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Topic:Proteomics
Proteomics is the large-scale study of proteins, particularly their structures and functions, in horses. This field encompasses the analysis of the complete set of proteins expressed by the equine genome, known as the proteome. Proteomics research in horses aims to understand the diverse roles of proteins in various biological processes, including growth, development, and response to environmental stimuli. Techniques such as mass spectrometry and protein microarrays are commonly employed to identify and quantify proteins, assess post-translational modifications, and investigate protein-protein interactions. This page compiles peer-reviewed research studies and scholarly articles that explore the methodologies, findings, and implications of proteomics in equine health and disease.
Differential expression of equine muscle biopsy proteins during normal training and intensified training in young standardbred horses using proteomics technology. The major aim of the present study was to investigate the proteome of standardbred horses at different stages of training and intensified training. We searched for biomarkers using small skeletal muscle biopsies of live animals. 2D gel electrophoresis and mass spectrometry were successfully applied to investigate training-induced differential expression of equine muscle biopsy proteins. Despite the poor resolution of the equine genome and proteome, we were able to identify the proteins of 20 differential spots representing 16 different proteins. Evaluation of those proteins complies with adapt...
Purification and partial characterization of proteinase inhibitors of equine seminal plasma. The aims of the study were: 1/ to isolate and identify equine seminal plasma proteinase inhibitors, 2/ to evaluate their inhibitory potential, and 3/ to test a correlation between protein concentration in seminal plasma supernatant (obtained after precipitation with 36% ammonium sulfate) and stallion sexual maturity. Seminal plasma proteins obtained from six stallions were chromatographed in a Superose 12 (FPLC system) column followed by C(18) HPLC reverse-phase. Inhibition of trypsin amidase activity was evaluated in the collected fractions. Active proteins with a molecular mass of 6.3-7.0 KD...
Application of fluorogenic derivatization-liquid chromatography-tandem mass spectrometric proteome method to skeletal muscle proteins in fast Thoroughbred horses. To extend the applicability of the fluorogenic derivatization-high performance liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS) method, which consists of fluorogenic derivatization (FD), separation by liquid chromatography (LC), and identification by LC-tandem mass spectrometric (MS/MS) proteomic analysis, we applied it to Thoroughbred horse muscle. With the optimization of the protein extraction and separation procedure, reproducible chromatograms were obtained and the changes in protein expressions during exercise were able to be analyzed. To quantify the changed protein expressi...
Kininogen in autoimmune uveitis: decrease in peripheral blood stream versus increase in target tissue. Equine recurrent uveitis (ERU) is an incurable disease affecting the inner eye that leads to blindness, through activated T cells that pass the blood-retinal barrier and destroy the retina. Serum markers are a desirable choice for monitoring development of disease, as serum is easy accessible and the markers could serve to predict the beginning of disease or an imminent relapse. Methods: In this study, serum proteomes (depleted of high-abundance serum proteins) of horses with ERU and healthy controls were compared with the 2-D DIGE (two-dimensional gel electrophoresis) technique to identify di...
Equine alpha S1-casein: characterization of alternative splicing isoforms and determination of phosphorylation levels. alpha(S1)-Casein was isolated from Haflinger mare's milk by hydrophobic interaction chromatography and displayed great micro-heterogeneity by 2-dimensional electrophoresis, probably because of a variable degree of phosphorylation and alternative splicing events. The aim of the present work was to investigate the complexity of the mare's alpha(S1)-casein. The different isoforms present in milk were submitted to a double treatment of dephosphorylation, first by using alkaline phosphatase and then acid phosphatase to achieve complete dephosphorylation. The apoforms were then analyzed by electrosp...
Multiplexed LC-MS/MS analysis of horse plasma proteins to study doping in sport. The development of protein biomarkers for the indirect detection of doping in horse is a potential solution to doping threats such as gene and protein doping. A method for biomarker candidate discovery in horse plasma is presented using targeted analysis of proteotypic peptides from horse proteins. These peptides were first identified in a novel list of the abundant proteins in horse plasma. To monitor these peptides, an LC-MS/MS method using multiple reaction monitoring was developed to study the quantity of 49 proteins in horse plasma in a single run. The method was optimised and validated, ...
Identification and isolation of cDNA clones encoding the abundant secreted proteins in the saliva proteome of Culicoides nubeculosus. Culicoides spp. are vectors of several infectious diseases of veterinary importance and a major cause of allergy in horses and other livestock. Their saliva contains a number of proteins which enable blood feeding, enhance disease transmission and act as allergens. We report the construction of a novel cDNA library from Culicoides nubeculosus linked to the analysis of abundant salivary gland proteins by mass spectrometry. Fifty-four novel proteins sequences are described including those of the enzymes maltase, hyaluronidase and two serine proteases demonstrated to be present in Culicoides sali...
