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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.
Characterization of equine CSN1S2 variants considering genetics, transcriptomics, and proteomics. Currently, research interest is increasing in horse milk composition and its effect on human health. Despite previously published studies describing the presence of intra- and interbreed variability of equine milk components, no investigations have focused on the genetic background of this variation. Among horse caseins and the genes encoding them, least is known about the structure and expression of the α-S2 casein gene, CSN1S2. Herein, based on direct sequencing of the equine CSN1S2 coding sequence, we describe the presence of 51-bp insertion-deletion (in/del) polymorphism, which significan...
Characterisation of lubricin in synovial fluid from horses with osteoarthritis. The glycoprotein lubricin contributes to the boundary lubrication of the articular cartilage surface. The early events of osteoarthritis involve the superficial layer where lubricin is synthesised. Objective: To characterise the glycosylation profile of lubricin in synovial fluid from horses with osteoarthritis and study secretion and degradation of lubricin in an in vitro inflammation cartilage model. Methods: In vitro study. Methods: Synovial fluid samples collected from horses with joints with normal articular cartilage and structural osteoarthritic lesions; with and without osteochondral f...
Proteomic Profiling of Cranial (Superior) Cervical Ganglia Reveals Beta-Amyloid and Ubiquitin Proteasome System Perturbations in an Equine Multiple System Neuropathy. Equine grass sickness (EGS) is an acute, predominantly fatal, multiple system neuropathy of grazing horses with reported incidence rates of ∼2%. An apparently identical disease occurs in multiple species, including but not limited to cats, dogs, and rabbits. Although the precise etiology remains unclear, ultrastructural findings have suggested that the primary lesion lies in the glycoprotein biosynthetic pathway of specific neuronal populations. The goal of this study was therefore to identify the molecular processes underpinning neurodegeneration in EGS. Here, we use a bottom-up approach be...
Characterization of neopeptides in equine articular cartilage degradation. Osteoarthritis is characterized by a loss of extracellular matrix that leads to cartilage degradation and joint space narrowing. Specific proteases, including the aggrecanases ADAMTS-4 and matrix metalloproteinase 3, are important in initiating and promoting cartilage degradation in osteoarthritis. This study investigated protease-specific and disease-specific cleavage patterns of particular extracellular matrix proteins by comparing new peptide fragments, neopeptides, in specific exogenous protease-driven digestion of a crude cartilage proteoglycan extract and an in-vitro model of early osteo...
The ubiquitin proteasome system plays a role in venezuelan equine encephalitis virus infection. Many viruses have been implicated in utilizing or modulating the Ubiquitin Proteasome System (UPS) to enhance viral multiplication and/or to sustain a persistent infection. The mosquito-borne Venezuelan equine encephalitis virus (VEEV) belongs to the Togaviridae family and is an important biodefense pathogen and select agent. There are currently no approved vaccines or therapies for VEEV infections; therefore, it is imperative to identify novel targets for therapeutic development. We hypothesized that a functional UPS is required for efficient VEEV multiplication. We have shown that at non-tox...
Proteomics of endometrial fluid after dexamethasone treatment in mares susceptible to endometritis. Corticotherapy is a common treatment in mares susceptible to endometritis. Isoflupredone improves pregnancy rates and affects the protein profile of endometrial fluid in comparison to untreated mares. Dexamethasone decreases postbreeding fluid accumulation and uterine edema; however, its effects on the protein profile of the endometrial fluid have not yet been studied. The aim of the present study was to verify the effect of dexamethasone on the protein profile of endometrial fluid, in the presence or absence of infection, from mares susceptible to persistent postbreeding endometritis. Nine su...
An inflammatory equine model demonstrates dynamic changes of immune response and cartilage matrix molecule degradation in vitro. The molecular aspects of inflammation were investigated in equine articular cartilage explants using quantitative proteomics. Articular cartilage explants were stimulated with interleukin (IL)-1β in vitro for 25 days, and proteins released into cell culture media were chemically labeled with isobaric mass tags and analyzed by liquid chromatography-tandem mass spectrometry. A total of 127 proteins were identified and quantified in media from explants. IL-1β-stimulation resulted in an abundance of proteins related to inflammation, including matrix metalloproteinases, acute phase proteins, comp...
