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.
Braus BK, Miller I, Kummer S, Kleinwort KJH, Hirmer S, Hauck SM, McMullen RJ, Kerschbaumer M, Deeg CA.Immune mediated keratitis (IMMK) is primarily a non-ulcerative keratitis in horses causing intermittent ocular pain, eventually resulting in visual impairment. Affected horses typically respond to immunomodulatory treatment. However, the underlying cause of the disease remains enigmatic. The current study was undertaken to investigate the presence of autoantibodies in horses with immune mediated keratitis. Using 28 horses with IMMK and 27 healthy controls screening for serum autoantibodies against the corneal proteome using indirect immunofluorescence, one-dimensional (1DE) and two-dimensional...
Sedykh SE, Purvinish LV, Monogarov AS, Burkova EE, Grigor'eva AE, Bulgakov DV, Dmitrenok PS, Vlassov VV, Ryabchikova EI, Nevinsky GA.Exosomes are 40-100 nm nanovesicles containing RNA and different proteins. Exosomes containing proteins, lipids, mRNAs, and microRNAs are important in intracellular communication and immune function. Exosomes from different sources are usually obtained by combination of centrifugation and ultracentrifugation and according to published data can contain from a few dozens to thousands of different proteins. Crude exosome preparations from milk of eighteen horses were obtained for the first time using several standard centrifugations. Exosome preparations were additionally purified by FPLC gel fi...
Skiöldebrand E, Ekman S, Mattsson Hultén L, Svala E, Björkman K, Lindahl A, Lundqvist A, Önnerfjord P, Sihlbom C, Rüetschi U.Clinical tools to diagnose the early changes of osteoarthritis (OA) that occur in the articular cartilage are lacking. Objective: We sought to identify and quantify a novel cartilage oligomeric matrix protein (COMP) neoepitope in the synovial fluid from the joints of healthy horses and those with different stages of OA. Methods: In vitro quantitative proteomics and assay development with application in synovial fluids samples obtained from biobanks of well-characterised horses. Methods: Articular cartilage explants were incubated with or without interleukin-1β for 25 days. Media were analysed...
Journal of proteomicsJanuary 3, 2017
Volume 154 102-108 doi: 10.1016/j.jprot.2016.12.015
Degroote RL, Uhl PB, Amann B, Krackhardt AM, Ueffing M, Hauck SM, Deeg CA.The membrane protein expression repertoire of cells changes in course of activation. In equine recurrent uveitis (ERU), a spontaneous autoimmune disease in horses with relapsing and ultimately blinding inner eye inflammation, CD4+ T lymphocytes are the crucial pathogenic cells activated in the periphery directly prior to an inflammatory episode. In order to find relevant changes in the membrane proteome associated to disease, we sorted CD4+ lymphocytes and compared protein abundance from the generated proteome datasets of both healthy horses and ERU cases. We detected formin like 1, a key play...
Wong KS, Chan GHM, Ho ENM, Wan TSM.Growth hormone (GH), or somatotropin, is a protein that may enhance physical performance and facilitate growth and wound healing. For this reason, growth hormones and their recombinant analogues are prohibited in human sports by the World Anti-Doping Agency (WADA) and in horseracing under Article 6 of the International Agreement on Breeding, Racing and Wagering published by the International Federation of Horse Racing Authorities (IFHA). Identifying the illicit use of GHs in both human athletes and racehorses is challenging, especially when differentiating between endogenous and exogenous GHs,...
Loux SC, Scoggin KE, Troedsson MH, Squires EL, Ball BA.The cervical mucus plug (CMP) is believed to play an integral role in the maintenance of pregnancy in the mare, primarily by inhibiting microbial entry. Unfortunately, very little is known about its composition or origin. To determine the proteomic composition of the CMP, we collected CMPs from mares (n = 4) at 9 months of gestation, and proteins were subsequently analyzed by nano-LC-MS/MS. Results were searched against EquCab2.0, and proteomic pathways were predicted by Ingenuity Pathway Analysis. Histologic sections of the CMP were stained with H&E and PAS. To identify the origin of ...
