Project description:Collagen type II-induced arthritis (CIA) is an autoimmune disease that is accompanied by a complex host systemic response, which includes inflammatory and autoimmune reactions. Since to develop CIA is required the injection of antigen in complete Freund’s adjuvant (CFA), which is a water-in-mineral-oil emulsion containing killed Mycobacteria, systemic response proteins related with inflammation can confound the detection or diagnosis of arthritis. CIA in CD38 deficient mice (CD38 KO) is milder than that in C57BL/6 (B6 WT) mice. Protein extracts from spleen were subjected to 2D-DiGE and MS-MALDI-TOF/TOF analysis to identify proteins that were differentially expressed in CD38 KO and B6 WT mice with arthritis, with inflammation, or non-immunized. Differential protein expression was validated by ELISA, or Western-blotting.
Project description:Collagen-type-II-induced arthritis (CIA) is an autoimmune disease, which involves a complex host systemic response including inflammatory and autoimmune reactions. CIA in C57BL/6 mice resembles human rheumatoid arthritis (RA) in terms of its disease course, histological findings, and in its response to commonly used anti-arthritic drugs. In this study, the goal has been to identify proteins from serum that change in their abundance in CD38-KO versus WT mice which may reflect their distinct response to an antigen-challenge that induces the development of an autoimmune disease. CIA is milder in CD38-/- than in Wild-type (WT) mice. We analyzed the sera from CD38-/- versus WT mice either with arthritis (CIA+), with no arthritis (CIA-), or with inflammation (Complete Freund’s adjuvant (CFA)-treated mice). To decrease the dynamic concentration range of serum a combinatorial ligand library composed of hexapeptides was used (called ProteoMiner). ProteoMiner-equalized serum samples were then subjected to 2D-DiGE and MS-MALDI-TOF/TOF analyses to identify proteins that changed in their relative abundances. Multivariate analyses revealed that a multi-protein signature (n = 28) was able to discriminate CIA+ from CIA- mice, and WT from CD38-/- mice within each condition. Likewise, a distinct multi-protein signature (n = 16) was identified which differentiated CIA+ CD38-/- mice from CIA+ WT mice, and lastly, a third multi-protein signature (n = 18) indicated that CD38-/- and WT mice could be segregated in response to CFA treatment This approach allows the identification of multiple protein species, or proteoforms of a given protein in a single analysis, and therefore, to focus the interest in fully characterize just the protein species that differ in abundance.
Project description:Trees establish a symbiotic relationship with specialized soil fungi, called ectomycorrhizae, which is essential for nutrition, growth and health of temperate forest ecosystems. Understanding the mechanisms governing the establishment and functioning of ectomycorrhiza is important because of the role of forests in sequestering CO2 and also to develop ways to optimize tree productivity and sustainability. Here, we investigated the response of an oak species to ectomycorrhiza formation using a two dimensional differential in gel electrophoresis (2D-DIGE) and MALDI-TOF/TOF mass spectrometry proteomics approach. At the root level, changes in the abundance of 34 unique oak proteins were detected and revealed proteins involved in carbon and energy metabolism, protein processing and degradation, response to oxidative stress, lipid metabolism/transport, nitrogen and phosphorous assimilation and cell wall modification. Proteins supporting the importance of the secretory pathway functioning, in particular of the endoplasmic reticulum, during ectomycorrhiza functioning were identified. These proteins were identified as components of the endoplasmic reticulum folding/chaperoning machinery and proteins involved in the ER quality control system. This study constitutes an important contribution for the understanding of the mechanisms underlying the response of plants to ectomycorrhizal symbiosis establishment.
Project description:This study was aimed to elucidate a global antigenic profile of Mycoplasma bovis (M. bovis) with immunoproteomics, immunoinformatics, and gene expression approaches. The extracts of whole-cell proteins and TX-114 membrane fraction of a Chinese strain M. bovis HB0801 were separated with two dimensional gel electrophoresis (2-DE) and proteins reacting with antisera to M. bovis from experimentally infected calves were detected by MALDI-TOF MS.