Comprehensive protein profiling of synovial fluid in osteoarthritis following protein equalization. The aim of the study was to characterise the protein complement of synovial fluid (SF) in health and osteoarthritis (OA) using liquid chromatography mass spectrometry (LC-MS/MS) following peptide-based depletion of high abundance proteins. Methods: SF was used from nine normal and nine OA Thoroughbred horses. Samples were analysed with LC-MS/MS using a NanoAcquityâ„¢ LC coupled to an LTQ Orbitrap Velos. In order to enrich the lower-abundance protein fractions protein equalisation was first undertaken using ProteoMinerâ„¢. Progenesis-QIâ„¢ LC-MS software was used for label-free quantification. ...
Identification of Equine Lactadherin-derived Peptides That Inhibit Rotavirus Infection via Integrin Receptor Competition. Human rotavirus is the leading cause of severe gastroenteritis in infants and children under the age of 5 years in both developed and developing countries. Human lactadherin, a milk fat globule membrane glycoprotein, inhibits human rotavirus infection in vitro, whereas bovine lactadherin is not active. Moreover, it protects breastfed infants against symptomatic rotavirus infections. To explore the potential antiviral activity of lactadherin sourced by equines, we undertook a proteomic analysis of milk fat globule membrane proteins from donkey milk and elucidated its amino acid sequence. Alignm...
Proteomic alteration of equine monocyte-derived macrophages infected with equine infectious anemia virus. Similar to the well-studied viruses human immunodeficiency virus (HIV)-1 and simian immunodeficiency virus (SIV), equine infectious anemia virus (EIAV) is another member of the Lentivirus genus in the family Retroviridae. Previous studies revealed that interactions between EIAV and the host resulted in viral evolution in pathogenicity and immunogenicity, as well as adaptation to the host. Proteomic analysis has been performed to examine changes in protein expression and/or modification in host cells infected with viruses and has revealed useful information for virus-host interactions. In this ...
Equine sperm-neutrophil binding. When mares are inseminated repeatedly, protein molecules from the seminal plasma (SP) prevent sperm-neutrophil binding and reduced fertility. The molecule(s) responsible for sperm-neutrophil binding is not known and the identification of beneficial SP proteins is complicated by their large numbers and abundant variation. We examined several important aspects of sperm-neutrophil binding to ultimately facilitate the identification and isolation of the molecule(s) responsible. First, we raised anti-equine P-selectin antibodies to determine the involvement of this adhesion molecule in sperm-neutro...
Differences in serum protein 2D gel electrophoresis patterns of Przewalski’s (Mongolian wild horse) and thoroughbred horses. The objective of this study was to assess differences in serum protein expression profiles of Przewalski's (Mongolian wild horse) and thoroughbred horses using proteome analysis. The serum proteins were separated by two-dimensional electrophoresis (2-DE) and five different gene products were identified. Proteins represented by the five spots were identified by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS)/MS technology. The identities of all proteins were deduced based on their similarity to proteins in the human plasma protein database. Three pr...
Distinctive proteolytic activity of cell envelope proteinase of Lactobacillus helveticus isolated from airag, a traditional Mongolian fermented mare’s milk. Airag is a traditional fermented milk of Mongolia that is usually made from raw mare's milk. Lactobacillus helveticus is one of the lactic acid bacteria most frequently isolated from airag. In this study, we investigated the genetic and physiological characteristics of L. helveticus strains isolated from airag and clarified their significance in airag by comparing them with strains from different sources. Six strains of L. helveticus were isolated from five home-made airag samples collected from different regions of Mongolia. The optimal temperature for acidification in skim milk was 30 to 35Â...
Investigation of the stallion sperm proteome by mass spectrometry. Stallion spermatozoa continue to present scientific and clinical challenges with regard to the biological mechanisms responsible for their survival and function. In particular, deeper understanding of sperm energy metabolism, defence against oxidative damage and cell-cell interactions should improve fertility assessment and the application of advanced reproductive technologies in the equine species. In this study, we used highly sensitive LC-MS/MS technology and sequence database analysis to identify and characterise the proteome of Percoll-isolated ejaculated equine spermatozoa, with the aim ...
