Two-dimensional Gel Electrophoresis Research Articles

Synovial Fluid Proteomics and Serum Metabolomics Reveal Molecular and Metabolic Changes in Osteoarthritis

Background: Osteoarthritis (OA) is a common joint disorder with a complex and multifactorial pathogenesis. Proteomics analysis using two-dimensional gel electrophoresis (2DE) and mass spectrometry (MS) enables high-throughput identification of differentially expressed proteins related to OA. However, the etiology, pathophysiology, and early diagnostic markers of OA are still poorly understood. Methods: Synovial fluid protein biomarkers were compared between OA patients and healthy controls. It was fractionated using DEAE cellulose and Sephadex G-200 columns, followed by SDS‒PAGE and 2D-PAGE for visualization and identification. Mass spectrometry and Mascot were used for protein analysis, and serum metabolite profiles were also investigated using 1D 1H CPMG NMR spectra. Multivariate data analysis, including PCA and PLS-DA, was performed to detect metabolic differences between groups. Results: Proteomics analysis revealed differential expression of synovial fluid proteins, such as serine protease inhibitors, complement components, and apolipoproteins, which may be involved in inflammation and cartilage breakdown. Additionally, serum metabolite profiles differed significantly between OA patients and controls, involving amino acid, lipid, glucose, and energy metabolism. The pathway analysis indicated disruption of the metabolic pathways associated with these metabolites. Conclusions: This study provides insights into the molecular and metabolic changes in OA. Protein biomarkers and serum metabolite alterations enhance the understanding of OA pathogenesis and offer potential opportunities for early diagnosis and disease management. Further validation and translation of these findings into clinical applications are needed for improved OA detection and intervention strategies.

Proteomic analysis of host cell protein fouling during bioreactor harvesting.

Chinese hamster ovary (CHO) cells are among the most common cell lines used for therapeutic protein production. Membrane fouling during bioreactor harvesting is a major limitation for the downstream purification of therapeutic proteins. Host cell proteins (HCP) are the most challenging impurities during downstream purification processes. The present work focuses on identification of HCP foulants during CHO bioreactor harvesting using reverse asymmetrical commercial membrane BioOptimal™ MF-SL. In order to investigate foulants and fouling behavior during cell clarification, for the first time a novel backwash process was developed to effectively elute almost all the HCP and DNA from the fouled membrane filter. The isoelectric points (pIs) and molecular weights (MWs) of major HCP in the bioreactor harvest and fouled on the membrane were successfully characterized using two-dimensional gel electrophoresis (2D SDS-PAGE). In addition, a total of 8 HCP were identified using matrix-assisted laser desorption/ionization-mass spectroscopy (MALDI-MS). The majority of these HCP are enzymes or associated with exosomes, both of which can form submicron-sized particles which could lead to the plugging of the filters.

Proteomic Analysis and Immunoprofiling of Persian Horned Viper Venom, Pseudocerastes Persicus, from Central Part of Iran.

Numerous species of venomous snakes of medical importance exist in Iran. Pseudocerastes persicus (P. persicus), one of the medically important snakes, also called the Persian horned viper, has a geographical spread that extends to the east, southwest, and central areas of Iran and is endemic across the wider region. As a result, this species is responsible for many snakebite occurrences. Venom from P. persicus found in the central province of Semnan contains phospholipase A2 and L-amino acid oxidase activities, and high toxic potency. The venom was fractionated by reverse-phase high-performance liquid chromatography (HPLC) and analyzed by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and two-dimensional electrophoresis. Using liquid chromatography with tandem mass spectrometry (LC-MS/MS), a range of components were identified, consistent with the biochemical and toxicological properties of the venom. Proteins identified from 2D electrophoresis and shotgun methods included metallo- and serine proteases, phospholipases, oxidases, and Kunitz trypsin inhibitors, along with many other components at lower qualitative abundance. This study provides a more detailed understanding of the protein profile of Iranian P. persicus venom, which can be effective in the production of an effective antidote against it. The analysis of the resulting data shows that there is a wide range of proteins in the venom of the Persian horned viper. This information can provide a better understanding of how venom is neutralized by polyclonal antivenom. Considering the wide presence of this snake and its related species in Iran and surrounding countries, knowing the venom protein profile of this family can be of great support to antivenom producers such as Razi Vaccine & Serum Research Institute in the preparation of regional antivenoms.

