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Apoptosis ensures removal of damaged cells and helps shape organs during development by removing excessive cells. To prevent the intracellular content of the apoptotic cells causing damage to surrounding cells, apoptotic cells are quickly cleared by engulfment. Tight regulation of apoptosis and engulfment is needed to prevent several pathologies such as cancer, neurodegenerative and autoimmune diseases. There is increasing evidence that the engulfment machinery can regulate the execution of apoptosis. However, the underlying molecular mechanisms are poorly understood. We show that dynein mediates cell non-autonomous cross-talk between the engulfment and apoptotic programs in the Caenorhabditis elegans germline. Dynein is an ATP-powered microtubule-based molecular motor, built from several subunits. Dynein has many diverse functions including transport of cargo around the cell. We show that both dynein light chain 1 (DLC-1) and dynein heavy chain 1 (DHC-1) localize to the nuclear membrane inside apoptotic germ cells in C. elegans. Strikingly, lack of either DLC-1 or DHC-1 at the nuclear membrane inhibits physiological apoptosis specifically in mutants defective in engulfment. This suggests that a cell fate determining dialogue takes place between engulfing somatic sheath cells and apoptotic germ cells. The underlying mechanism involves the core apoptotic protein CED-4/Apaf1, as we find that DLC-1 and the engulfment protein CED-6/GULP are required for the localization of CED-4 to the nuclear membrane of germ cells. A better understanding of the communication between the engulfment machinery and the apoptotic program is essential for identifying novel therapeutic targets in diseases caused by inappropriate engulfment or apoptosis.

Mastitis is a major challenge to bovine health. The detection of sensitive markers for mastitis in dairy herds is of great demand. Suitable biomarkers should be measurable in milk and should report pathogen-specific changes at an early stage to support earlier diagnosis and more efficient treatment. However, the identification of sensitive biomarkers in milk has remained a challenge, in part due to their relatively low concentration in milk. In the present study, we used a selected reaction monitoring (SRM) mass spectrometry approach, which allowed the absolute quantitation of 13 host response proteins in milk for the first time. These proteins were measured over a 54-h period upon an in vivo challenge with cell wall components from either gram-negative (lipopolysaccharide from Escherichia coli; LPS) or gram-positive bacteria (peptidoglycan from Staphylococcus aureus; PGN). Whereas our data clearly demonstrate that all challenged animals have consistent upregulation of innate immune response proteins after both LPS and PGN challenge, the data also reveal clearly that LPS challenge unleashes faster and shows a more intense host response compared with PGN challenge. Biomarker candidates that may distinguish between gram-negative and gram-positive bacteria include α-2 macroglobulin, α-1 antitrypsin, haptoglobin, serum amyloid A3, cluster of differentiation 14, calgranulin B, cathepsin C, vanin-1, galectin 1, galectin 3, and IL-8. Our approach can support further studies of large cohorts of animals with natural occurring mastitis, to validate the relevance of these suggested biomarkers in dairy production.

The aim of the present study was to investigate how maternal diet can influence the adipose tissue of goat kids. Omental adipose tissue proteomes of goat-kids from mothers fed with diet enriched with stearic acid (ST-kids), fish oil (FO-kids) and standard diets (CTRL) were determined by quantitative iTRAQ 2D-LC-MS/MS analysis. Twenty proteins were found to be differentially expressed in suckling kids' omental adipose tissue. Stearic acid induces changes in a higher number of proteins when compared to fish oil. Eleven proteins, namely AARS, ECl1, PMSC2, CP, HSPA8, GPD1, RPL7, OGDH, RPL24, FGA and RPL5 were decreased in ST-kids only. Four proteins, namely DLST, EEF1G, BCAP31 and RALA were decreased in FO-kids only, and one, NUCKS1, was increased. Four proteins, namely PMSC1, PPIB, TUB5 x 2 and EIF5A1, were be less abundant in both ST- and FO- kids. Most of the protein whose abundance was decreased in ST kids (10 out of 15) are involved in protein metabolism and catabolism pathways. Qualitative gene expression analysis confirmed that all the proteins identified by mass spectrometry, with the exception of FGA, were produced by adipose tissue. Quantitative gene expression analysis demonstrated that two proteins, namely CP, a minor acute phase protein, and ECl1, involved in fatty acid beta oxidation, were downregulated at mRNA level as well. ECl1 gene expression was downregulated in ST-kids AT as compared to Ctrl-kids and CP was downregulated in both ST- and FO-kids. The present results demonstrate that it is possible to influence adipose goat-kid proteome by modifying the maternal diet.

In horses, pathological healing with formation of exuberant granulation tissue (EGT) is a particular problem in limb wounds, whereas body wounds tend to heal without complications. Chronic inflammation has been proposed to be central to the pathogenesis of EGT. This study aimed to investigate levels of inflammatory acute phase proteins (APPs) in interstitial fluid from wounds in horses. A novel approach for absolute quantification of proteins, selected reaction monitoring (SRM)-based mass spectrometry in combination with a quantification concatamer (QconCAT), was used for the quantification of five established equine APPs (fibrinogen, serum amyloid A, ceruloplasmin, haptoglobin, and plasminogen) and three proposed equine APPs (prothrombin, α-2-macroglobulin, and α-1-antitrypsin). Wound interstitial fluid was recovered by large pore microdialysis from experimental body and limb wounds from five horses at days 1, 2, 7, and 14 after wounding and healing without (body) and with (limb) the formation of EGT. The QconCAT included proteotypic peptides representing each of the protein targets and was used to direct the design of a gene, which was expressed in Escherichia coli in a media supplemented with stable isotopes for metabolically labeling of standard peptides. Co-analysis of wound interstitial fluid samples with the stable isotope-labeled QconCAT tryptic peptides in known amounts enabled quantification of the APPs in absolute terms. The concentrations of fibrinogen, haptoglobin, ceruloplasmin, prothrombin, and α-1-antitrypsin in dialysate from limb wounds were significantly higher than in dialysate from body wounds. This is the first report of simultaneous analysis of a panel of APPs using the QconCAT-SRM technology. The microdialysis technique in combination with the QconCAT-SRM-based approach proved useful for quantification of the investigated proteins in the wound interstitial fluid, and the results indicated that there is a state of sustained inflammation in equine wounds healing with formation of EGT.