Omics technologies provide new insights into the molecular physiopathology of equine osteochondrosis. Osteochondrosis (OC(D)) is a juvenile osteo-articular disorder affecting several mammalian species. In horses, OC(D) is considered as a multifactorial disease and has been described as a focal disruption of endochondral ossification leading to the development of osteoarticular lesions. Nevertheless, OC(D) physiopathology is poorly understood. Affected horses may present joint swelling, stiffness and lameness. Thus, OC(D) is a major concern for the equine industry. Our study was designed as an integrative approach using omics technologies for the identification of constitutive defects in epiphy...
Development of a method for absolute quantification of equine acute phase proteins using concatenated peptide standards and selected reaction monitoring. 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 to 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 synthesized and expressed as an isotope-labeled chimaeric protein in Escherichia coli. The QconCAT tryptic peptides were analyzed on a ...
The use of liquid chromatography tandem mass spectrometry to detect proteins in saliva from horses with and without systemic inflammation. 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, haptoglob...
Proteomic analysis reveals age-related changes in tendon matrix composition, with age- and injury-specific matrix fragmentation. Energy storing tendons, such as the human Achilles and equine superficial digital flexor tendon (SDFT), are highly prone to injury, the incidence of which increases with aging. The cellular and molecular mechanisms that result in increased injury in aged tendons are not well established but are thought to result in altered matrix turnover. However, little attempt has been made to fully characterize the tendon proteome nor determine how the abundance of specific tendon proteins changes with aging and/or injury. The aim of this study was, therefore, to assess the protein profile of normal SDFTs ...
In situ cell surface proteomics reveals differentially expressed membrane proteins in retinal pigment epithelial cells during autoimmune uveitis. Retinal pigment epithelium (RPE) builds the outer blood-retinal barrier of the eye and plays an important role in pathogenesis of the sight threatening disease equine recurrent uveitis (ERU). ERU is a spontaneous autoimmune mediated inflammatory disease characterised by the breakdown of the outer blood-retinal barrier and an influx of autoaggressive T-cells into the inner eye. Therefore, identification of molecular mechanisms contributing to changed function of blood-retinal barrier in ERU is important for the understanding of pathophysiology. Cell surface proteins of RPE collected from health...
Proteomic analysis of equine amniotic membrane: characterization of proteins. Human amniotic membrane (AM) has been used as a biomaterial for surgical wound skin and ocular surface reconstruction for several years. Currently, equine AM has been used for corneal reconstruction in several animal species, and appears to have the same properties as human AM. Despite the observed positive healing abilities of this tissue in horses with ulcerative keratitis the proteins of equine AM have not been described. Objective: To identify proteins known to be associated with corneal healing from frozen equine AM. Methods: Placentas were acquired from healthy live foal births from a lo...
Proteomics in farm animals models of human diseases. The need to provide in vivo complex environments to understand human diseases strongly relies on the use of animal models, which traditionally include small rodents and rabbits. It is becoming increasingly evident that the few species utilised to date cannot be regarded as universal. There is a great need for new animal species that are naturally endowed with specific features relevant to human diseases. Farm animals, including pigs, cows, sheep and horses, represent a valid alternative to commonly utilised rodent models. There is an ample scope for the application of proteomic techniques in f...
Involvement of mitochondrial dysfunction and ER-stress in the physiopathology of equine osteochondritis dissecans (OCD). Osteochondrosis (OC) is a developmental bone disorder affecting several mammalian species including the horse. Equine OC is described as a focal disruption of endochondral ossification, leading to osteochondral lesions (osteochondritis dissecans, OCD) that may release free bodies within the joint. OCD lesions trigger joint swelling, stiffness and lameness and affects about 30% of the equine population. OCD is considered as multifactorial but its physiopathology is still poorly understood and genes involved in genetic predisposition are still unknown. Our study compared two healthy and two OC-a...