Comparative hemolymph proteomic analyses of the freezing and resistance-freezing Ostrinia furnacalis (Guenée)

The Asian corn borer, Ostrinia furnacalis (Guenée) (Lepidoptera: Crambidae), is one of the most harmful pests of maize in Asia. It poses a significant threat to maize production, causing economic losses due to its strong ecological adaptation. In this study, we compared and analyzed the hemolymph proteome between freezing and resistance-freezing O. furnacalis strains using two-dimensional gel electrophoresis to gain insights into the mechanisms of cold resistance. The results revealed that 300–400 hemolymph protein spots were common, with 24 spots showing differences between the two strains. Spectrometry analysis revealed 21 protein spots, including 17 upregulated spots and 4 downregulated ones. The expression of upregulation/downregulation proteins plays a crucial role in the metabolism, energy supply, and defense reaction of insects. Proteomics research not only provides a method for investigating protein expression patterns but also identifies numerous attractive candidates for further exploration.

Comparative proteomics reveals different protein expression in platelets in patients with alcoholic liver cirrhosis

BackgroundThe role of platelets in disease progression as well as the function of platelets as part of the haemostatic and immunological system in patients with liver cirrhosis is only incompletely understood. This is partly due to difficulties in assessing platelet function. Proteome analyses of platelets have been used to further investigate the role of platelets in other diseases.AimTo assess possible changes in the platelet proteome during different stages of alcohol induced liver cirrhosis compared to healthy donors.Patients and methodsA 45 ml blood sample was drawn from 18 participants aged 18–80 years evenly divided into three groups of healthy donors, patients with less advanced alcohol induced liver cirrhosis (Child-Pugh < 7) and patients with advanced liver cirrhosis (Child-Pugh > 10). The blood was processed to isolate platelets and perform subsequent two-dimensional gel-electrophoresis using a SYPRO™ Ruby dye. After computational analysation significantly in- or decreased protein spots (defined as a two-fold abundance change between different study cohorts and ANOVA < 0.05) were identified via liquid chromatography–mass spectrometry (LCMS) and searching against human protein databases.ResultsThe comparative analysis identified four platelet proteins with progressively decreased protein expression in patients with liver cirrhosis. More specifically Ras-related protein Rab-7a (Rab-7a), Ran-specific binding protein 1 (RANBP1), Rho GDP-dissociation inhibitor 1 (RhoGDI1), and 14–3-3 gamma.ConclusionThere is significant change in protein expression in the platelet proteome throughout the disease progression of alcohol induced liver cirrhosis. The identified proteins are possibly involved in haemostatic and immunoregulatory function of platelets.

Nematode egg parasitic fungus, Purpureocillium lilacinum: efficacy of indigenous strains for the management of Meloidogyne incognita in chickpea

BackgroundNematode egg parasitic fungus, Purpureocillium lilacinum is the most effective biocontrol agent and has been widely used commercially in many countries for the management of root-knot nematode, Meloidogyne incognita. Availability of indigenous potential strains specific to an agro ecosystem is very crucial for their successful commercial exploitation for suppression of nematode population. Hence, an attempt was made to isolate, characterize, evaluate and identify potential indigenous strains of P. lilacinum from pulse ecosystem for root-knot nematode management in chickpea.ResultsThe fungal colony was initially white and when spore was formed it turned into pink colour in 72 to 96 h. Hyphae was hyaline and septate, conidiophore was blunt, and phialides were with wide base and long neck bearing round to oval conidia in chains. Molecular identification of the species, P. lilacinum was carried out based on ITS1-5.8S-ITS2 region of the genomic DNA. In vitro bioassay of cultural filtrates on juvenile mortality revealed that maximum percentage of mortality was observed in IIPR-Pl-11 (88.36%). Spectrophotometric assay on chitinolytic activity showed that the strain IIPR-Pl-11 produced significantly high chitinolytic activity, chitinase enzyme and total protein content (0.139, 51.1 and173.75 µg/ml at 5days); (0.245, 90.1 and 272.67 µg/ml at 10 days) and (0.273, 100.4 and 306.25 µg/ml), respectively, at 15 days of culturing in colloidal chitin-enriched medium C. 2D gel electrophoresis of the crude chitinase suspension showed the presence of chitinase (32, 46 kDa size) in the sample from chitinase-induced medium C. In vitro bioassay of the cultural filtrates of the fungus grown in chitin-enriched medium C on inhibition on egg hatching revealed that the highest percent inhibition on egg hatching showed by IIPR-Pl-8 strain at 5 days of inoculation (42.6%) and IIPR-Pl-11 at 10 and 20 days of inoculation (62.80 and 93.50%), respectively. In vivo pot experiment revealed that among all strains, IIPR-Pl-11 was efficient in promoting plant growth very effectively by reducing gall number (41.3 per plant), egg mass (28.3 per plant) and soil population (284.3 per 200cc of soil) compared to control.ConclusionP. lilacinum strain IIPR-Pl-11 was the highest potential strain from pulse rhizosphere for the management of root-knot nematode, M. incognita in chickpea.