Mass spectrometry (MS) has become essential for efficient and accurate quantification of proteins and proteomes and, thus, a key technology throughout all biosciences. However, validated MS methods are still scarce for meat quality research applications. The objective of this work was to develop and compare two targeted proteomic methods, namely, selected reaction monitoring (SRM) and sequential window acquisition of all theoretical spectra (SWATH), for the quantification of 11 bovine muscle proteins that may be indicators of meat color. Both methods require evaluation of spectra from proteotypic and quantotypic peptides, and we here report our evaluation of which peptides and MS parameters are best suited for robust quantification of these 11 proteins. We observed that the SRM approach provides better reproducibility, linearity, and sensitivity than SWATH and is therefore ideal for targeted quantification of low-abundance proteins, while the SWATH approach provides a more time-efficient method for targeted protein quantification of high-abundance proteins and, additionally, supports the search for novel biomarkers.

Administration of colostrum to the newborn calf before gut closure is pivotal to its health, because of the transfer of passive immunity. Traditionally, passive immunity has been attributed to the transfer of immunoglobulins although it is increasingly clear that multiple other factors contribute, including innate immune proteins, developmental factors, immunomodulatory factors, and the presence of cellular immunity. The objective of this study was to produce a comprehensive comparison of the bovine colostrum proteome and the milk proteome by applying 2-dimensional liquid chromatography-tandem mass spectrometry. Further, the objectives were to rank proteins mutually and generate protein ratios from the spectral counts of the 2 proteomes and ELISA to gain insight into which proteins could be of most relevance to neonatal calf health. To obtain an in-depth picture of the bovine colostrum and milk proteome, we compared the contents of different fractions from bovine colostrum and milk from our 2 previous studies. A total of 140 colostrum fluid-phase proteins and 103 milk fluid-phase proteins were detected. In the cellular fraction, 324 and 310 proteins were detected in colostrum and milk, respectively. In total, 514 proteins were detected, of which 162 were in the fluid phase. Of these, 50 proteins were exclusively seen in colostrum, 13 were exclusively seen in milk, and 99 were common to colostrum and milk. Ranking of proteins mutually and calculating protein ratios based on spectral counts and ELISA resulted in new information on how proteins were associated with the fluid or cellular fraction of the samples. Interestingly, despite lower counts/concentrations than the classical proteins such as immunoglobulins, β-lactoglobulin, and lactotransferrin, several proteins appeared in higher or similar colostrum:milk spectral count ratios as these. Using this approach indicated, for example, that osteopontin, haptoglobin, milk amyloid A, and gelsolin may be interesting molecules to study in detail in their relation to calf health. Although the sensitivity, identification, and ranking of proteins varied between the 2 methods, and proteome analysis clearly suffers from low sensitivity, we believe that this idea and approach of generating ratios and ranking proteins can contribute new information and perspectives on how to prioritize the importance of multiple proteins, beyond immunoglobulins, in relation to neonatal calf health.

Progress in mass spectrometry-based methods for veterinary research and diagnostics is lagging behind compared to the human research, and proteome data of domestic animals is still not well represented in open source data repositories. This is particularly true for the equine species. Here we present a first Equine PeptideAtlas encompassing high-resolution tandem mass spectrometry analyses of 51 samples representing a selection of equine tissues and body fluids from healthy and diseased animals. The raw data were processed through the Trans-Proteomic Pipeline to yield high quality identification of proteins and peptides. The current release comprises 24,131 distinct peptides representing 2636 canonical proteins observed at false discovery rates of 0.2 % at the peptide level and 1.4 % at the protein level. Data from the Equine PeptideAtlas are available for experimental planning, validation of new datasets, and as a proteomic data mining resource. The advantages of the Equine PeptideAtlas are demonstrated by examples of mining the contents for information on potential and well-known equine acute phase proteins, which have extensive general interest in the veterinary clinic. The extracted information will support further analyses, and emphasises the value of the Equine PeptideAtlas as a resource for the design of targeted quantitative proteomic studies.

Intrauterine growth restriction (IUGR) is characterized by reduced growth and weight of the foetus, mainly due to the lack of nutrients and oxygen. Animals affected by IUGR show changes in specific brain areas and several neuronal processes. Female offspring affected by IUGR show increased survival and development compared to males. The objective of this study was to analyse changes in the hippocampus proteome in male and female piglets affected by IUGR. Seven pregnant Iberian sows were fed from Day 35 of pregnancy onwards at 50% of their requirements. At Day 100 of pregnancy, foetuses were obtained and classified by sex and weight, as mild IUGR (Normal Body Weight) versus severe IUGR (Low Body Weight). Hippocampi were dissected and the proteomes analysed by SWATH-MS DIA. In this study, 1497 proteins were identified of which 260 were quantitatively analysed. All differential proteins were more abundant in females versus males and were involved in protein synthesis, neuronal development, metabolism, antiapoptotic signalling and vesicular transport. Our findings support that female foetuses tolerate nutrient limitation better than males, especially under mild IUGR. Under severe IUGR, females still seems to maintain normal lipid metabolism and antiapoptotic signalling, which may be related to the increased female survival. SIGNIFICANCE: In the last years, proteomics have been used to evidence differences related to sex in non-reproductive organs. Intrauterine Growth Restriction (IUGR) can affect female and male offspring differently. Female offspring has stronger protective strategies compared to males, enhancing growth and postnatal survival. Most studies regarding this issue have focused on metabolic organs (i.e. liver). However, the predominance of neurodevelopmental disorders in males suggests that the central nervous system in female offspring adapt better to nutritional stress conditions than that of males. Based on the differential protein expression in hippocampal samples, our work demonstrates that female foetuses indeed adapt better to IUGR than males, especially under mild IUGR conditions. In severe IUGR conditions, differences between males and females were not so evident, but even in this case, the remaining differences suggest increased survival in females than in males.

Proteomics has become an important analytical tool in the field of farm animals and their products. A general overview on approaches, sample types, and current animal protein and peptide databases is given. Food quality is closely connected with animal health; therefore this introductory chapter summarizes where proteomics can be of help to investigate food issues related to the live animal. This comprises animal health–related aspects, mainly stress, welfare, and influence of management, and production-related issues. The latter refers to breeds and traits, the influence of nutrition and metabolic aspects, as well as studies to detect fraud (use of growth promoters) and to improve reproduction. Though not all aspects are equally well studied by proteomics yet, approaches in this field show great promise.