Unraveling the equine lymphocyte proteome: differential septin 7 expression associates with immune cells in equine recurrent uveitis. Equine recurrent uveitis is a spontaneous, lymphocyte-driven autoimmune disease. It affects horses worldwide and presents with painful remitting-relapsing inflammatory attacks of inner eye structures eventually leading to blindness. Since lymphocytes are the key players in equine recurrent uveitis, we were interested in potential changes of their protein repertoire which may be involved in disease pathogenesis. To create a reference for differential proteome analysis, we first unraveled the equine lymphocyte proteome by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis ...
The Equine PeptideAtlas: a resource for developing proteomics-based veterinary research. Progress in MS-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 MS 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. T...
Proteomic analysis of tendon extracellular matrix reveals disease stage-specific fragmentation and differential cleavage of COMP (cartilage oligomeric matrix protein). During inflammatory processes the extracellular matrix (ECM) is extensively remodeled, and many of the constituent components are released as proteolytically cleaved fragments. These degradative processes are better documented for inflammatory joint diseases than tendinopathy even though the pathogenesis has many similarities. The aims of this study were to investigate the proteomic composition of injured tendons during early and late disease stages to identify disease-specific cleavage patterns of the ECM protein cartilage oligomeric matrix protein (COMP). In addition to characterizing fragme...
Carprofen inhibits the release of matrix metalloproteinases 1, 3, and 13 in the secretome of an explant model of articular cartilage stimulated with interleukin 1β. Arthritic diseases are characterized by the degradation of collagenous and noncollagenous extracellular matrix (ECM) components in articular cartilage. The increased expression and activity of matrix metalloproteinases (MMPs) is partly responsible for cartilage degradation. This study used proteomics to identify inflammatory proteins and catabolic enzymes released in a serum-free explant model of articular cartilage stimulated with the pro-inflammatory cytokine interleukin 1β (IL-1β). Western blotting was used to quantify the release of selected proteins in the presence or absence of the cyc...
Thiol-disulfide proteins of stallion epididymal spermatozoa. Thiol groups of cysteine residues represent redox centers involved in multiple biological functions. It has been postulated that changes in the redox status of mammalian epididymal spermatozoa contribute to the sperm maturation process. The present work shows the thiol-disulfide protein profile of stallion epididymal spermatozoa achieved by two-dimension electrophoresis and MALDI-TOF/TOF mass spectrometry of proteins labeled with a thiol-reactive fluorescent tag, monobromobimane. Our results have shown the formation of disulfide bonds in several sperm protein fractions during the epididymal ma...
Expression changes and novel interaction partners of talin 1 in effector cells of autoimmune uveitis. Autoimmune uveitis is characterized by crossing of blood-retinal barrier (BRB) by autoaggressive immune cells. Equine recurrent uveitis (ERU) is a valuable spontaneous model for autoimmune uveitis and analyses of differentially expressed proteins in ERU unraveled changed protein clusters in target tissues and immune system. Healthy eyes are devoid of leukocytes. In ERU, however, leukocytes enter the inner eye and subsequently destroy it. Molecular mechanisms enabling cell migration through BRB still remain elusive. Previously, we detected decreased talin 1 expression in blood-derived granulocy...
Identification of autoantigens in body fluids by combining pull-downs and organic precipitations of intact immune complexes with quantitative label-free mass spectrometry. Most autoimmune diseases are multifactorial diseases and are caused by the immunological reaction against a number of autoantigens. Key for understanding autoimmune pathologies is the knowledge of the targeted autoantigens, both initially and during disease progression. We present an approach for autoantigen identification based on isolation of intact autoantibody-antigen complexes from body fluids. After organic precipitation of high molecular weight proteins and free immunoglobulins, released autoantigens were identified by quantitative label-free liquid chromatography mass spectrometry. We ...
Biochemical characterization of stallion prostasomes and comparison to their human counterparts. Release of nanometer-sized prostasomes into human and equine semen suggests essential functions in their relationships with sperm cells and the fertilization process. The two types of prostasomes displayed ultrastructural similarities, albeit the human prostasomes were somewhat larger than the stallion prostasomes. A high ratio of saturated fatty acids was characteristic for the two prostasome types. Electrophoretic separation systems revealed an equine prostasomal pattern different from that of human. The 21 distinctive low molecular weight protein spots in the 2D-gel (with no counterparts in...