Protein expression profile of Gasterophilus intestinalis larvae causing horse gastric myiasis and characterization of horse immune reaction. Little information is available on the immunological aspect of parasitic Gasterophilus intestinalis (Diptera, Oestridae) larvae causing horse gastric myiasis. The objectives of this research were to analyze the protein content of larval crude extracts of the migrating second and third larvae (L2 and L3) of G. intestinalis in order to characterize the immune response of horses. Results: The proteomic profile of L2 and L3, investigated by using one and two dimensional approaches, revealed a migration pattern specific to each larval stage. Furthermore, Western blots were performed with horse sera...
Ocular immunology in equine recurrent uveitis. Equine recurrent uveitis (ERU) is a disease with high prevalence and relevance for the equine population, since it results in blindness. Over the last decade, important advancements have been made in our understanding of the underlying immune responses in this disease. ERU is mediated by an autoaggressive Th1 response directed against several retinal proteins. Interphotoreceptor-retinoid binding protein (IRBP) and cellular retinaldehyde-binding protein (CRALBP) are capable to induce ERU-like disease in experimental horses, with the unique possibility to activate relapses in a well-defined mann...
Virion associated proteins of equine rhinitis B virus 1 (ERBV1): the non-structural protein 3C(pro) co-purifies with virions. Equine rhinitis B virus (ERBV), genus Erbovirus, is most closely related to the Cardiovirus genus in the family Picornaviridae. The structural proteins (VP1-4) of erboviruses are not well described, but are predicted by sequence to be 35, 29, 26 and 7 kDa. Methods for the purification of cardioviruses (polyethylene glycol, trypsin treatment) were used to characterise the structural proteins of ERBV1. Only one of the virus proteins detected was an expected molecular mass, and this 26 kDa protein was identified as VP3 by N-terminal amino acid sequencing. N-terminal sequencing of the 56 and a 29 ...
A proteomic approach for studying the pathogenesis of spontaneous equine recurrent uveitis (ERU). Equine recurrent uveitis (ERU) is a wide spread disease of the eye, which is the main cause for blindness in horses worldwide. Meanwhile, ERU is also accepted as the only reliable spontaneous model for human autoimmune uveitis. We identified and characterized novel autoantigens by analyzing the autoantibody-binding pattern from ERU cases to the retinal proteome. Cellular retinaldehyde-binding protein (CRALBP) and malate dehydrogenase (MDH) were detected as novel ERU autoantigens by this approach. B- and T-cell autoreactivity was detected to both autoantigens in ERU cases. The evaluation of the...
Proteomic analysis and immunogenicity of secreted proteins from Rhodococcus equi ATCC 33701. Rhodococcus equi is one of the most important causes of mortality in foals between 1 and 6 months of age. Although rare, infection also occurs in a variety of other mammals including humans, often following immunosuppression of various causes. Secreted proteins are known to mediate important pathogen-host interactions and consequently are favored candidates for vaccine development as they are the most easily accessible microbial antigens to the immune system. Here, we describe the results of a proteomic analysis based on SDS-PAGE, immunoblot and mass spectrometry, which was carried out aiming ...
Analysis of major proteins and fat fractions associated with mare’s milk fat globules. Although several studies have aimed to identify mare's milk proteins, only the major whey proteins and some caseins have yet been characterized. Incomplete sequencing of the equine genome and the difficulty of recovering highly hydrophobic proteins mean that little is known to date about the proteins associated with milk fat globules, which have been shown to play an important role in newborns' defense mechanisms. The fat fraction, in particular the distribution of unsaturated fatty acids, has been more extensively studied, but complex lipids are only partially elucidated. This study reports a...
Biomarkers for ragwort poisoning in horses: identification of protein targets. Ingestion of the poisonous weed ragwort (Senecio jacobea) by horses leads to irreversible liver damage. The principal toxins of ragwort are the pyrrolizidine alkaloids that are rapidly metabolised to highly reactive and cytotoxic pyrroles, which can escape into the circulation and bind to proteins. In this study a non-invasive in vitro model system has been developed to investigate whether pyrrole toxins induce specific modifications of equine blood proteins that are detectable by proteomic methods. Results: One dimensional gel electrophoresis revealed a significant alteration in the equine pl...
Proteins associated with the early intrauterine equine conceptus. A critical period of early gestation in the mare involves the immobilization (fixation) of the encapsulated conceptus at around days 16-17. We compared the major proteins in the normal equine embryonic capsule and endometrial secretions around the period of fixation with those from pregnancies in the process of termination induced by administration of an analogue of prostaglandin F(2 alpha) (PGF(2 alpha)). Uterocalin and beta(2)-microglobulin (beta(2)M) associated with the embryonic capsule were proteolytically converted to smaller forms during the fixation period. These conversions were simil...
