Animal board invited review: advances in proteomics for animal and food sciences.

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.

Identification of hepatic biomarkers for physiological imbalance of dairy cows in early and mid lactation using proteomic technology.

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 ± 22d in milk (DIM); n=14] and mid (159 ± 39 DIM; n=15) lactation were nutrient restricted for 4d to increase PI by supplementing the ration with 60% wheat straw. Liver biopsies were collected -1 and 3d relative to restriction. Before restriction, an index for PI was calculated based on plasma nonesterified fatty acids, ß-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.

Liver protein expression in young pigs in response to a high-fat diet and diet restriction.

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<0.05) the subcutaneous and visceral fat deposition by 45 and 56%, respectively. However, the young pigs on the high-fat diet had normal glucose tolerance and liver lipid content despite a general increase (P<0.05) in plasma lipids (i.e., NEFA, triglycerides, phospholipids, total cholesterol, and lipoproteins). In addition, diet restriction in young pigs induced a modest BW loss (0.7 kg/d; P<0.01) through increased fat mobilization whereas the concentrations of plasma phospholipids, total cholesterol, and low-density lipoprotein decreased (P<0.05) by 37, 36, and 38%, respectively. Data from the proteome analysis indicate that the liver response to a high-fat diet in young pigs is similar to that of humans in terms of increased fatty acid oxidation whereas the liver response to diet restriction is similar to humans in terms of increased gluconeogenesis and glycogenolysis and decreased urea synthesis. Our results suggest that 5 liver proteins, namely acyl-CoA synthetase long-chain 1, sterol carrier protein 2, apolipoprotein C-III, liver fatty acid binding protein, and acyl-CoA-binding protein, play a role in intracellular lipid transport and export in young pigs. In contrast to humans, our results indicate that young pigs are resistant to fat-induced liver lipid accumulation whereas diet restriction decreases fatty acid oxidation and the subsequent ketogenesis in the liver. Consequently, the liver response in adolescents to a high fat diet and diet restriction-induced BW loss cannot reliably be reproduced in young pigs.

Liver protein expression in dairy cows with high liver triglycerides in early lactation.

Fatty liver is a frequent subclinical health disorder in dairy cows that may lead to disorders related to the liver function. However, the effect of triglyceride (TG) accumulation on liver metabolic pathways is still unclear. The objective was, therefore, to characterize quantitative differences in the liver proteome between early lactation dairy cows with a low or high liver TG content. The liver proteome analysis indicated that a high liver TG content in early lactation dairy cows is associated with increased oxidation of saturated fatty acids, oxidative stress, and urea synthesis and decreased oxidation of unsaturated fatty acids. Furthermore, liver gluconeogenesis is apparently not impaired by an increased liver TG content. Based on correlations between liver proteins and plasma components, we suggest that future studies investigate the sensitivity and specificity of plasma aspartate aminotransferase, ß-hydroxybutyrate, total bilirubin, total bile acids, and γ-glutamyltransferase for potential use as blood-based biomarkers for early detection of fatty liver in dairy cows. Our study is the first to study the proteome of dairy cows with naturally occurring fatty liver in early lactation.

Changes in protein abundance between tender and tough meat from bovine longissimus thoracis muscle assessed by isobaric Tag for Relative and Absolute Quantitation (iTRAQ) and 2-dimensional gel electrophoresis analysis.

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.

Proteomic characterization of a temperature-sensitive conditional lethal in Drosophila melanogaster.