We investigated the liver response in young pigs to a high-fat diet (containing 25% animal fat) and diet restriction (equivalent to 60% of maintenance) using differential proteome analysis. The objective was to investigate whether young pigs can be used to model the liver response in adolescents to a high-fat diet and diet restriction-induced BW loss. The high-fat diet increased (P

Background The objective assessment of animal stress and welfare requires proper laboratory biomarkers. In this work, we have analyzed the changes in serum composition in gilts after switching their housing, from pen to individual stalls, which is generally accepted to cause animal discomfort. Results Blood and saliva samples were collected a day before and up to four days after changing the housing system. Biochemical analyses showed adaptive changes in lipid and protein metabolism after the housing switch, whereas cortisol and muscular markers showed a large variability between animals. 2D-DIGE and iTRAQ proteomic approaches revealed variations in serum protein composition after changing housing and diet of gilts. Both techniques showed alterations in two main homeostatic mechanisms: the innate immune and redox systems. The acute phase proteins haptoglobin, apolipoprotein A-I and α1-antichymotrypsin 3, and the antioxidant enzyme peroxiredoxin 2 were found differentially expressed by 2D-DIGE. Other proteins related to the innate immune system, including lactotransferrin, protegrin 3 and galectin 1 were also identified by iTRAQ, as well as oxidative stress enzymes such as peroxiredoxin 2 and glutathione peroxidase 3. Proteomics also revealed the decrease of apolipoproteins, and the presence of intracellular proteins in serum, which may indicate physical injury to tissues. Conclusions Housing of gilts in individual stalls and diet change increase lipid and protein catabolism, oxidative stress, activate the innate immune system and cause a certain degree of tissue damage. We propose that valuable assays for stress assessment in gilts may be based on a score composed by a combination of salivary cortisol, lipid metabolites, innate immunity and oxidative stress markers and intracellular proteins. Electronic supplementary material The online version of this article (doi:10.1186/s12917-016-0887-1) contains supplementary material, which is available to authorized users.

The aim of this study was to find potential biomarkers for meat tenderness in bovine Longissimus thoracis muscle and to compare results from isobaric Tag for Relative and Absolute Quantitation (iTRAQ) and 2-dimensional gel electrophoresis (2-DE) analysis. The experiment included 4 tender and 4 tough samples, based on shear force measurements at 7 d postmortem, from young Norwegian red (NRF) bulls, taken at 1 h postmortem. A number of the proteins which have previously been related to tenderness were found to change in abundance between tender and tough samples, both in iTRAQ (P < 0.1) and 2-DE analysis (P < 0.05). Furthermore, 3 proteins that have not previously been related to tenderness were found to change significantly in abundance between tender and tough meat samples in the present study. These include proteins related to control of flux through the tricarboxylate cycle [2-oxoglutarate dehydrogenase complex component E2 (OGDC-E2)], apoptosis (galectin-1) and regulatory role in the release of Ca(2+) from intracellular stores (annexin A6). Even though the overlap in significantly changing proteins was relatively low between iTRAQ and 2-DE analysis, certain proteins predicted to have the same function were found in both analyses and showed similar changes between the groups, such as structural proteins and proteins related to apoptosis and energy metabolism.

The community working on model organisms is growing steadily and the number of model organisms for which proteome data are being generated is continuously increasing. To standardize efforts and to make optimal use of proteomics data acquired from model organisms, a new Human Proteome Organisation (HUPO) initiative on model organism proteomes (iMOP) was approved at the HUPO Ninth Annual World Congress in Sydney, 2010. iMOP will seek to stimulate scientific exchange and disseminate HUPO best practices. The needs of model organism researchers for central databases will be better represented, catalyzing the integration of proteomics and organism-specific databases. Full details of iMOP activities, members, tools and resources can be found at our website http://www.imop.uzh.ch/ and new members are invited to join us.

iMOP--the Initiative on Model Organism Proteomes--was accepted as a new HUPO initiative at the Ninth HUPO meeting in Sydney in 2010. A goal of iMOP is to integrate research groups working on a great diversity of species into a model organism community. At the Tenth HUPO meeting in Geneva this variety was reflected in the iMOP session on Tuesday September 6, 2011. The presentations covered the quantitative proteome database PaxDb, proteomics projects studying farm animals, Arabidopsis thaliana, as well as host-pathogen interactions.

Our current knowledge of human biology is often based on studying a wide range of animal species. In particular, for understanding human diseases, the development of adequate animal models is of immediate importance. Although genetic strains and transgenic animal model organisms like fruit fly (Drosophila), zebrafish and rodents are highly informative about the function of single genes and proteins, these organisms do not always closely reflect human biology, and alternative animal models are thus in great demand. The pig is a non-primate mammal that closely resembles man in anatomy, physiology and genetics. Pigs, although not easily kept for laboratory research, are, however, readily available for biomedical research through the large scale industrial production of pigs produced for human consumption. Recent research has facilitated the biological experimentation with pigs, and helped develop the pig into a novel model organism for biomedical research. This toolbox includes the near completion of the pig genome, catalogues of genes and genetic variation in pigs, extensive characterization of pig proteomes and transcriptomes, as well as the development of transgenic disease models. The aim of this review is to highlight the current progress of these ongoing areas of research, which are mandatory for successful development of biomedical pig models that are in demand for understanding human biology in health and disease.