Isani G, Ferlizza E, Cuoghi A, Bellei E, Monari E, Bianchin Butina B, Castagnetti C.Characterisation of the physiologic equine amniotic fluid (AF) proteome is a prerequisite to study its changes during diseases and discover new biomarkers. The aim of this study was to identify by a proteomic approach the most abundant proteins of equine AF. AF samples were collected at parturition from 24 healthy mares that delivered healthy foals. All samples were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) on 4-12% gels. A pool of the 24 samples, after SDS-PAGE, was cut in 25 slices, trypsin-digested and analysed by mass spectrometry (MS) for protein i...
Yang Y, Zheng N, Wang W, Zhao X, Zhang Y, Han R, Ma L, Zhao S, Li S, Guo T, Zang C, Wang J.Glycosylation of proteins has been implicated in various biological functions and has received much attention; however, glycoprotein components and inter-species complexity have not yet been elucidated fully in milk proteins. N-linked glycosylation sites and glycoproteins in milk fat globule membrane (MFGM) fractions were investigated by combining N-glycosylated peptides enrichment and high-accuracy Q Exactive identification, to map the N-glycoproteome profiles in Holstein and Jersey cows, buffaloes, yaks, goats, camels, horses, and humans. A total of 399 N-glycoproteins with 677 glycosylation...
Smits K, Nelis H, Van Steendam K, Govaere J, Roels K, Ververs C, Leemans B, Wydooghe E, Deforce D, Van Soom A.The equine oviduct plays a pivotal role in providing the optimal microenvironment for early embryonic development, but little is known about the protein composition of the oviducal fluid in the horse. The aim of the present study was to provide a large-scale identification of proteins in equine oviducal fluid and to determine the effects of ovulation and pregnancy. Four days after ovulation, the oviducts ipsilateral and contralateral to the ovulation side were collected from five pregnant and five non-pregnant mares. Identification and relative quantification of proteins in the oviducal fluid ...
Wood PL, Scoggin K, Ball BA, Troedsson MH, Squires EL.Using a nontargeted lipidomics analysis of equine sperm and seminal plasma, we were able to characterize a diverse array of individual lipids including ethanolamine and choline ether lipids and seminolipids essential to membrane raft function. We also detected, for the first time in sperm, the presence of (O-acyl)-ω-hydroxy-fatty acids (OAHFA) with up to 52 carbon chain lengths, which were localized to the head and not the tail of sperm. The only previous identification of OAHFAs has been in meibomian glands and their sebaceous secretions. The identities of these lipid amphiphiles were valida...
Bundgaard L, Bendixen E, Sørensen MA, Harman VM, Beynon RJ, Petersen LJ, Jacobsen S.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...
Matta C, Zhang X, Liddell S, Smith JR, Mobasheri A.There is insufficient knowledge about the chondrocyte membranome and its molecular composition. Objective: To develop a Triton X-114 based separation technique using nanoLC-MS/MS combined with shotgun proteomics to identify chondrocyte membrane proteins. Methods: Articular chondrocytes from equine metacarpophalangeal joints were separated into hydrophobic and hydrophilic fractions; trypsin-digested proteins were analysed by nanoLC-MS/MS. Results: A total of 315 proteins were identified. The phase extraction method yielded a high proportion of membrane proteins (56%) including CD276, S100-A6 an...
Thorpe CT, Peffers MJ, Simpson D, Halliwell E, Screen HR, Clegg PD.Tendon is a simple aligned fibre composite, consisting of collagen-rich fascicles surrounded by a softer interfascicular matrix (IFM). The composition and interactions between these material phases are fundamental in ensuring tissue mechanics meet functional requirements. However the IFM is poorly defined, therefore tendon structure-function relationships are incompletely understood. We hypothesised that the IFM has a more complex proteome, with faster turnover than the fascicular matrix (FM). Using laser-capture microdissection and mass spectrometry, we demonstrate that the IFM contains more ...
Mori M, Ichibangase T, Yamashita S, Kijima-Suda I, Kawahara M, Imai K.In the doping tests currently used in horse racing, prohibited substances or their metabolites are usually directly detected in urine or blood samples. However, despite their lasting pharmaceutical effects, some prohibited substances are rapidly eliminated from horse urine and blood, making them difficult to detect. Therefore, new indirect biomarkers for doping, such as plasma proteins that are increased by the prohibited substances, have recently attracted much attention. Here, a fluorogenic derivatization-liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS) method was adopted for hor...