Genetic variation that is expressed only under specific environmental conditions can contribute to additional adverse effects of inbreeding if environmental conditions change. We present a proteomic characterization of a conditional lethal found in an inbred line of Drosophila melanogaster. The lethal effect is apparent as a large increase in early mortality at the restrictive temperature (29 degrees C) as opposed to normal survival at the permissive temperature (20 degrees C). The increased mortality in response to the restrictive temperature is probably caused by a single recessive major locus. A quantitative trait locus (QTL) region segregating variation affecting the lethal effect has been identified, allowing for a separation of primary/causal effects and secondary consequences in the proteome expression patterns observed. In this study, the proteomic response to the restrictive temperature in the lethal-line (L-line) was compared with the response in an inbred-control-line (IC-line) and an outbred-control-line (OC-line). Quantitative protein changes were detected using isobaric tags for relative and absolute quantitation (iTRAQ) two-dimensional liquid chromatography-tandem mass spectrometry. In all, 45 proteins were found to be significantly differently regulated in response to the restrictive temperature in the L-line as compared with the IC-line. No proteins were significantly differently regulated between the IC-line and the OC-line, verifying that differential protein regulation was specific to a genetic defect in the L-line. Proteins associated with oxidative phosphorylation and mitochondria were significantly overrepresented within the list of differentially expressed proteins. Proteins related to muscle contraction were also found to be differentially expressed in the L-line in response to the restrictive temperature, supporting phenotypic observations of moribund muscle hyper-contraction.

Identification of myofibrillar substrates for µ-calpain.

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.

Proteome analysis applied to meat science: characterizing postmortem changes in porcine muscle.

The aim of this work was to test the application of two-dimensional electrophoresis (2DE)-based proteome analysis in studying muscle tissues and meat of pork, and to use this technology to characterize as many of the changes that occur in pig muscle proteins during post mortem storage of the carcass as possible. For this purpose, 2DE proved to be a powerful tool, as it is far more sensitive and shows a higher resolving power than conventional SDS-PAGE, allowing for the precise and semiquantitative recognition of approximately 1000 individual muscle proteins in every 2DE display. In this study, we have chosen to analyze the subset of muscle proteins that have molecular masses of 5-200 kDa, and can be reproducibly separated in the pH span of 4-9. By comparing 2DE patterns of muscle samples taken immediately after slaughter (time 0), as well as those taken 4, 8, 24, and 48 h post mortem, we have estimated the relative changes of individual muscle proteins during the post mortem storage of the carcass. In this paper, of these changes we report the 15 most notable.

Protein radicals in the reaction between H2O2-activated metmyoglobin and bovine serum albumin.

Hydrogen peroxide activation of MMb with and without the presence of BSA gave rise to rapid formation of hyper-valent myoglobin species, myoglobin ferryl radical (*MbFe(IV) = O) and/or ferrylmyoglobin (MbFe(IV) = O). Reduction of MbFe(IV) = O showed first-order kinetics for a 1-2 times stoichiometric excess of H2O2 to MMb while a 3-10 times stoichiometric excess of H2O2 resulted in a biphasic reaction pattern. Radical species formed in the reaction between MMb, H2O2 and BSA were influenced by [H2O2] as measured by electron spin resonance (ESR) spectroscopy and resulted in the formation of cross-linking between BSA and myoglobin which was confirmed by SDS-PAGE and subsequent amino acid sequencing. Moreover, dityrosine was formed in the initial phases of the reaction for all concentrations of H2O2. However, initially formed dityrosine was subsequently utilized in reactions employing stoichiometric excess of H2O2 to MMb. The observed breakdown of dityrosine was ascribed to additional radical species formed from the interaction between H2O2 and the hyper-valent iron-center of H2O2-activated MMb.

The anodic hemoglobin of Anguilla anguilla. Molecular basis for allosteric effects in a root-effect hemoglobin.