The synapsin (syn) protein family comprises the neuron-specific phosphoproteins syn I, II, and III, all found in at least two isoforms (Südhof et al. 1989; Porton et al. 1999). Mature syns localize to the membrane of neurotransmitter-releasing synaptic vesicles; they are expected to have regulatory roles in linking the vesicles to the cytoskeleton, supported by the identified abilities of the syns to bind both phospholipids (Schiebler et al. 1986) and tubulin/actin (Baines and Bennett 1986; Bähler and Greengard 1987). Originating from an alternatively spliced common transcript, syn Ia is the full-length protein, whereas the isoform syn Ib holds a truncated and altered carboxy terminal. The peptide chain of syn I is divided into five domains. The common domains are designated A-D; the last and divergent domain is E in syn Ia and F in syn Ib (Südhof et al. 1989). Of these domains, A, C, and E/F show the highest degree of conservation between species and within the protein family. The central neurobiological functions of syn I are well illustrated by knock-out mice consistently found to display epilepsy (Li et al. 1995; Rosahl et al. 1995). More recently, and supportive of the mice experiments, a nonsense mutation leading to a truncated form of syn I, without domain D and E/F, was identified in a family with frequent cases of X-linked epilepsy (Garcia et al. 2004). This study reports molecular cloning and characterization of the coding sequence of the porcine ortholog of syn I, including identification and verification at the protein level of an alanine-encoding insert Udgivelsesdato: December The synapsin (syn) protein family comprises the neuron-specific phosphoproteins syn I, II, and III, all found in at least two isoforms (Südhof et al. 1989; Porton et al. 1999). Mature syns localize to the membrane of neurotransmitter-releasing synaptic vesicles; they are expected to have regulatory roles in linking the vesicles to the cytoskeleton, supported by the identified abilities of the syns to bind both phospholipids (Schiebler et al. 1986) and tubulin/actin (Baines and Bennett 1986; Bähler and Greengard 1987). Originating from an alternatively spliced common transcript, syn Ia is the full-length protein, whereas the isoform syn Ib holds a truncated and altered carboxy terminal. The peptide chain of syn I is divided into five domains. The common domains are designated A-D; the last and divergent domain is E in syn Ia and F in syn Ib (Südhof et al. 1989). Of these domains, A, C, and E/F show the highest degree of conservation between species and within the protein family. The central neurobiological functions of syn I are well illustrated by knock-out mice consistently found to display epilepsy (Li et al. 1995; Rosahl et al. 1995). More recently, and supportive of the mice experiments, a nonsense mutation leading to a truncated form of syn I, without domain D and E/F, was identified in a family with frequent cases of X-linked epilepsy (Garcia et al. 2004). This study reports molecular cloning and characterization of the coding sequence of the porcine ortholog of syn I, including identification and verification at the protein level of an alanine-encoding insert

Complete list of differentially expressed proteins identified by DIGE at D3 and D5 versus D1 (basal conditions) in H and HS groups (XLS file). Increased proteins at D3 are represented as positive fold-changes (FC) and decreased proteins as negative FC. Proteins are ordered by number of spot. (DOCX 16 kb)

List of differentially expressed proteins identified and quantified by iTRAQ at D3 and D5 versus D1 (basal conditions) in H and HS groups (XLS file). Increased proteins at D3 are represented as positive fold-changes (FC) and decreased proteins as negative FC. Proteins are classified according to their biological function and ordered by Mascot score. (DOCX 41 kb)

Biological research of Sus scrofa, the domestic pig, is of immediate relevance for food production sciences, and for developing pig as a model organism for human biomedical research. Publicly available data repositories play a fundamental role for all biological sciences, and protein data repositories are in particular essential for the successful development of new proteomic methods. Cumulative proteome data repositories, including the PeptideAtlas, provide the means for targeted proteomics, system wide observations, and cross species observational studies, but pigs have so far been underrepresented in existing repositories. We here present a significantly improved build of the Pig PeptideAtlas, which includes pig proteome data from 25 tissues and three body fluid types mapped to 7139 canonical proteins. The content of the Pig PeptideAtlas reflects actively ongoing research within the veterinary proteomics domain, and this manuscript demonstrates how the expression of isoform-unique peptides can be observed across distinct tissues and body fluids. The Pig PeptideAtlas is a unique resource for use in animal proteome research, particularly biomarker discovery and for preliminary design of SRM assays, which are equally important for progress in research that supports farm animal production and veterinary health, as for developing pig models with relevance to human health research. This article is protected by copyright. All rights reserved.

Experimental design of the 2D-DIGE experiment (word file). The pooled D1 sample was labelled with Cy2 and used as internal standard in all four gels. Dye-swap was performed and samples were randomly paired. (DOCX 12 kb)

Bovine lactoferrin (bLF) may modulate neonatal intestinal inflammation. Previous studies in intestinal epithelial cells (IECs) indicated that moderate bLF doses enhance proliferation whereas high doses trigger inflammation. To further elucidate cellular mechanisms, we profiled the porcine IEC proteome after stimulation with bLF at 0, 0.1, 1 and 10 g/L by LC-MS-based proteomics. Key pathways were analyzed in the intestine of formula-fed preterm pigs with and without supplementation of 10 g/L bLF. Levels of 123 IEC proteins were altered by bLF. Low bLF doses (0.1–1 g/L) up-regulated 11 proteins associated with glycolysis, energy metabolism and protein synthesis, indicating support of cell survival. In contrast, a high bLF dose (10 g/L) up-regulated three apoptosis-inducing proteins, down-regulated five anti-apoptotic and proliferation-inducing proteins and 15 proteins related to energy and amino acid metabolism, and altered three proteins enhancing the hypoxia inducible factor-1 (HIF-1) pathway. In the preterm pig intestine, bLF at 10 g/L decreased villus height/crypt depth ratio and up-regulated the Bax/Bcl-2 ratio and HIF-1α, indicating elevated intestinal apoptosis and inflammation. In conclusion, bLF dose-dependently affects IECs via metabolic, apoptotic and inflammatory pathways. It is important to select an appropriate dose when feeding neonates with bLF to avoid detrimental effects exerted by excessive doses. Biological significance The present work elucidates dose-dependent effects of bLF on the proteomic changes of IECs in vitro supplemented with data from a preterm pig study confirming detrimental effects of enteral feeding with the highest dose of bLF (10 g/L). The study contributes to further understanding on mechanisms that bLF, as an important milk protein, can regulate the homeostasis of the immature intestine. Results from this study urge neonatologists to carefully consider the dose of bLF to supplement into infant formula used for preterm neonates.