Molecular Insights into the Breast and Prostate Cancer Cells in Response to the Change of Extracellular Zinc.

Zinc dyshomeostasis is manifested in breast and prostate cancer cells. This study attempted to uncover the molecular details prodded by the change of extracellular zinc by employing a panel of normal and cancerous breast and prostate cell lines coupled with the top-down proteomics with two-dimensional gel electrophoresis followed by liquid chromatography-tandem mass spectrometry. The protein samples were generated from MCF-7 breast cancer cells, MCF10A normal breast cells, PC3 prostate cancer cells, and RWPE-1 normal prostate cells with or without exogenous zinc exposure in a time course (T0 and T120). By comparing the cancer cells vs respective normal epithelial cells without zinc treatment (T0), differentially expressed proteins (23 upregulated and 18 downregulated in MCF-7 cells; 14 upregulated and 30 downregulated in PC3 cells) were identified, which provides insights into the intrinsic differences of breast and prostate cancer cells. The dynamic protein landscapes in the cancer cells prodded by the extracellular zinc treatment reveal the potential roles of the identified zinc-responsive proteins (e.g., triosephosphate isomerase, S100A13, tumour proteins hD53 and hD54, and tumour suppressor prohibitin) in breast and prostate cancers. This study, for the first time, simultaneously investigated the two kinds of cancer cells related to zinc dyshomeostasis, and the findings shed light on the molecular understanding of the breast and prostate cancer cells in response to extracellular zinc variation.

Two-Dimensional Gel Electrophoresis in Studies of Flower and Leaf Proteome of Common Buckwheat.

Two-dimensional gel electrophoresis (2-DE) is a proteomic tool used for the separation of protein mixtures according to protein isoelectric point and molecular mass. Although gel-free quantitative and qualitative proteomic study techniques are now available, 2-DE remains a useful analytical tool. The presented protocol was performed to analyze the flower and leaf proteome of common buckwheat using 24cm immobilized pH gradient strips (pH4-7) and visualization of proteins on gels via colloidal Coomassie G-250 staining.

Applications of Proteomics in Probiotics Having Anticancer and Chemopreventive Properties.

Proteomics has grown in importance in molecular sciences because it gives vital information on protein identification, expression levels, and alteration. Cancer is one of the world's major causes of death and is the major focus of much research. Cancer risk is determined by hereditary variables as well as the body's immunological condition. Probiotics have increasing medical importance due to their therapeutic influence on the human body in the prevention and treatment of numerous chronic illnesses, including cancer, with no adverse effects. Several anticancer, anti-inflammatory, and chemopreventive probiotics are studied using different proteomic approaches like two-dimensional gel electrophoresis, liquid chromatography-mass spectrometry, and matrix-assisted laser desorption/ionization mass spectrometry. To gain relevant information about probiotic characteristics, data from the proteomic analysis are evaluated and processed using bioinformatics pipelines. Proteomic studies showed the significance of different proteomic approaches in characterization, comparing strains, and determination of oxidative stress of different probiotics. Moreover, proteomic approaches identified different proteins that are involved in glucose metabolism and the formation of cell walls or cell membranes, and the differences in the expression of critical enzymes in the HIF-1 signaling pathway, starch, and sucrose metabolism, and other critical metabolic pathways.