Richards SL, Cawley AT, Cavicchioli R, Suann CJ, Pickford R, Raftery MJ.Over recent years threats to racing have expanded to include naturally occurring biological molecules, such as peptides and proteins, and their synthetic analogues. Traditionally, antibodies have been used to enable detection of these compounds as they allow purification and concentration of the analyte of interest. The rapid expansion of peptide-based therapeutics necessitates a similarly rapid development of suitable antibodies or other means of enrichment. Potential alternative enrichment strategies include the use of aptamers, which offer the significant advantage of chemical synthesis onc...
Cieslak J, Pawlak P, Wodas L, Borowska A, Stachowiak A, Puppel K, Kuczynska B, Luczak M, Marczak L, Mackowski M.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...
Svala E, Jin C, Rüetschi U, Ekman S, Lindahl A, Karlsson NG, Skiöldebrand E.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...
McGorum BC, Pirie RS, Eaton SL, Keen JA, Cumyn EM, Arnott DM, Chen W, Lamont DJ, Graham LC, Llavero Hurtado M, Pemberton A, Wishart TM.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...
Peffers MJ, Thornton DJ, Clegg PD.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...
Amaya M, Keck F, Lindquist M, Voss K, Scavone L, Kehn-Hall K, Roberts B, Bailey C, Schmaljohn C, Narayanan A.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...
Arlas TR, Wolf CA, Petrucci BP, Estanislau JF, Gregory RM, Jobim MI, Mattos RC.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...
Svala E, Löfgren M, Sihlbom C, Rüetschi U, Lindahl A, Ekman S, Skiöldebrand E.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...
Peffers MJ, McDermott B, Clegg PD, Riggs CM.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 NanoAcquity™ LC coupled to an LTQ Orbitrap Velos. In order to enrich the lower-abundance protein fractions protein equalisation was first undertaken using ProteoMiner™. Progenesis-QI™ LC-MS software was used for label-free quantification. ...
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...
Du C, Liu HF, Lin YZ, Wang XF, Ma J, Li YJ, Wang X, Zhou JH.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 ...
Alghamdi AS, Madill S, Foster DN, Troedsson MH.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...
Barsuren E, Namkhai B, Kong HS.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...
Miyamoto M, Ueno HM, Watanabe M, Tatsuma Y, Seto Y, Miyamoto T, Nakajima H.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 35...
Swegen A, Curry BJ, Gibb Z, Lambourne SR, Smith ND, Aitken RJ.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 ...
Desjardin C, Riviere J, Vaiman A, Morgenthaler C, Diribarne M, Zivy M, Robert C, Le Moyec L, Wimel L, Lepage O, Jacques C, Cribiu E, Schibler L.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...
Puppione DL, Whitelegge JP, Yam LM, Bassilian S, Schumaker VN, MacDonald MH.In pigs, humans, chimpanzees and probably other great apes, a cysteine at residue 6 enables apolipoprotein A-II to form a homodimer. However, the apoA-IIs of other primates, lacking a cysteine residue, are monomeric. We have already reported that horse apoA-IIs form homodimers due also to a cysteine at residue 6. In this study, we wanted to determine whether other equine apoA-IIs might be monomeric. The high density lipoproteins were ultracentrifugally isolated from the plasmas of a horse (Equus caballus), a donkey (Equus asinus) and five wild equines: two types of zebras (Equus zebra hartmann...
Koivunen AL, Maisi P, Fang W, Sandholm M.To clarify the role of proteolytic enzymes in the pathogenesis of chronic obstructive pulmonary disease (COPD) in horses, and to investigate new possibilities for treatment of this disease by interfering in the proteolytic process. Methods: Effect of antiproteolytic activity of selected protease inhibitors on tracheal aspirates was studied in vitro, and the inhibition profiles were compared with those of purified proteases. Methods: Respiratory tract secretions with antiproteolytic activity from 9 horses with COPD. Methods: Caseinolytic agar-diffusion assay. Results: The protease-inhibition pr...