The functional and structural basis for the Root effect has been investigated in the anodic hemoglobin of the European eel, Anguilla anguilla. This hemoglobin exhibits a large Bohr effect, which is accounted for by oxygen-linked binding of seven to eight protons in the presence of GTP at pH 7.5. Oxygen equilibrium curves show nonlinear lower asymptote of Hill plots, indicating the occurrence of heme-heme interactions within the T state. Analysis of the curves according to the co-operon model (Brunori, M., Coletta, M., and Di Cera, E. (1986) Biophys. Chem. 23, 215-222) reveals that T state cooperativity is positive at high pH and in the stripped hemoglobin (where the T --> R allosteric transition is operative) and negative at low pH and in the presence of organic phosphate (where the molecule is locked in the low affinity structure), indicating site heterogeneity. The complete amino acid sequence of eel anodic hemoglobin has been established and compared with that of other fish hemoglobins. The presence of the Root effect correlates with a specific configuration of the alpha1beta2 switch interface, which at low pH would stabilize subunit ligation in the T state without changing the quaternary structure. We propose that the major groups involved in the binding of oxygen-linked protons in eel anodic hemoglobin are located on the beta chain and comprise His-HC3 at the C terminus, His-FG4 at the switch interface, and Lys-EF6 and the N terminus at the phosphate-binding site.

Location of the major epsilon-(gamma-glutamyl)lysyl cross-linking site in transglutaminase-modified human plasminogen.

Tissue and plasma transglutaminases cross-link human plasminogen into high molecular weight complexes (Bendixen, E., Borth, W., and Harpel, P. C. (1993) J. Biol. Chem. 268, 21962-21967). A major cross-linking site in plasminogen involved in the tissue transglutaminase-mediated polymerization process has been identified. The epsilon-(gamma-glutamyl)lysyl bridges of the polymer are formed between Lys-298 and Gln-322. Both the acyl donor Gln residue and the acyl acceptor Lys residue are located in the kringle 3 domain of plasminogen, i.e. cross-linking of plasminogen by tissue transglutaminase involves neither the catalytic domain nor the lysine-dependent binding sites of plasminogen. This study documents that kringle 3 contains a novel functional site with the potential to participate in transglutaminase-mediated cross-linking interactions with plasma, cell-surface, and extracellular proteins.

Transglutaminases catalyze cross-linking of plasminogen to fibronectin and human endothelial cells.

We have previously reported that apolipoprotein (a) is a substrate for transglutaminases. We now demonstrate that plasminogen which is homologous to apolipoprotein (a), is also modified by these enzymes. Transglutaminases from different sources mediated the incorporation of monodansyl-cadaverine into plasminogen, indicating the presence of reactive glutamine(s) in plasminogen. Reactive lysines were also identified using the lysine-decorating peptide dansyl-PGGQQIV. In addition, transglutaminases catalyzed the formation of plasminogen homopolymers and plasminogen-fibronectin heteropolymers. Human umbilical vein endothelial cells cross-linked plasminogen into high molecular mass aggregates. Cross-linked plasminogen was cell associated, and no cross-linking of plasminogen was seen in the fluid-phase. Large molecular mass plasminogen generated on the human umbilical vein endothelial cell (HUVEC) surface could not be eluted with epsilon-aminocapoic acid and was activatable by tissue plasminogen activator. These results suggest that, following non-covalent association of plasminogen with the HUVEC surface, cell surface-associated transglutaminase catalyzes cross-linking of plasminogen into large molecular mass aggregates that can be converted into functional plasmin. It is proposed that transglutaminases may function to localize plasminogen to cell surfaces and matrices of tissues.

Complete primary structure of bovine beta 2-glycoprotein I: localization of the disulfide bridges.

The complete primary structure of bovine beta 2-glycoprotein I was determined by a combination of cDNA and peptide sequencing. Bovine beta 2-glycoprotein I was purified from citrated plasma, and by sequencing selected peptides, the complete disulfide bridge patterns of the 11 disulfide bridges were established as well as the positions of the five asparagine-linked carbohydrate groups. Bovine beta 2-glycoprotein I comprises five mutually homologous domains or Short Consensus Repeats, each containing two disulfide bridges, except for the fifth most C-terminal domain which diverges from the Short Consensus Repeat consensus by containing an additional disulfide bridge. In the four N-terminal domains, the first and third and the second and fourth half-cystines are disulfide-linked, while in the fifth domain the first and fourth, the second and fifth, and the third and sixth half-cystines are disulfide-linked.