Inflachromene ameliorates Parkinson's disease by targeting Nrf2-binding Keap1.

Parkinson's disease (PD) is the most common neurodegenerative disease characterized by movement disorder. Despite current therapeutic efforts, PD progression and the loss of dopaminergic neurons in the substantia nigra remain challenging to prevent due to the complex and unclear molecular mechanism involved. We adopted a phenotype-based drug screening approach with neuronal cells to overcome these limitations. In this study, we successfully identified a small molecule with a promising therapeutic effect for PD treatment, called inflachromene (ICM), through our phenotypic screening strategy. Subsequent target identification using fluorescence difference in two-dimensional gel electrophoresis (FITGE) revealed that ICM ameliorates PD by targeting a specific form of Keap1. This interaction led to upregulating various antioxidants, including HO-1, NQO1, and glutathione, ultimately alleviating PD symptoms. Furthermore, ICM exhibited remarkable efficacy in inhibiting the loss of dopaminergic neurons and the activation of astrocytes and microglia, which are critical factors in PD pathology. Our findings suggest that the phenotypic approach employed in this study identified that ICM has potential for PD treatment, offering new hope for more effective therapeutic interventions in the future.

Improved methods for total and chloroplast protein extraction from Cajanus species for two-dimensional gel electrophoresis and mass spectrometry.

The recent advances in pigeon pea genomics, including high-quality whole genome and chloroplast genome sequence information helped develop improved varieties. However, a comprehensive Cajanus proteome, including the organelle proteome, is yet to be fully mapped. The spatial delineation of pigeon pea proteins at sub-cellular levels and inter-organelle communication could offer valuable insights into its defense mechanism against various stresses. However, the major bottleneck in the proteomic study is the lack of a suitable method of protein extraction and sample preparation compatible with two-dimensional gel electrophoresis (2D-PAGE), liquid chromatography-mass spectrometry (LCMS), or matrix-assisted laser desorption ionization-time of flight (MALDi-ToF). Our study introduces two efficient methods, one for isolating total proteins and another for organelle (chloroplast) proteins from various Cajanus spp. For total protein extraction, we have optimized a protocol using phenol in combination with a reducing agent (DTT) and protease inhibitor cocktail, also washing (6-7 times) with ice-cold acetone after overnight protein precipitation of total proteins. Our modified extraction method using phenol for total leaf protein yielded approximately 2-fold more proteins than the previously reported protocols from C. cajan (3.18 ± 0.11 mg/gm) and C. scarabaeoides (2.06 ± 0.08 mg/gm). We have also optimized a protocol for plastid protein extraction, which yielded 1.33 ± 0.25 mg/10 gm plastid proteins from C. cajan and 0.88 ± 0.19 mg/10 gm plastid proteins from C. scarabaeoides. The 2D-PAGE analysis revealed 678 ± 08 reproducible total protein spots from C. cajan and 597 ± 22 protein spots from C. scarabaeoides. Similarly, we found 566 ± 10 and 486 ± 14 reproducible chloroplast protein spots in C. cajan and C. scarabaeoides, respectively. We confirmed the plastid protein fractions through immunoblot analysis using antibodies against LHCb1/LHCⅡ type Ⅰ protein. We found both methods suitable for 2D-PAGE and mass spectrometry (MS). This is the first report on developing protocols for total and chloroplastic protein extraction of Cajanus spp. suitable for advanced proteomics research.

Anti-filarial efficacy of Centratherum anthelminticum: unravelling the underlying mechanisms through biochemical, HRAMS proteomics and MD simulation approaches.