Cieslak J, Pawlak P, Wodas L, Borowska A, Stachowiak A, Puppel K, Kuczynska B, Luczak M, Marczak L, Mackowski M.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...
Onzere CK, Fry LM, Bishop RP, Silva MG, Bastos RG, Knowles DP, Suarez CE.Theileria equi is a widely distributed apicomplexan parasite that causes severe hemolytic anemia in equid species. There is currently no effective vaccine for control of the parasite and understanding the mechanism that T. equi utilizes to invade host cells may be crucial for vaccine development. Unlike most apicomplexan species studied to date, the role of micronemes in T. equi invasion of host cells is unknown. We therefore assessed the role of the T. equi claudin-like apicomplexan microneme protein (CLAMP) in the invasion of equine erythrocytes as a first step towards understanding the role...
Yu F, Chen Y, Liu B, Wang T, Ding Z, Yi Z, Zhu Y, Li J.Health monitoring is critical for newborn animals due to their vulnerability to diseases. Urine can be not only a useful and non-invasive tool (free-catch samples) to reflect the physiological status of animals but also to help monitor the progression of diseases. Proteomics involves the study of the whole complement of proteins and peptides, including structure, quantities, functions, variations and interactions. In this study, urinary proteomics of neonatal donkeys were characterized and compared to the profiles of adult donkeys to provide a reference database for healthy neonatal donkeys. T...
Gaitskell-Phillips G, Martín-Cano FE, da Silva-Álvarez E, Tapia JA, Silva A, Gil MC, Ortega-Ferrusola C, Peña FJ.Although recent research has addressed the impact of cryopreservation on the stallion sperm proteome, studies addressing the stallion sperm phosphoproteome are lacking. In the present study, the data set of proteomes of fresh and cryopreserved spermatozoa were reanalyzed, showing that cryopreservation caused significant changes in the phosphoproteome. The phosphoproteins reduced most significantly by cryopreservation were Ca2+binding tyrosine phosphorylation regulated, protein kinase cAMP-activated catalytic subunit beta (CABYR), mitochondria eating protein (SPATA18), A kinase anchoring protei...
Lawson EF, Grupen CG, Baker MA, Aitken RJ, Swegen A, Pollard CL, Gibb Z.Lipids are dynamic biological molecules that play key roles in metabolism, inflammation, cell signalling and structure. They are biologically significant in the physiology of conception and reproduction. Many of the mechanisms surrounding equine conception and the early feto-maternal dialogue are yet to be understood at a biochemical level. Recently, lipidomic technologies have advanced considerably and analytical strategies have been enhanced and diversified. Consequently, in-depth lipidomic exploration now has the potential to reveal new lipid biomarkers and biochemical relationships that im...
Amaral A, Fernandes C, Szóstek-Mioduchowska A, Rebordão MR, Skarzynski DJ, Ferreira-Dias G.Endometrosis is a reproductive pathology that is responsible for mare infertility. Our recent studies have focused on the involvement of neutrophil extracellular traps enzymes, such as elastase (ELA), in the development of equine endometrosis. Noscapine (NOSC) is an alkaloid derived from poppy opium with anticough, antistroke, anticancer, and antifibrotic properties. The present work investigates the putative inhibitory in vitro effect of NOSC on collagen type I alpha 2 chain () mRNA and COL1 protein relative abundance induced by ELA in endometrial explants of mares in the follicular or mid-lu...
Bridges TS, Davidson TR, Chamberlain CS, Geisert RD, Spicer LJ.The objective of the present study was to evaluate changes in equine follicular fluid insulin-like growth factor binding protein (IGFBP) proteolytic activity as well as steroid, IGF, and IGFBP concentrations during follicular development in the mare. Mares (n = 14) were classified as either in the follicular phase (n = 8) or luteal phase (n = 6). Follicles (n = 92) were categorized as small (6 to 15 mm; n = 54), medium (16 to 25 mm; n = 23), or large (> 25 mm; n = 15), and follicular fluid was collected. Estradiol and androstenedione levels in follicular fluid were greater (P < 0.05), an...
