactome protein network created from the initial list of differential proteins. To complement the proteomic analysis, serum biochemical parameters and levels of know biomarkers of cardiac function were measured. Several proteins with biomarker potential were identified, such as inter-alpha-trypsin inhibitor heavy chain H4, microfibril-associated glycoprotein 4 and apolipoprotein A-IV, which were validated using an independent method (Western blotting) and showed high specificity and sensitivity according to the receiver operating characteristic curve analysis. Bioinformatics analysis revealed involvement of different pathways in iDCM, such as complement cascade activation, lipoprotein particles dynamics, elastic fibre formation, GPCR signalling and respiratory electron transport chain. SIGNIFICANCE: Idiopathic dilated cardiomyopathy is a severe primary myocardial disease of unknown cause, affecting both humans and dogs. This study is a contribution to the canine heart disease research by means of proteomic and bioinformatic state of the art analyses, following similar approach in human iDCM research. Importantly, we used serum as non-invasive and easily accessible biological source of information and contributed to the scarce data on biofluid proteome research on this topic. Bioinformatics analysis revealed biological pathways modulated in canine iDCM with potential of further targeted research. Also, several proteins with biomarker potential have been identified and successfully validated.

Surface Proteome Biotinylation Combined with Bioinformatic Tools as a Strategy for Predicting Pathogen Interacting Proteins.

Constant advancements in methodology and mass spectrometry instrumentation, genome sequencing and bioinformatic tools have enabled the identification of numerous pathogen proteomes. Identifying the pathogen interacting proteins by means of high-throughput techniques is key for understanding pathogen invasion and survival mechanisms and in such a way proposing specific proteins as pharmaceutical targets. Herein we describe the methodology for the enrichment and identification of pathogen surface proteome using cell surface protein biotinylation followed by LC-MS/MS and bioinformatic analyses of such data. This strategy is to be employed for the determination of protein subcellular localization and prediction of potential pathogen interacting proteins.

Integrated dataset on acute phase protein response in chicken challenged with Escherichia coli lipopolysaccharide endotoxin.

Data herein describe the quantitative changes in the plasma proteome in chickens challenged with lipopolysaccharide (LPS), a bacterial endotoxin known to stimulate the host innate immune system obtained by shotgun quantitative proteomic tandem mass tags approach using high-resolution Orbitrap technology. Statistical and bioinformatic analyses were performed to specify the effect of bacterial endotoxin. Plasma from chicken (N=6) challenged with Escherichia coli (LPS) (2 mg/kg body weight) was collected pre (0 h) and at 12, 24, 48, and 72 h post injection along with plasma from a control group (N=6) challenged with sterile saline. Protein identification and relative quantification were performed using Proteome Discoverer, and data were analysed using R. Gene Ontology terms were analysed by the Cytoscape application ClueGO based on Gallus gallus GO Biological Process database, and refined by REVIGO. Absolute quantification of several acute phase proteins, e.g. alpha-1-acid glycoprotein (AGP), serum amyloid A (SAA) and ovotrensferrin (OVT) was performed by immunoassays to validate the LC-MS results. The data contained within this article are directly related to our research article"Quantitative proteomics using tandem mass tags in relation to the acute phase protein response in chicken challenged with Escherichia coli lipopolysaccharide endotoxin" [1]. The raw mass spectrometric data generated in this study were deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD009399 (http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD009399).