Traditionally, Centratherum anthelminticum (CA) has been reported to be a potent anti-filarial, however no reports are available detailing its mechanism of action against filarial parasites. In this study, we have evaluated the anti-filarial activity of CA against lymphatic filarial parasites Setaria cervi using ex vivo biochemical, proteomics and in silico approaches. The motility and viability of the parasites decreased significantly after treatment with CA concentrations of ≥125 μg mL-1. An increase in lipid peroxidation (51.92%), protein carbonylation (48.99%), NADPH oxidase (88.88%) activity and decrease in the glutathione (GSH) (-39.23%), glutathione reductase (GR) (-60.17%), and glutathione S-transferase (GST) (-50.48%) activity was also observed after CA treatment. The proteomics analysis was performed by two-dimensional gel electrophoresis and high-resolution accurate mass spectrometry (HRAMS). In total, 185 proteins were differentially expressed (DEPs) following CA treatment. The major DEPs were mostly involved in tRNA processing, biosynthetic processes, metabolic activities, protein transport, the tricarboxylic acid cycle, protein translation, and stress response. The UPLC-ESI-MS/MS analysis of CA extract revealed the presence of 40 bioactive compounds. Further the docking analysis showed 10 CA bioactive compounds to have high binding affinity towards antioxidant proteins of filarial parasites. Additionally, MD simulation studies showed stable interactions (RMSF ≤ 10 Å) of 3-O-methylquercitin, quinic acid, gentisic acid, and vanillin with filarial antioxidant enzymes/proteins. To our knowledge, this is the first report detailing the molecular mechanism of anti-filarial activity of CA, which can be further evaluated for the development of new anti-filarial formulations.

Multi-omics Investigations in Endocrine Systems and Their Clinical Implications.

Innovative techniques such as the "omics" can be a powerful tool for the understanding of intracellular pathways involved in homeostasis maintenance and identification of new potential therapeutic targets against endocrine-metabolic disorders. Over the last decades, proteomics has been extensively applied in the study of a wide variety of human diseases, including those involving the endocrine system. Among the most endocrine-related disorders investigated by proteomics in humans are diabetes mellitus and thyroid, pituitary, and reproductive system disorders. In diabetes, proteins implicated in insulin signaling, glucose metabolism, and β-cell activity have been investigated. In thyroid diseases, protein expression alterations were described in thyroid malignancies and autoimmune thyroid illnesses. Additionally, proteomics has been used to investigate the variations in protein expression in adrenal cancers and conditions, including Cushing's syndrome and Addison's disease. Pituitary tumors and disorders including acromegaly and hypopituitarism have been studied using proteomics to examine changes in protein expression. Reproductive problems such as polycystic ovarian syndrome and endometriosis are two examples of conditions where alterations in protein expression have been studied using proteomics. Proteomics has, in general, shed light on the molecular underpinnings of many endocrine-related illnesses and revealed promising biomarkers for both their detection and treatment. The capacity of proteomics to thoroughly and objectively examine complex protein mixtures is one of its main benefits. Mass spectrometry (MS) is a widely used method that identifies and measures proteins based on their mass-to-charge ratio and their fragmentation pattern. MS can perform the separation of proteins according to their physicochemical characteristics, such as hydrophobicity, charge, and size, in combination with liquid chromatography. Other proteomics techniques include protein arrays, which enable the simultaneous identification of several proteins in a single assay, and two-dimensional gel electrophoresis (2D-DIGE), which divides proteins depending on their isoelectric point and molecular weight. This chapter aims to summarize the most relevant proteomics data from targeted tissues, as well as the daily rhythmic variation of relevant biomarkers in both physiological and pathophysiological conditions within the involved endocrine system, especially because the actual modern lifestyle constantly imposes a chronic unentrained condition, which virtually affects all the circadian clock systems within human's body, being also correlated with innumerous endocrine-metabolic diseases.

Differential Protein Expression between the Motor and Sensory Fascicles in Rat Femoral Nerve Injury.