A massive use of antibiotics in industrial pig production is a major cause of the rapidly rising bacterial resistance to antibiotics. An enhanced understanding of infectious diseases and of host-microbe interactions has the potential to explore alternative ways to improve pig health and reduce the need for antibiotics. Host-microbe interactions depend on host-expressed glycans and microbe-carrying lectins. In this study, a G > A (nucleotide 307) missense mutation in the porcine α1,2fucosyltransferase 1 gene (FUT1), which has been reported to prevent infections by the common porcine enteric pathogen F18 fimbriated Escherichia coli, provided a unique opportunity to study glycan structures potentially involved in intestinal infections. N- and O-Linked glycans of the intestinal mucosa proteins were characterized in detail using LC-MS/MS. Relative abundances of all glycans were determined and compared between four heterozygous pigs (FUT1-307A/G) and four age-matched homozygous pigs from the same 2 litters carrying the missense FUT1 gene constellation (FUT1-307A/A). None of the characterized 48 N-linked glycans was found to be regulated by the FUT1 missense mutation, while 11 of the O-linked glycans showed significantly altered abundances between the two genotypes. The overall abundance of H-antigen carrying structures was decreased fivefold, while H-antigen precursors and sialylated structures were relatively more abundant in pigs with the FUT1 missense mutation. These results provide insight into the role of FUT1 on intestinal glycosylation, improve our understanding of how variation in FUT1 can modulate host-microbe interactions, and suggest that the FUT1 genetic variant may help to improve pig gut health.

In horses, pathological healing with formation of exuberant granulation tissue (EGT) is a particular problem in limb wounds, whereas body wounds tend to heal without complications. Chronic inflammation has been proposed to be central to the pathogenesis of EGT. This study aimed to investigate levels of inflammatory acute phase proteins (APPs) in interstitial fluid from wounds in horses. A novel approach for absolute quantification of proteins, selected reaction monitoring (SRM)-based mass spectrometry in combination with a quantification concatamer (QconCAT), was used for the quantification of five established equine APPs (fibrinogen, serum amyloid A, ceruloplasmin, haptoglobin, and plasminogen) and three proposed equine APPs (prothrombin, α-2-macroglobulin, and α-1-antitrypsin). Wound interstitial fluid was recovered by large pore microdialysis from experimental body and limb wounds from five horses at days 1, 2, 7, and 14 after wounding and healing without (body) and with (limb) the formation of EGT. The QconCAT included proteotypic peptides representing each of the protein targets and was used to direct the design of a gene, which was expressed in Escherichia coli in a media supplemented with stable isotopes for metabolically labeling of standard peptides. Co-analysis of wound interstitial fluid samples with the stable isotope-labeled QconCAT tryptic peptides in known amounts enabled quantification of the APPs in absolute terms. The concentrations of fibrinogen, haptoglobin, ceruloplasmin, prothrombin, and α-1-antitrypsin in dialysate from limb wounds were significantly higher than in dialysate from body wounds. This is the first report of simultaneous analysis of a panel of APPs using the QconCAT-SRM technology. The microdialysis technique in combination with the QconCAT-SRM-based approach proved useful for quantification of the investigated proteins in the wound interstitial fluid, and the results indicated that there is a state of sustained inflammation in equine wounds healing with formation of EGT.

The gut epithelium formed between an organism and the environment plays an essential role in host-microbe interactions, yet remains one of the least characterized mammalian tissues. Especially the membrane proteins, which are critical to bacterial adhesion, are understudied, because these proteins are low in abundance, and large amounts of sample is needed for their preparation and for undertaking MS-based analysis. The aim of this study was to evaluate three different methods for isolation and preparation of pig intestinal epithelial cells for MS-based analysis of the proteome. Samples were analyzed by LC and electrospray QTOF-MS. The methods were evaluated according to efficiency, purity, transmembrane protein recovery, as well as for suitability to large-scale preparations. Our data clearly demonstrate that mucosal shaving is by far the best-suited method for in-depth MS analysis in terms of ease and speed of sample preparation, as well as protein recovery. In comparison, more gentle methods where intestinal epithelial cells are harvested by shaking are more time consuming, result in lower protein yield, and are prone to increased technical variation due to multiple steps involved.

We announce the draft genome sequence of Clostridium tyrobutyricum strain DIVETGP. This strain was isolated from cow's milk used for Grana Padano cheese production. The genome was obtained using Illumina HiSeq technology and comprises 45 contigs for 3,018,999 bp, with a G+C content of 30.8%.

The extensive use of a limited number of elite bulls in cattle breeding can lead to rapid spread of recessively inherited disorders. A recent example is the globally distributed syndrome Complex Vertebral Malformation (CVM), which is characterized by misshapen and fused vertebrae around the cervico-thoracic junction. Here, we show that CVM is caused by a mutation in the Golgi-resident nucleotide-sugar transporter encoded by SLC35A3. Thus, the disease showed complete cosegregation with the mutation in a homozygous state, and proteome patterns indicated abnormal protein glycosylation in tissues of affected animals. In addition, a yeast mutant that is deficient in the transport of UDP-N-acetylglucosamine into its Golgi lumen can be rescued by the wild-type SLC35A3 gene, but not by the mutated gene. These results provide the first demonstration of a genetic disorder associated with a defective SLC35A3 gene, and reveal a new mechanism for malformation of the vertebral column caused by abnormal nucleotide-sugar transport into the Golgi apparatus.

Characterising the function of genes is a major challenge in the post-genomic era. Post-genomic tools and technologies have dramatically changed the experimental approaches by which complex biological systems can be characterised. Proteomics is an important cornerstone in functional genome characterisation, and like all other functional genomics tools, including transcriptomics and metabolomics, the aim of proteome studies is to translate genome information into useful biological insight, that will allow scientists to build and test better hypotheses, with the ultimate goal to find better solutions to challenges in food production, medicine and environmental management. In agricultural sciences as well as in all other life sciences, the implementation of proteomics and the other post-genomic tools is an important step towards achieving better product quality and a more sustainable animal production. The aim of this review is to introduce the developing field of proteomics, and to discuss the use of proteomics in meat science projects. The most frequently used technologies for characterising cellular protein expression patterns will be introduced, and some early examples of applying proteomics to meat quality research will be discussed.

To identify myofibrillar substrates of μ-calpain under post-mortem conditions, a combination of SDS–PAGE, two-dimensional gel electrophoresis (2DE) and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) was used. Purified myofibrils were incubated with μ-calpain under post-mortem-simulated conditions for two or four days at 4 °C. The resulting protein changes were analyzed by SDS–PAGE and 2DE. The μ-calpain-mediated protein changes were identified by peptide-mass mapping using MALDI-TOF MS and revealed that desmin, actin, myosin heavy chain, myosin light chain I, troponin T, tropomyosin α1, tropomyosin α4, thioredoxin and CapZ are all degraded in vitro by μ-calpain. The findings that actin and myosin heavy chain are substrates of μ-calpain were rather surprising, as it has previously been reported that these proteins are resistant to μ-calpain degradation. However, both actin and myosin heavy chain are poor substrates compared with desmin.