Tesena P, Yingchutrakul Y, Roytrakul S, Wongtawan T, Angkanaporn K.Equine Glandular Gastric Disease (EGGD) is a common disease in sport horses. This disease might be associated with usage of nonsteroidal anti-inflammatory drugs (NSAIDs) for treating inflammatory diseases. Although gastroscopy has been an effective method for diagnosis, but a less invasive, and inexpensive method is preferred. This study used proteomic technology to identify candidate serum proteins that might be used as markers of NSAIDs induced EGGD. Five Thoroughbred horses were given high doses of NSAID, phenylbutazone to treat lameness. The experiment was divided into three periods: (i) P...
Potempa J, Wunderlich JK, Travis J.Three structurally related but functionally different serpins from horse plasma were isolated and characterized. In spite of their identical N-terminal sequences, which show some similarity to that of human alpha 1-proteinase inhibitor, the reactive-centre loops of each of these proteins show extensive variation. Only inhibitor I, with a P1 methionine residue, resembles human alpha 1-PI with regard to (a) similarity of amino acid sequence in the vicinity of the reactive-site peptide bond, (b) broad inhibitory specificity, (c) sensitivity to oxidative inactivation and (d) high rate of reactivit...
Poltep K, Tesena P, Yingchutrakul Y, Taylor J, Wongtawan T.Silent gastric ulceration occurs without evidence of clinical signs and is common in horses. There is currently no a simple and effective method to diagnose this disease. Proteomics can be used to identify serum biomarkers, but the most abundant serum protein, albumin, could conceal candidate biomarkers. Therefore, it is recommended to remove albumin before a proteomic study; however, there is no specific albumin depletion kit or standard protocol available for horse samples. The objectives of this study were to optimise a protocol to remove equine serum albumin and to use albumin-depleted ser...
Barello C, Garoffo LP, Montorfano G, Zava S, Berra B, Conti A, Giuffrida MG.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...
Journal of proteomicsAugust 5, 2020
Volume 228 103927 doi: 10.1016/j.jprot.2020.103927
Chiaradia E, Pepe M, Sassi P, Mohren R, Orvietani PL, Paolantoni M, Tognoloni A, Sforna M, Eveque M, Tombolesi N, Cillero-Pastor B.Osteochondrosis is a developmental orthopedic disease affecting growing cartilage in young horses. In this study we compared the proteomes of equine chondrocytes obtained from healthy and osteochondrotic cartilage using a label-free mass spectrometry approach. Quantitative changes of some proteins selected for their involvement in different functional pathways highlighted by the bioinformatics analysis, were validated by western blotting, while biochemical alterations of extracellular matrix were confirmed via Raman spectroscopy analysis. In total 1637 proteins were identified, of which 59 wer...
Fernández-Hernández P, Marinaro F, Sánchez-Calabuig MJ, García-Marín LJ, Bragado MJ, González-Fernández L, Macías-García B.Equine fertilization cannot be performed in the laboratory as equine spermatozoa do not cross the oocyte's zona pellucida . Hence, a more profound study of equine oviductal fluid (OF) composition at the pre-ovulatory and post-ovulatory stages could help in understanding what components are required to achieve fertilization in horses. Our work aimed to elucidate the proteomic composition of equine OF at both stages. To do this, OF was obtained postmortem from oviducts of slaughtered mares ipsilateral to a pre-ovulatory follicle ( = 4) or a recent ovulation ( = 4); the samples were kept at -80°...
Szober CM, Hauck SM, Euler KN, Fröhlich KJ, Alge-Priglinger C, Ueffing M, Deeg CA.The purpose of this study was to characterize the cell surface proteome of native compared to cultured equine retinal pigment epithelium (RPE) cells. The RPE plays an essential role in visual function and represents the outer blood-retinal barrier. We are investigating immunopathomechanisms of equine recurrent uveitis, an autoimmune inflammatory disease in horses leading to breakdown of the outer blood-retinal barrier and influx of autoreactive T-cells into affected horses' vitrei. Cell surface proteins of native and cultured RPE cells from eye-healthy horses were captured by biotinylation, an...