Equine recurrent uveitis–a spontaneous horse model of uveitis. Equine recurrent uveitis (ERU) is an autoimmune disease that occurs with a high prevalence (10%) in horses. ERU represents the only reliable spontaneous model for human autoimmune uveitis. We already identified and characterized novel autoantigens (malate dehydrogenase, recoverin, CRALBP) by analyzing the autoantibody-binding pattern of horses affected by spontaneous recurrent uveitis (ERU) to the retinal proteome. CRALBP also seems to be relevant to human autoimmune uveitis. Proteomic screening of vitreous and retinal samples from ERU diseased cases in comparison to healthy controls has led t...
Differentiation and identification of recombinant human erythropoietin and darbepoetin Alfa in equine plasma by LC-MS/MS for doping control. Recombinant human erythropoietin (rhEPO) and darbepoetin alfa (DPO) are protein-based drugs for the treatment of anemia in humans by stimulating erythrocyte production. However, these agents are abused in human and equine sports due to their potential to enhance performance. This paper describes the first method for differentiation and identification of rhEPO and DPO in equine plasma by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The method comprised analyte extraction and enrichment by immunoaffinity separation with anti-rhEPO antibodies, dual digestion by trypsin an...
The effect of pre-polymeric solution and subsequent encapsulation in hydrogel membranes on the stability and biological activity of horse myoglobins. Proteins are biological macromolecules which have a unique spatial conformation. Once this 3D spatial conformation is affected the protein's biological stability and activity can be severely limited. For these reasons, this investigation focuses on the effects of pre-polymeric solution components on the behavior of proteins to be encapsulated by the entrapment technique in anionic, cationic, and neutral hydrogel membranes. Equine skeletal muscle myoglobin (MMb), and equine heart myoglobin (HMb) were employed as model molecules. Three hydrogel morphologies were examined: methacrylic acid-poly(e...
Identification of abundant proteins and potential allergens in Culicoides nubeculosus salivary glands. IgE-mediated type 1 hypersensitivity reactions to the bites of insects are a common cause of skin disease in horses. Insect bite hypersensitivity (IBH) is most frequently associated with bites of Culicoides spp. and occurs in all parts of the world where horses and Culicoides coexist. The main allergens that cause IBH are probably some of the abundant proteins in the saliva of Culicoides associated with blood feeding. Western blots of Culicoides proteins separated by 1D gel-electrophoresis detected strong IgE responses in all horses with IBH to antigens in protein extracts from wild caught Cul...
Detection of an isoform of alpha(1)-antitrypsin in serum samples from foals with gastric ulcers. The objective of this study was to find serum indicators of gastric ulcers in foals. By using two-dimensional electrophoresis of serum proteins, three distinct spots were detected in samples from foals with gastric ulcers detected endoscopically. One of them appeared with high frequency and was identified by partial digestion with trypsin and subsequent nano-electrospray ionisation-tandem mass spectrometry (nanoesi-ms/ms) analysis as an alpha(1)-antitrypsin. Western blot analysis, using an antibody against human alpha(1)-antitrypsin, revealed at least two bands, of molecular weight 58 kDa and ...
Ruthenium anticancer drugs and proteins: a study of the interactions of the ruthenium(III) complex imidazolium trans-[tetrachloro(dimethyl sulfoxide)(imidazole)ruthenate(III)] with hen egg white lysozyme and horse heart cytochrome c. The interactions with protein targets of the ruthenium(III) complex imidazolium trans-[tetrachloro(dimethyl sulfoxide)(imidazole)ruthenate(III)], NAMI-A, an effective anticancer and antimetastatic agent now in clinical trials, deserve great attention as they are believed to be at the basis of the mechanism of action of this innovative molecule. Here, we report on the reactions of NAMI-A with two well-known model proteins, namely, hen egg white lysozyme and horse heart cytochrome c; these reactions were investigated by a variety of physicochemical methods, including optical spectroscopy, (1)H N...
Susceptibility of mammalian deoxyribonucleases I (DNases I) to proteolysis by proteases and its relationships to tissue distribution: biochemical and molecular analysis of equine DNase I. Equine (Equus caballus) deoxyribonuclease I (DNase I) was purified from the parotid gland, and its 1295-bp cDNA was cloned. The mature equine DNase I protein consisted of 260 amino acid residues. The enzymatic properties and structural aspects of the equine enzyme were closely similar to those of other mammalian DNases I. Mammalian DNases I are classified into three types--pancreatic, parotid and pancreatic-parotid-based on their tissue distribution; as equine DNase I showed the highest activity in the parotid gland, it was confirmed to be of the parotid-type. Comparison of the susceptibility ...