It is important to distinguish between motor and sensory fascicles of the peripheral nerves for nerve alignment in surgery. However, there are no biomarkers currently available for effective identification of motor or sensory fascicles. The objective of this study was to identify differentially expressed proteins between motor and sensory fascicles of rats in response to injury. The study was carried out using a rat femoral nerve injury model. A proteomic analysis was performed to detect differential protein expression using samples of bilateral motor and sensory branches of intact and injured rat femoral nerves through fluorescent two-dimensional difference gel electrophoresis and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Chi-square tests and t-tests were performed for comparison between motor or sensory nerve groups. The data identified six proteins that were differentially expressed between motor and sensory fascicles (>1.5-fold, P < 0.05), including apolipoprotein E, neurofilament light polypepticle, TEC kinase, serine protease inhibitor A3N, peroxiredoxin-2, and TPM1. The proteomic results were consistent with the mRNA expression levels of these genes as determined by quantitative reverse transcription polymerase chain reaction. Our study suggests that these proteins may play roles in nerve regeneration and repair. Importantly, apolipoprotein E and Serpina3n may serve as specific biomarkers for distinguishing motor and sensory fascicles of the peripheral nerves for nerve alignment in surgery.

Electrophoretic Mobility Assay to Separate Supercoiled, Catenated, and Knotted DNA Molecules.

Two-dimensional (2D) agarose gel electrophoresis is the method of choice to analyze DNA topology. The possibility to use E. coli strains with different genetic backgrounds in combination with nicking enzymes and different concentrations of norfloxacin improves the resolution of 2D gels to study the electrophoretic behavior of three different families of DNA topoisomers: supercoiled DNA molecules, post-replicative catenanes, and knotted DNA molecules. Here, we describe the materials and procedures required to optimize their separation by 2D gels. Understanding the differences in their electrophoretic behavior can help explain some important physical characteristics of these different types of DNA topoisomers. Key features • Preparative method to enrich DNA samples of supercoiled, catenated, and knotted families of topoisomers, later analyzed by 2D gels (or other techniques, e.g., microscopy). • 2D gels facilitate the separation of the topoisomers of any given circular DNA molecule. • Separation of DNA molecules with the same molecular masses but different shapes can be optimized by modifying the conditions of 2D gels. • Evaluating the roles of electric field and agarose concentration on the electrophoretic mobility of DNA topoisomers sheds light on their physical characteristics.

Two-Dimensional Gel Electrophoresis for Protein Separation of Plant Samples.

This chapter provides a description of the procedure for two-dimensional electrophoresis that can be performed for any given gel size and isoelectric focusing range. This will enable the operator to recognize critical steps and gain sufficient information to generate 2D images suitable for computer-assisted analysis of 2D-gel, as well as mass spectrometry analysis for protein identification and characterization.

Proteomic Analysis of Gingival Crevicular Fluid During Tooth Eruption

Objective: Gingival crevicular fluid (GCF) is a biological fluid that has the unique capacity to reflect changes in periodontium to its protein composition, making it ideal for potential biomarkers. There is limited information about the mechanism of tooth eruption, for which GCF might provide valuable knowledge. This study aimed to provide a proteomic approach to investigate the composition of GCF obtained from two different supraosseous tooth eruption stages of permanent molars changes.&#x0D; Methods: GCF samples were taken from a total of 26 healthy children, whose permanent molar just emerged from the gingiva (5-8 years old) and the occlusal equilibrium stage (9-13 years old). Proteins were extracted with Bio-Rad Rehydration Buffer followed by ZebaTM Spin Desalting Column. GCF samples were separated with two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) followed by mass spectrometry- based protein identification.&#x0D; Results: A new optimized protocol with enriched protein extraction from GCF samples was developed. Several proteins related to tooth eruption were detected. It was determined that keratin type II cytoskeletal 4 and keratin type I cytoskeletal 9 decreased and albumin increased in GCF protein content in erupting teeth compared to those in the occlusal equilibrium phase.&#x0D; Conclusion: This methodology, which we have applied for 2D-PAGE of GCF, can also be a source for other studies. There is huge diagnostic potential in mass spectrometry technologies, this study can be carried forward by using other approaches.

The evolutionary loss of the Eh1 motif in FoxE1 in the lineage of placental mammals.