Mendes MA, Chies JM, de Oliveira Dias AC, Filho SA, Palma MS.Most commercial recombinant proteins used as molecular biology tools, as well as many academically made preparations, are generally maintained in the presence of high glycerol concentrations after purification to maintain their biological activity. The present study shows that larger proteins containing high concentrations of glycerol are not amenable to analysis using conventional electrospray ionization mass spectrometry (ESI-MS) interfaces. In this investigation the presence of 25% (v/v) glycerol suppressed the signals of Taq DNA polymerase molecules, while 1% (v/v) glycerol suppressed the ...
Green MK, Johnston MV, Larsen BS.To elucidate the role of high mass accuracy in mass spectrometric peptide mapping and database searching, selected proteins were subjected to tryptic digestion and the resulting mixtures were analyzed by electrospray ionization on a 7 Tesla Fourier transform mass spectrometer with a mass accuracy of 1 ppm. Two extreme cases were examined in detail: equine apomyoglobin, which digested easily and gave very few spurious masses, and bovine alpha-lactalbumin, which under the conditions used, gave many spurious masses. The effectiveness of accurate mass measurements in minimizing false protein match...
Lepczyński A, Ożgo M, Dratwa-Chałupnik A, Robak P, Pyć A, Zaborski D, Herosimczyk A.The main objectives of the study were to: (1) deeply analyse the serum protein composition of Equus caballus, (2) assess the effectiveness of the high-abundant protein depletion and improve the concentration of medium- and low-abundant proteins. The analysis were performed on the blood plasma of three healthy part-Arabian mares. The implementation of two-dimensional electrophoresis and matrix-assisted laser desorption/ionisation - time of flight mass spectrometry allowed us to establish a horse plasma proteome map. Serum proteins were resolved at pH 4 to 7, followed by 12% SDS-PAGE. As a resul...
Mazerbourg S, Zapf J, Bar RS, Brigstock DR, Monget P.We recently showed that insulin-like growth factor-binding protein-4 (IGFBP-4) proteolytic degradation in ovine preovulatory ovarian follicles is IGF-dependent and regulated by the heparin-binding domain (HBD) from IGFBP-3 and from connective tissue growth factor (CTGF), heparan/heparin-interacting protein (HIP), and vitronectin. The present study investigated regulation of IGFBP-4 proteolytic degradation in porcine, bovine, and equine ovarian preovulatory follicles. Follicular fluid from such preovulatory follicles contains proteolytic activity, degrading exogenous IGFBP-4. An excess of IGF-I...
McCoy AM, Arrington J, Yau PM.The protein content of amnion is thought to be the primary contributor to its efficacy as a biological dressing for wounds. Protein elution into antibiotic processing media has been reported, but the effect of antiseptic-based processing methods is unknown. Amniotic membranes were collected from eight healthy mares. Samples were collected after removal of gross debris. Tissues were subsequently divided and processed with either 0.05% chlorhexidine or 2% iodine/0.25% acetic acid. After protein extraction and trypsin digestion, the proteins were labeled with 8-plex iTRAQ tags, combined, and anal...
Lancheros-Buitrago J, Rodriguez-Villamil P, Gregory J, Bastos H, Camacho CA, Caballeros JE, Cazales N, Barros E, José de Jesus Silva M, Pimentel A....Characterization of the uterine proteome before the entry of the conceptus to the uterus is essential to know the factors involved in the physiological events of gestation. The objective of the study was to compare proteomic profile of uterine fluid collected on day 5 post ovulation of cyclic and inseminated mares. Samples of endometrial secretion were recovered over 2 cycles during the fifth day post ovulation. The first cycle constituted the Cyclic group and in the following cycle, the same mares were inseminated and considered as the Inseminated group. All the samples were subjected to two-...
Riveroll AL, Skyba-Lewin S, Lynn KD, Mubyeyi G, Abd-El-Aziz A, Kibenge FST, Kibenge MJT, Cohen AM, Esparza-Gonsalez B, McD￮ L, Montelpare WJ.Adipose-derived stem cells (ADSCs) are used in tissue regeneration therapies. The objective of this study is to identify stable reference genes (RGs) for use in gene expression studies in a characterized equine adipose-derived mesenchymal stem cell (EADMSC) differentiation model. ADSCs were differentiated into adipocytes (ADs) or osteoblasts (OBs), and the proteomes from these cells were analyzed by liquid chromatography tandem mass spectrometry. Proteins that were stably expressed in all three cells types were identified, and the mRNA expression stabilities for their corresponding genes were ...