Muscle proteins are generally believed to be key players in free radical processes that eventually lead to oxidative deterioration or modifications of meat proteins resulting in alterations in functionality, for example, gel-forming ability, emulsification properties, and water-binding capacity. This study addresses protein oxidation in chicken muscles using a combined immunologic and proteomic approach and identifies specific proteins that contain carbonyls and/or 3-nitrotyrosine (3-NT). Whereas α-enolase was the predominant carbonyl-reactive species among the water-soluble muscle proteins, several other proteins (actin, heat shock protein 70, and creatine kinase) contained carbonyls and/or 3-nitrotyrosine. Finally, this approach was used to demonstrate differential susceptibility of water-soluble muscle proteins toward oxidation in chickens fed a low-antioxidant diet compared with chickens fed a diet supplemented with antioxidant-rich fruits/vegetables. Keywords: Protein oxidation; carbonyls; 3-nitrotyr...

Colostrum intake is a key factor for newborn ruminant survival because the placenta does not allow the transfer of immune components. Therefore, newborn ruminants depend entirely on passive immunity transfer from the mother to the neonate, through the suckling of colostrum. Understanding the importance of specific colostrum proteins has gained significant attention in recent years. However, proteomics studies of sheep colostrum and their uptake in neonate lambs has not yet been presented. The aim of this study was to describe the proteomes of sheep colostrum and lamb blood plasma, using sodium dodecyl sulfate-PAGE for protein separation and in-gel digestion, followed by liquid chromatography-tandem mass spectrometry of resulting tryptic peptides for protein identification. An isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomics approach was subsequently used to provide relative quantification of how neonatal plasma protein concentrations change as an effect of colostrum intake. The results of this study describe the presence of 70 proteins in the ovine colostrum proteome. Furthermore, colostrum intake resulted in an increase of 8 proteins with important immune functions in the blood plasma of lambs. Further proteomic studies will be necessary, particularly using the selected reaction monitoring approach, to describe in detail the role of specific colostrum proteins for immune transfer to the neonate. Colostrum intake is a key factor for newborn ruminant survival because the placenta does not allow the transfer of immune components. Therefore, newborn ruminants depend entirely on passive immunity transfer from the mother to the neonate, through the suckling of colostrum. Understanding the importance of specific colostrum proteins has gained significant attention in recent years. However, proteomics studies of sheep colostrum and their uptake in neonate lambs has not yet been presented. The aim of this study was to describe the proteomes of sheep colostrum and lamb blood plasma, using sodium dodecyl sulfate-PAGE for protein separation and in-gel digestion, followed by liquid chromatography-tandem mass spectrometry of resulting tryptic peptides for protein identification. An isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomics approach was subsequently used to provide relative quantification of how neonatal plasma protein concentrations change as an effect of colostrum intake. The results of this study describe the presence of 70 proteins in the ovine colostrum proteome. Furthermore, colostrum intake resulted in an increase of 8 proteins with important immune functions in the blood plasma of lambs. Further proteomic studies will be necessary, particularly using the selected reaction monitoring approach, to describe in detail the role of specific colostrum proteins for immune transfer to the neonate.

Animal production and health (APH) is an important sector in the world economy, representing a large proportion of the budget of all member states in the European Union and in other continents. APH is a highly competitive sector with a strong emphasis on innovation and, albeit with country to country variations, on scientific research. Proteomics (the study of all proteins present in a given tissue or fluid - i.e. the proteome) has an enormous potential when applied to APH. Nevertheless, for a variety of reasons and in contrast to disciplines such as plant sciences or human biomedicine, such potential is only now being tapped. To counter such limited usage, 6 years ago we created a consortium dedicated to the applications of Proteomics to APH, specifically in the form of a Cooperation in Science and Technology (COST) Action, termed FA1002 - Proteomics in Farm Animals: www.cost-faproteomics.org. In 4 years, the consortium quickly enlarged to a total of 31 countries in Europe, as well as Israel, Argentina, Australia and New Zealand. This article has a triple purpose. First, we aim to provide clear examples on the applications and benefits of the use of proteomics in all aspects related to APH. Second, we provide insights and possibilities on the new trends and objectives for APH proteomics applications and technologies for the years to come. Finally, we provide an overview and balance of the major activities and accomplishments of the COST Action on Farm Animal Proteomics. These include activities such as the organization of seminars, workshops and major scientific conferences, organization of summer schools, financing Short-Term Scientific Missions (STSMs) and the generation of scientific literature. Overall, the Action has attained all of the proposed objectives and has made considerable difference by putting proteomics on the global map for animal and veterinary researchers in general and by contributing significantly to reduce the East-West and North-South gaps existing in the European farm animal research. Future activities of significance in the field of scientific research, involving members of the action, as well as others, will likely be established in the future. European Science Foundation (Brussels, Belgium) info:eu-repo/semantics/publishedVersion

The aim of this study was the development of a quantitative assay that could support future studies of a panel of acute phase proteins (APPs) in the horse. The assay was based on a quantification concatamer (QconCAT) coupled with selected reaction monitoring methodology. Thirty-two peptides, corresponding to 13 putative or confirmed APPs for the Equus caballus (equine) species were selected for the design of a QconCAT construct. The gene encoding the QconCAT was synthesised and expressed as an isotope-labelled chimaeric protein in Escherichia coli. The QconCAT tryptic peptides were analysed on a triple-quadrupole instrument, and the quantotypic properties were assessed in equine serum, wound tissue and wound interstitial fluid. Reasonable quantotypic performance was found for 12, 14, and 14 peptides in serum, wound tissue, and interstitial fluid, respectively. Seven proteins were quantified in absolute terms in serum collected from a horse before and after the onset of a systemic inflammatory condition, and the observed protein concentrations were in close agreement with previous data. We conclude, that this QconCAT is applicable for concurrent quantitative analysis of multiple APPs in serum and may also support future studies of these proteins in other types of tissues and body fluids from the horse.

