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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.
Comparison of genomic and proteomic data in recurrent airway obstruction affected horses using Ingenuity Pathway Analysis®. Recurrent airway obstruction (RAO) is a severe chronic respiratory disease affecting horses worldwide, though mostly in the Northern hemisphere. Environmental as well as genetic factors strongly influence the course and prognosis of the disease. Research has been focused on characterization of immunologic factors contributing to inflammatory responses, on genetic linkage analysis, and, more recently, on proteomic analysis of airway secretions from affected horses. The goal of this study was to investigate the interactions between eight candidate genes previously identified in a genetic linkage...
Immunoprecipitation of equine CD molecules using anti-human MABs previously analyzed by flow cytometry and immunohistochemistry. Earlier studies investigating the cross-reactivity of antibodies submitted to the HLDA8 had used flow cytometry as a method of choice to screen mAbs for reactivity with equine leukocytes, including two-color flow-cytometry to characterize the lymphocyte population they detect. In addition, immuno-histochemistry (IHC) was used to detect distribution of positive cells in lymphoid tissue sections. In this study we performed immunoprecipitation (IP) to complement the previous results and add valuable information regarding the molecules detected by the cross-reacting antibodies. Surface molecules f...
Latherin and other biocompatible surfactant proteins. Horses and other equids are unusual in producing protein-rich sweat for thermoregulation, a major component of which is latherin, a highly surface-active, non-glycosylated protein that is a member of the PLUNC (palate, lung and nasal epithelium clone) family. Latherin produces a significant reduction in water surface tension at low concentrations (≤1 mg/ml), and probably acts as a wetting agent to facilitate evaporative cooling through a thick, waterproofed pelt. Latherin binds temporarily to hydrophobic surfaces, and so may also have a disruptive effect on microbial biofilms. It may conseq...
Neorickettsia risticii surface-exposed proteins: proteomics identification, recognition by naturally-infected horses, and strain variations. Neorickettsia risticii is the Gram-negative, obligate, and intracellular bacterial pathogen responsible for Potomac horse fever (PHF): an important acute systemic disease of horses. N. risticii surface proteins, critical for immune recognition, have not been thoroughly characterized. In this paper, we identified the 51-kDa antigen (P51) as a major surface-exposed outer membrane protein of older and contemporary strains of N. risticii through mass spectrometry of streptavidin-purified biotinylated surface-labeled proteins. Western blot analysis of sera from naturally-infected horses demonstrate...
Two-dimensional polyacrylamide gel electrophoresis of equine seminal plasma proteins and their relation with semen freezability. The objective was to evaluate protein profiles of equine seminal plasma using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and to determine whether any of these proteins were related to semen freezability. Seminal plasma was collected from 10 stallions, of high and low semen freezability, housed at the State Stud of Lower Saxony, and routinely used in AI programs. Twenty-five protein spots were identified from the two-dimensional gel (12%), seven of which were present in all samples (all proteins were identified by MALDI-MS). Matrix-assisted laser desorption/ionization mass spe...
A mass spectrometric analysis of 4-hydroxy-2-(E)-nonenal modification of cytochrome c. Cytochrome c is a key mitochondrial respiratory protein that is particularly susceptible to modification during oxidative stress. The nature of this susceptibility is linked to the mitochondrial membrane being rich in esterified linoleic acid, which predisposes this organelle to the formation of lipid peroxidation products such as 4-hydroxy-2-(E)-nonenal (4-HNE). To better understand the nature of cytochrome c modification by 4-HNE, we initiated an in vitro study utilizing a combination of MALDI-TOF mass spectrometry, LC-ESI-MS/MS and isotope labeling to monitor 4-HNE modification of cytochrom...
High throughput proteomic analysis of the secretome in an explant model of articular cartilage inflammation. This study employed a targeted high-throughput proteomic approach to identify the major proteins present in the secretome of articular cartilage. Explants from equine metacarpophalangeal joints were incubated alone or with interleukin-1beta (IL-1β, 10ng/ml), with or without carprofen, a non-steroidal anti-inflammatory drug, for six days. After tryptic digestion of culture medium supernatants, resulting peptides were separated by HPLC and detected in a Bruker amaZon ion trap instrument. The five most abundant peptides in each MS scan were fragmented and the fragmentation patterns compared to m...
Functional modelling of an equine bronchoalveolar lavage fluid proteome provides experimental confirmation and functional annotation of equine genome sequences. The equine genome sequence enables the use of high-throughput genomic technologies in equine research, but accurate identification of expressed gene products and interpreting their biological relevance require additional structural and functional genome annotation. Here, we employ the equine genome sequence to identify predicted and known proteins using proteomics and model these proteins into biological pathways, identifying 582 proteins in normal cell-free equine bronchoalveolar lavage fluid (BALF). We improved structural and functional annotation by directly confirming the in vivo expressio...
Confirmatory analysis of continuous erythropoietin receptor activator and erythropoietin analogues in equine plasma by LC-MS for doping control. Continuous erythropoietin receptor activator (CERA) is the third generation of recombinant human erythropoietin (rhEPO) medication that retains the effect of promoting red blood cell production but has longer duration of action in the body. CERA, rhEPO, and darbepoetin alpha (DPO) can be misused to enhance performance in both human and equine athletes. To deter such misuse, a very selective and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method has now been developed for identification of CERA, rhEPO, and DPO in equine plasma. The method employs a new signature tryptic...