Pintado CO, Friend M, Llanes D.This paper describes two anti-glycoprotein IIb/IIIa or CD41/61 murine monoclonal antibodies (Co.35E4 and Co.2oA1). The cellular distribution and apparent molecular weight of the antigen detected by these antibodies is consistent with their reaction with ruminant and equine glycoprotein IIb/IIIa. Biochemical analysis of the equine molecule using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) revealed bands of 24, 100 and 110 kDa under reducing conditions and 115 and 80 kDa under nonreducing conditions. Biochemical analysis of ruminant antigen revealed that the 24 kDa band...
Liu L, Yu W, Cai K, Ma S, Wang Y, Ma Y, Zhao H. () is a zoonotic opportunistic pathogen that can cause life-threatening infections. The rapid evolution of multidrug-resistant and the fact that there is no currently licensed effective vaccine against warrant the need for vaccine development. Reverse vaccinology (RV), which involves screening a pathogen's entire genome and proteome using various web-based prediction tools, is considered one of the most effective approaches for identifying vaccine candidates. Here, we performed a pangenome analysis to determine the core proteins of . We then used the RV approach to examine the subcellular l...
Araujo-Viel MS, Juliano L, Prado ES.The synthetic tridecapeptide Gly-Leu-Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg was used as a model substrate for horse urinary and porcine pancreatic kallikreins. The Met-Lys bond is hydrolyzed selectively by both enzymes. Oxidation of the methionine residue to sulfoxide made the peptide resistant to both kallikreins. Substitution of either the methionine or lysine residues by norleucine led to peptides in which the Nle-Lys or the Met-Nle bonds, respectively, were susceptible to the urinary kallikrein. The esterolytic and Met-Lys bond-splitting activities of both enzymes were inhibited simil...
Roelfstra L, Deeg CA, Hauck SM, Buse C, Membrez M, Betschart B, Pfister K.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...
Zhang XJ, Li HL, Deng DY, Ji C, Yao XD, Liu JX.Tetanus is still a major cause of human deaths in several developing countries. In particular, the neonatal form remains a significant public health problem. According to the World Health Organization, administration of tetanus toxoid is recommended for neonatal tetanus patients. Furthermore, tetanus antitoxin or anti-tetanus immunoglobulin (Ig) are used for mild case or intensive care. This paper discusses a novel purification technique for improving equine anti-tetanus Ig production. First, equine plasma dealt with two steps salting out with ammonium sulfate; second, ultrafiltration concentr...
Pemberton AD, Miller HR, John HA, Scudamore CL.1. Antiproteinase deficiency can result in excessive proteinase-induced tissue damage. The major anti-elastase (Spi1) protein of equine alpha 1-proteinase inhibitor (alpha 1-PI) was isolated from the plasma/serum of three common haplotypes (I, L and U). 2. The N-terminal amino acid sequences of the three inhibitors were identical, but were only approx 65-77% homologous with two other published equine Spi1 sequences. 3. All three inhibitors complexed quickly and irreversibly with equine leucocyte proteinase 2A (kass = 2 x 10(7) M-1 sec-1). They were also efficient inhibitors of chymase (rat mas...
Hernández-Avilés C, Ramírez-Agámez L, Weintraub ST, Scoggin CF, Davis BW, Raudsepp T, Varner DD, Love CC.Thoroughbred stallions that carry a double-homozygous genotype A/A-A/A for SNPs rs397316122 and rs69101140 in exon 5 of the FKBP6 gene (chr13; EquCab3.0) are uniquely subfertile due to impaired acrosomal exocytosis (IAE). In this study, the sperm proteome in frozen/thawed semen from subfertile Thoroughbred stallions was studied and compared to that of frozen/thawed sperm from fertile Thoroughbred stallions. A total of 2,220 proteins was identified, of which 140 proteins were found to be differentially abundant in sperm from the subfertile stallions compared to that of fertile stallions (83 les...