The objective of the study was to assess global expression of proteins in equine saliva using liquid chromatography tandem mass spectrometry (LC-MS/MS). Saliva was obtained from seven horses with and six horses without evidence of systemic inflammatory disease. Tryptic peptides from saliva were analysed by LC-MS/MS. Of 195 unique proteins identified, 57 were detected only in saliva samples from horses with systemic inflammation (in two to six of the seven horses). Among the differentially expressed proteins were several acute phase proteins (APPs) such as serum amyloid A, fibrinogen, haptoglobin, and alpha1-acid glycoprotein. The study is the first to describe detection of inflammatory proteins in horse saliva. The proteins detected were similar to those described in saliva from cattle, small ruminants and pigs. Detection of APPs in horses with systemic inflammation suggests that saliva may be used for non-invasive disease monitoring in horses as in humans, pigs and dogs.

The influence of the halothane gene on performance, carcass composition, chemical composition of carcass fractions, and muscle fibre characteristics of M. semitendinosus (ST) was examined in 14 litters of two pigs of either NN (halothane free) or Nn genotype (heterozygous for the halothane gene) (within litter comparison). The pigs were offspring of Duroc×Pietran boars (Nn) mated to Landrace×Yorkshire females (NN). The feed intake of Nn pigs was 8% (P

Adipose tissue is not only a tissue where energy is stored but is also involved in regulating several body functions such as appetite and energy expenditure via its endocrine activity. Moreover, it thereby modulates complex processes like reproduction, inflammation and immune response. The products secreted from adipose tissue comprise hormones and cytokines that are collectively termed as adipocytokines or "adipokines"; the discovery and characterization of new proteins secreted by adipose tissue is still ongoing and their number is thus increasing. Adipokines act in both endocrine manner as well as locally, as autocrine or paracrine effectors. Proteomics has emerged as a valuable technique to characterize both cellular and secreted proteomes from adipose tissues, including those of main cellular fractions, i.e. the adipocytes or the stromal vascular fraction containing mainly adipocyte precursors and immune cells. The scientific interest in adipose tissue is largely based on the worldwide increasing prevalence of obesity in humans; in contrast, obesity is hardly an issue for farmed animals that are fed according to their well-defined needs. Adipose tissue is nevertheless of major importance in these animals, as the adipose percentage of the bodyweight is a major determinant for the efficiency of transferring nutrients from feed into food products and thus for the economic value from meat producing animals. In dairy animals, the importance of adipose tissue is based on its function as stromal structure for the mammary gland and on its role in participating in and regulating of energy metabolism and other functions. Moreover, as pig has recently become an important model organism to study human diseases, the knowledge of adipose tissue metabolism in pig is relevant for the study of obesity and metabolic disorders. We herein provide a general overview of adipose tissue functions and its importance in farm animals. This review will summarize recent achievements in farm animal adipose tissue proteomics, mainly in cattle and pigs, but also in poultry, i.e. chicken and in farmed fish. Proteomics advancement in adipocyte cell lines, have also been included. Adipose tissue is not only a tissue where energy is stored but is also involved in regulating several body functions such as appetite and energy expenditure via its endocrine activity. Moreover, it thereby modulates complex processes like reproduction, inflammation and immune response. The products secreted from adipose tissue comprise hormones and cytokines that are collectively termed as adipocytokines or "adipokines"; the discovery and characterization of new proteins secreted by adipose tissue is still ongoing and their number is thus increasing. Adipokines act in both endocrine manner as well as locally, as autocrine or paracrine effectors. Proteomics has emerged as a valuable technique to characterize both cellular and secreted proteomes from adipose tissues, including those of main cellular fractions, i.e. the adipocytes or the stromal vascular fraction containing mainly adipocyte precursors and immune cells. The scientific interest in adipose tissue is largely based on the worldwide increasing prevalence of obesity in humans; in contrast, obesity is hardly an issue for farmed animals that are fed according to their well-defined needs. Adipose tissue is nevertheless of major importance in these animals, as the adipose percentage of the bodyweight is a major determinant for the efficiency of transferring nutrients from feed into food products and thus for the economic value from meat producing animals. In dairy animals, the importance of adipose tissue is based on its function as stromal structure for the mammary gland and on its role in participating in and regulating of energy metabolism and other functions. Moreover, as pig has recently become an important model organism to study human diseases, the knowledge of adipose tissue metabolism in pig is relevant for the study of obesity and metabolic disorders. We herein provide a general overview of adipose tissue functions and its importance in farm animals. This review will summarize recent achievements in farm animal adipose tissue proteomics, mainly in cattle and pigs, but also in poultry, i.e. chicken and in farmed fish. Proteomics advancement in adipocyte cell lines, have also been included.

Articles in International Journals Milk and dairy products are central elements in the human diet. It is estimated that 108 kg of milk per year are consumed per person worldwide. Therefore, dairy production represents a relevant fraction of the economies of many countries, being cattle, sheep, goat, water buffalo, and other ruminants the main species used worldwide. An adequate management of dairy farming cannot be achieved without the knowledge on the biological mechanisms behind lactation in ruminants. Thus, understanding the morphology, development and regulation of the mammary gland in health, disease and production is crucial. Presently, innovative and high-throughput technologies such as genomics, transcriptomics, proteomics and metabolomics allow a much broader and detailed knowledge on such issues. Additionally, the application of a systems biology approach to animal science is vastly growing, as new advances in one field of specialization or animal species lead to new lines of research in other areas or/and are expanded to other species. This article addresses how modern research approaches may help us understand long-known issues in mammary development, lactation biology and dairy production. Biological significance Dairy production depends upon the knowledge of the morphology and regulation of the mammary gland and lactation. High-throughput technologies allow a much broader and detailed knowledge on the biology of the mammary gland. This paper reviews the major contributions that genomics, transcriptomics, metabolomics and proteomics approaches have provided to understand the regulation of the mammary gland in health, disease and production. In the context of mammary gland “omics”-based research, the integration of results using a Systems Biology Approach is of key importance.