Major retinal autoantigens remain stably expressed during all stages of spontaneous uveitis. Equine recurrent uveitis (ERU) is a valuable model for autoimmune diseases, since it develops frequently and occurs spontaneously. We investigated the overall expression level of three major retinal autoantigens in normal retinas and various ERU stages. Analysis of retinal proteomes of both, healthy and diseased retinas revealed an almost unaffected expression of IRBP, S-antigen and cRALBP in ERU cases. Validation of these findings with western blots and immunohistochemistry confirmed constant to increased expression of these autoantigens, although loss of their physiological expression sites ...
Retinal Mueller glial cells trigger the hallmark inflammatory process in autoimmune uveitis. Spontaneous equine recurrent uveitis (ERU) is an incurable autoimmune disease affecting the eye. Although retinal-autoantigen specific T-helper 1 cells have been demonstrated to trigger disease progression and relapses, the molecular processes leading to retinal degeneration and consequent blindness remain unknown. To elucidate such processes, we studied changes in the total retinal proteome of ERU-diseased horses compared to healthy controls. Severe changes in the retinal proteome were found for several markers for blood-retinal barrier breakdown and whose emergence depended upon disease seve...
The primary structure of a low-Mr multiphosphorylated variant of beta-casein in equine milk. Highly phosphorylated casein with a low molecular mass was isolated from Haflinger mare's milk by RP-HPLC. It accounts for 4.0% of the casein content. Its mass was determined by LC-ESI-MS before and after treatment by alkaline phosphatase. The molecular mass found for the apo-form (10,591 +/- 2 Da) is in agreement with its primary structure, which was established by ESI-MS/MS from tryptic peptides. It appeared that this short protein (94 amino acid residues) is an internally truncated form of the full-length equine beta-casein (226 residues). This low-Mr variant of equine beta-casein displays ...
Identifying the future needs for long-term USDA efforts in agricultural animal genomics. Agricultural animal research has been immensely successful over the past century in developing technology and methodologies that have dramatically enhanced production efficiency of the beef, dairy, swine, poultry, sheep, and aquaculture industries. In the past two decades, molecular biology has changed the face of agricultural animal research, primarily in the arena of genomics and the relatively new offshoot areas of functional genomics, proteomics, transcriptomics, metabolomics and metagenomics. Publication of genetic and physical genome maps in the past 15 years has given rise to the possib...
Proteolytic maturation of replicase polyprotein pp1a by the nsp4 main proteinase is essential for equine arteritis virus replication and includes internal cleavage of nsp7. The positive-stranded RNA genome of the arterivirus Equine arteritis virus (order Nidovirales) encodes the partially overlapping replicase polyproteins pp1a (1727 aa) and pp1ab (3175 aa). Previously, three viral proteinases were reported to cleave these large polyproteins into 12 non-structural proteins (nsps). The chymotrypsin-like viral main proteinase residing in nsp4 is responsible for eight of these cleavages. Processing of the C-terminal half of pp1a (the nsp3-8 region) was postulated to occur following either of two alternative proteolytic pathways (the 'major' and 'minor' pathways). He...
Mass spectrometry studies of demetallation of haemin by recombinant horse L chain apoferritin and its mutant (E 53,56,57,60 Q). An essential difference between eukaryotic ferritins and bacterioferritins is that the latter contain naturally, in vivo haem as Fe-protoporphyrin IX. This haem is located in a hydrophobic pocket along the 2-fold symmetry axes and is liganded by two Met 52. However, in in vivo studies, a cofactor has been isolated in horse spleen apoferritin similar to protoporphyrin IX; in in vitro experiments, it has been shown that horse spleen apoferritin is able to interact with haem. Studies of haemin (Fe(III)-PPIX) incorporation into horse spleen apoferritin have been carried out, which show that the me...
Fine and domain-level epitope mapping of botulinum neurotoxin type A neutralizing antibodies by yeast surface display. Botulinum neurotoxin (BoNT), the most poisonous substance known, causes naturally occurring human disease (botulism) and is one of the top six biothreat agents. Botulism is treated with polyclonal antibodies produced in horses that are associated with a high incidence of systemic reactions. Human monoclonal antibodies (mAbs) are under development as a safer therapy. Identifying neutralizing epitopes on BoNTs is an important step in generating neutralizing mAbs, and has implications for vaccine development. Here, we show that the three domains of BoNT serotype A (BoNT/A) can be displayed on the...