Forkhead box E1 (FoxE1) protein is a transcriptional regulator known to play a major role in the development of the thyroid gland. By performing sequence alignments, we detected a deletion in FoxE1, which occurred in the evolution of mammals, near the point of divergence of placental mammals. This deletion led to the loss of the majority of the Eh1 motif, which was important for interactions with transcriptional corepressors. To investigate a potential mechanism for this deletion, we analyzed replication through the deletion area in mammalian cells with two-dimensional gel electrophoresis, and in vitro, using a primer extension reaction. We demonstrated that the area of the deletion presented an obstacle for replication in both assays. The exact position of polymerization arrest in primer extension indicated that it was most likely caused by a quadruplex DNA structure. The quadruplex structure hypothesis is also consistent with the exact borders of the deletion. The exact roles of these evolutionary changes in FoxE1 family proteins are still to be determined.

Changes in the saliva proteome analysed by gel-proteomics in horses diagnosed with equine gastric ulcer syndrome (EGUS) at diagnosis and after successful treatment

Equine gastric ulcer syndrome (EGUS) is currently one of the more frequent diseases in horses. We aimed to identify changes in the salivary proteome in horses with EGUS at diagnosis and after successful treatment by using gel proteomics. Saliva samples were collected from nine horses with EGUS before and after treatment and nine matched healthy controls. SDS-PAGE (1DE) and two-dimensional gel electrophoresis (2DE) were performed, and significantly different protein bands and spots were identified by mass spectrometry. Horses with EGUS had increases in proteins such as adenosine deaminase (ADA), triosephosphate isomerase, keratins and immuno-globulin heavy constant mu and decreases in carbonic anhydrase (CA), albumin and prolactin-induced protein. These changes would indicate various physiopathological mechanisms involved in this disease, such as the activation of the immune system, decreased stomach defence mechanisms and inflammation. The treated horses presented lower expression levels of thioredoxin (TRX) after a successful treatment, in proteomics analysis and also measured with a commercially available ELISA kit. Overall, horses with EGUS have protein changes in their saliva when measured with gel proteomics compared with healthy horses, and they also showed changes after successful treatment. These proteins could be potential biomarkers for detection and monitoring treatment response in EGUS.

Immunoproteomics reveal increased serum IgG3/5 binding to Dermatophagoides and yeast protein antigens in severe equine asthma in a preliminary study.

Severe equine asthma (SEA) is a common, chronic respiratory disease of horses characterized by hyperreactivity to hay dust which has many similarities to severe neutrophilic asthma in humans. SEA-provoking antigens have not been comprehensively characterized, but molds and mites have been suggested as relevant sources. Here, we identified relevant antigen candidates using immunoproteomics with IgG isotype-binding analyses. Proteins from Dermatophagoides pteronyssinus (Der p) were separated by two-dimensional gel electrophoresis followed by immunoblotting (2D immunoblots) resulting in a characteristic pattern of 440 spots. After serum incubation, antibody (Ig)-binding of all Ig (Pan-Ig) and IgG isotypes (type-2-associated IgG3/5, type-1-associated IgG4/7) was quantified per each spot and compared between asthmatic and healthy horses' sera (n=5 per group). Ig binding differences were detected in 30 spots. Pan-Ig binding was higher with asthmatics compared to healthy horses' sera on four spots, and IgG3/5 binding was higher on 18 spots. Small IgG4/7 binding differences were detected on 10 spots with higher binding with asthmatics' sera on four but higher binding with healthy horses' sera on six spots. Proteins from the spots with group differences including mite and yeast proteins were identified by liquid chromatography mass spectrometry. The latter likely originated from the feeding substrate of the Der p culture. Prioritized antigen candidates amongst the proteins identified were Der p 1, Der p 11, group 15 allergens, myosin heavy chain, and uncharacterized Der p proteins. Additionally, yeast enolases, alcohol dehydrogenase (ADH), phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase, and heat shock proteins were prioritized. Eleven antigen candidates were tested for confirmation by ELISAs using the respective proteins separately. Differences in asthmatics vs. healthy horses' serum Ig binding to Der p 1, Der p 18, and three yeast enzymes (enolase, ADH, and PGK) confirmed these as promising antigens of immune responses in SEA. Antigens with relevance in SEA were newly identified by immunoproteomics, and yeast antigens were considered for SEA for the first time. Serum IgG3/5 binding to relevant antigens was increased in SEA and is a novel feature that points to increased type-2 responses in SEA but requires confirmation of the corresponding cellular responses.