Background/Aims The purpose of the present pilot study was to investigate the feasibility of combining large pore dermal microdialysis with shotgun proteomic analysis in human skin. Methods Dialysate was recovered from human skin by 2000 kDa microdialysis membranes from one subject at three different phases of the study; trauma due to implantation of the dialysis device, a post implantation steady-state period, and after induction of vasodilatation and plasma extravasation. For shotgun proteomics, the proteins were extracted and digested with trypsin. Peptides were separated by capillary and nanoflow HPLC systems, followed by tandem mass spectrometry (MS/MS) on a Quadrupole-TOF hybrid instrument. The MS/MS spectra were merged and mapped to a human target protein database to achieve peptide identification and protein inference. Results Results showed variation in protein amounts and profiles for each of the different sampling phases. The total protein concentration was 1.7, 0.6, and 1.3 mg/mL during the three phases, respectively. A total of 158 different proteins were identified. Immunoglobulins and the major classes of plasma proteins, including proteases, coagulation factors, apolipoproteins, albumins, and complement factors, make up the major load of proteins in all three test conditions. Conclusion Shotgun proteomics allowed the identification of more than 150 proteins in microdialysis samples from human skin. This highlights the opportunities of LC-MS/MS to study the complex molecular interactions in the skin.

Bovine milk is an agricultural product of tremendous value worldwide. It contains proteins, fat, lactose, vitamins, and minerals. It provides nutrition and immunological protection (e.g., in the gastrointestinal tract) to the newborn and young calf. It also forms an important part of human nutrition. The repertoire of proteins in milk (i.e., its proteome) is vast and complex. The milk proteome can be described in detail by mass spectrometry-based proteomics. However, the high concentration of dominating proteins in milk reduces mass spectrometry detection sensitivity and limits detection of low abundant proteins. Further, the general health and udder health of the dairy cows delivering the milk may influence the composition of the milk proteome. To gain a more exhaustive and true picture of the milk proteome, we performed an extensive preanalysis fractionation of raw composite milk collected from documented healthy cows in early lactation. Four simple and industrially applicable techniques exploring the physical and chemical properties of milk, including acidification, filtration, and centrifugation, were used for separation of the proteins. This resulted in 5 different fractions, whose content of proteins were compared with the proteins of nonfractionated milk using 2-dimensional liquid chromatography tandem mass spectrometry analysis. To validate the proteome analysis, spectral counts and ELISA were performed on 7 proteins using the ELISA for estimation of the detection sensitivity limit of the 2-dimensional liquid chromatography tandem mass spectrometry analysis. Each fractionation technique resulted in identification of a unique subset of proteins. However, high-speed centrifugation of milk to whey was by far the best method to achieve high and repeatable proteome coverage. The total number of milk proteins initially detected in nonfractionated milk and the fractions were 635 in 2 replicates. Removal of dominant proteins and filtering for redundancy across the different fractions reduced the number to 376 unique proteins in 2 replicates. In addition, 366 proteins were detected by this process in 1 replicate. Hence, by applying different fractionation techniques to milk, we expanded the milk proteome. The milk proteome map may serve as a reference for scientists working in the dairy sector.

Identification of biomarkers for degree of physiological imbalance (PI), a situation in which physiological parameters deviate from normal, is needed to reduce disease risk and improve production and reproduction performance of cows. The objective was to describe the liver proteome in early and mid lactation for cows with different degrees of PI with a special focus on biomarkers and pathways involved in periparturient disease complexes. Twenty-nine cows in early [49 ± 22 d in milk (DIM); n = 14] and mid (159 ± 39 DIM; n = 15) lactation were nutrient restricted for 4 d to increase PI by supplementing the ration with 60% wheat straw. Liver biopsies were collected −1 and 3 d relative to restriction. Before restriction, an index for PI was calculated based on plasma nonesterified fatty acids, β-hydroxybutyrate, and glucose concentrations. Within E and M cows, a subsets of 6 cow was classified as having either the greatest (PI) or least (normal; N) degree of PI and were used for isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative profiling in liver using liquid chromatography-tandem mass spectrometry. We identified pyruvate carboxylase and isocitrate dehydrogenase as potential hepatic biomarkers for PI for cows during early lactation and alcohol dehydrogenase-4 and methylmalonate-semialdehyde dehydrogenase for cows in mid lactation. This preliminary study identified new biomarkers in liver for PI and provided a better understanding of the differences in coping strategies used for cows in PI. Despite the small sample size (n = 3/group), the results lay a foundation for future research focused on the usefulness of the hepatic biomarkers for predicting PI and thereby cows at risk for disease during lactation.

Adipose tissue (AT) is considered a group of highly active endocrine organs, taking part in regulating several metabolic processes, including reproduction, inflammatory response, and production and secretion of signalling molecules with important biological roles, also known as adipokines. Different deposits of visceral and subcutaneous origin differ in mRNA abundance of several adipokines in sheep (Lemor et al., 2010); few studies have been carried out at a proteomic level in ruminant adipose tissue (Rajesh et al., 2012) and none in goats.

It is mandatory, after protein identification, to validate candidate markers in a larger amount of samples for biomarker discovery. The most used methods for validation of new markers are enzymatic or immunoassay-based commercial kits. However, relatively few kits are available for farm animals. Selected reaction monitoring (SRM), a targeted quantitative proteomic technique, may be used as an alternative to commercial kits for the measurement and validation of target proteins. Acute phase proteins (APPs) are widely recognized inflammation and infection biomarkers (Eckersall, 2010) and there is recent evidence that they can be also considered as welfare markers (Giannetto et al., 2011). During an acute stress situation, the levels of some interleukins (IL-1β, IL-6 and TNF-α) are increased (Elenkov and Chrousos, 2002) and consequently the concentration of the APPs varies dramatically. In pigs the most important APPs include haptoglobin (Hp), C-reactive protein (CRP), the inter-α-inhibitor-heavy chain 4 (ITIH4, also called Pig major acute phase protein, or Pig-MAP), serum amyloid A (SAA) and apolipoprotein A-I (Apo A-I), but also other proteins are well known to mark the acute phase response, as albumin, transferrin (Heegaard et al., 2011) and fetuin A (Brown et al., 1992).

The aim of this project is to develop and increase knowledge of key factors influencing the presence of clostridial spores in milk and factors affecting, directly or indirectly, technological characteristics and fermentation of milk, make it more or less susceptible to the development of defects (swelling). Aim of the project is to characterize by 1D/2D-gel electrophoresis and next-generation sequencing coupled to shotgun mass spectrometry the global microbiota isolated from ‘Grana Padano’ cheese. In particular the target is to identify the sub-proteome of Clostridium tyrobutyricum present in cheese and to investigate differences in proteins involved in the metabolic pathway of butyric acid in different stages of ripening and milk treatment (e.g. lysozyme treatment).