Hydrophilic interaction chromatography of intact, soluble proteins. The separation of intact proteins by means of Hydrophilic Interaction Chromatography (HILIC) was demonstrated with human apoA-I, recombinant human apoM, and equine cytochrome C. Five different commercially available HILIC columns were compared. Using one of these columns, different glycosylated isoforms of apoM were separated from each other and from the aglyco-form.
Comparison of methods for depletion of albumin and IgG from equine serum. Disease-specific biomarkers hold diagnostic promise in both human and veterinary medicine, but serum biomarkers in low concentrations may be masked by the presence of abundant proteins, mostly albumin and IgG. Methods to deplete albumin and IgG exist, but efficacy of these methods for depleting equine serum of these proteins has not been established. Objective: The aim of this study was to determine if albumin and IgG could be depleted from equine serum using several commercially available kits and procedures. Methods: One-dimensional gel electrophoresis followed by densitometry was used to de...
Novel keratins identified by quantitative proteomic analysis as the major cytoskeletal proteins of equine (Equus caballus) hoof lamellar tissue. The dermo-epidermal interface that connects the equine distal phalanx to the cornified hoof wall withstands great biomechanical demands, but is also a region where structural failure often ensues as a result of laminitis. The cytoskeleton in this region maintains cell structure and facilitates intercellular adhesion, making it likely to be involved in laminitis pathogenesis, although it is poorly characterized in the equine hoof lamellae. The objective of the present study was to identify and quantify the cytoskeletal proteins present in the epidermal and dermal lamellae of the equine hoof by ...
Deciphering membrane-associated molecular processes in target tissue of autoimmune uveitis by label-free quantitative mass spectrometry. Autoimmune uveitis is a blinding disease presenting with autoantibodies against eye-specific proteins as well as autoagressive T cells invading and attacking the immune-privileged target tissue retina. The molecular events enabling T cells to invade and attack the tissue have remained elusive. Changes in membrane protein expression patterns between diseased and healthy stages are especially interesting because initiating events of disease will most likely occur at membranes. Since disease progression is accompanied with a break-down of the blood-retinal barrier, serum-derived proteins mask the...
Biomarkers of in vivo fertility in sperm and seminal plasma of fertile stallions. The global proteome of sperm and seminal plasma of fertile stallions was investigated to determine whether associations with relative in vivo fertility exist. Seven stallions at stud in a commercial breeding station were collected throughout the breeding season and bred to a total of 164 mares to determine conception rates. On three occasions during the breeding season, raw semen was obtained from a regular collection for proteomic analysis using two-dimensional electrophoresis and also assessed for routine semen quality end points. First cycle conception rate was negatively related to ejacula...
Glycan profiling of a defect in decorin glycosylation in equine systemic proteoglycan accumulation, a potential model of progeroid form of Ehlers-Danlos syndrome. Defects in glycosylation of decorin can result in systemic hereditary disease. A mutation in the galactosyl transferase I gene is the underlying defect of a progeroid form of Ehlers-Danlos syndrome. We have previously described pathological changes in equine systemic proteoglycan accumulation (ESPA, formerly degenerative suspensory ligament desmitis) as consisting of excessive presence of decorin and other proteoglycans in organs and structures with a high content of connective tissue. Using liquid chromatography/mass spectrometry, and one- and two-dimensional immunoblotting we have determined...
Identification of phosphorylation sites of equine beta-casein isoforms. Equine beta-casein is phosphorylated at variable degrees and isoforms carrying 3 to 7 phosphate groups (3P-7P) have been found in milk, but the phosphorylated amino acid residues of each isoform are not yet identified. In the present work, the different phosphorylation variants were first isolated by ion-exchange chromatography and then hydrolysed by trypsin to generate caseinophosphopeptides (CPPs), each containing all the potential phosphorylation sites. The equine CPPs were prepared by metal oxide affinity chromatography, a method based on the affinity of phosphate groups towards titanium d...
Novel markers for tying-up in horses by proteomics analysis of equine muscle biopsies. The aim of the study was to identify new biomarkers for acute tying-up in horses. Skeletal muscle biopsies were taken from 3 horses suffering from acute tying-up and 3 healthy horses. We performed 2D gel electrophoresis and mass spectrometry for identification of proteins that are differentially expressed in tying-up. 2D gel electrophoresis of skeletal muscle sequential extracts yielded more than 350 protein spots on each gel, of which 14 were differentially expressed more than two-fold (p<0.05). In-gel digestion followed by peptide mass fingerprinting enabled identification of three significa...
Mass spectrometric investigations on lactate adduction to equine myoglobin. Research focused on determining the fundamental mechanisms by which lactate influences color stability has not considered a direct effect of lactate on myoglobin. Thus, the objective of this study was to use Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry to examine lactate adduction to myoglobin. Equine oxymyoglobin and equine carboxymyoglobin (0.15mM) were incubated with sodium lactate (200mM) at 4 degrees C, pH 5.6 in 50mM sodium citrate buffer or at 37 degrees C, pH 7.4 in 50mM sodium phosphate buffer, simulating typical meat storage and physiological condition...
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 ...