Project description:Polyandrous ant queens are inseminated early in their life and store sperm mixtures for a potential reproductive life of decades. However, they cannot re-mate later in life and are thus expected to control the loss of viable sperm because their life-time reproductive success is ultimately limited by the viability of sperm obtained in their single reproductive event. In the leaf-cutting ant Atta colombica, we have previously shown that sperm survival is lowered when getting in contact with seminal fluid of other males, and remains stable when in contact with secretions of the queen sperm storage organ (spermatheca). Here we aim to resolve the main protein-level interactions that mediate sperm competition dynamics and sperm preservation. We used artificial insemination and DIGE-based proteomics to identify proteomic changes when seminal fluid is exposed to spermathecal fluid. Seminal fluid was collected from mature males during field season experiments in Panama, by gently squeezing males’ abdomen until the ejaculate came out. Ejaculates from several males were kept together in ice and then centrifuged for 10 minutes at 13,500 g, the supernatant collected in a separate tube and then centrifuged again. The resulting supernatant was then transferred to another tube and stored at -20° C until further use. Four biological replicates of SF were collected from 4 colonies, each consisting of 400 µl SF from approximately 350 males. From each biological replicate three 100 µl aliquots were retrieved, and assigned to one of the following treatments: (1) un-inseminated controls, (2) inseminated SF retrieved after 30 min, and (3) inseminated SF retrieved after 12 h (Fig 1). Next, each of those aliquots was used to artificially inseminate 10 virgin queens (10 µl of aliquot per queen), for a total of 80 queens inseminated. Queens were allowed to recover for 30 min or 12 h before dissecting them to obtain the spermathecal content. All these spermathecal contents were then pooled per treatment and replicate, resulting in a total of 12 samples (3 treatments – un-inseminated SF, inseminated SF retrieved after 30 min inside the queen, and inseminated SF retrieved after 12 hours inside the queen - with 4 biological replicates). Proteins were precipitated in each sample by adding 4 volumes of ice-cold acetone for 4 hours at -20°C and centrifuged at 14,000 g for 10 minutes, after which supernatants were discarded and the protein pellets were stored at -80°C until further use. All of those 12 samples entered the DIGE analysis.
Project description:Initiation of adipocyte differentiation is promoted by the synergistic action of insulin/insulin-like growth factor, glucocorticoids, and agents activating cAMP-dependent signaling. The action of cAMP is mediated via PKA and Epac, where at least part of the PKA function relates to strong repression of Rho kinase activity, whereas Epac counteracts the reduction in insulin/insulin-like growth factor signaling associated with complete repression of Rho kinase activity. However, detailed knowledge of the Epac-dependent branch and the interplay with PKA is still limited. In the present study, we present a comprehensive evaluation of Epac-mediated processes and their interplay with PKA during the initiation of 3T3-L1 preadipocyte differentiation using a combination of proteomics, molecular approaches and bioinformatics. Proteomic analyses revealed 7 proteins specifically regulated in response to Epac activation, 4 in response to PKA activation, and 11 in response to the combined activation by Epac and PKA during the initial phase of differentiation. Network analyses indicated that the identified proteins are involved in pathways of importance for glucose metabolism, inositol metabolism and calcium-dependent signaling thereby adding a novel facet to our understanding of cAMP-mediated potentiation of adipocyte differentiation.
Project description:In this study, we performed the first dynamic proteome analysis of wheat seed germination using a two-dimensional differential gel electrophoresis (2D-DIGE)-based proteomic approach. A total of 166 differentially expressed protein (DEP) spots representing 73 unique proteins were identified.
Project description:Grapevine downy mildew is an important disease affecting crop production and causing severe losses. To identify genotype-dependent responses towards this pathogen and to explore the molecular mechanisms involved in grapevine-P. viticola resistance, we have conducted a proteomic analysis of leaf samples from resistant and susceptible grapevine genotypes prior and post-inoculation with the pathogen. Proteins were analyzed by quantitative two-dimensional differential gel electrophoresis (2D-DIGE). The analysis able to identified 50 unique proteins. Functional analysis showed that photosynthesis and metabolism were the main categories differentiating genotypes at 0h and that P. viticola-responsive proteins were mainly involved in photosynthesis, carbohydrate metabolism, stress and defense responses and redox homeostasis. ROS production, total antioxidant capacity and lipid peroxidation on both genotypes were determined and together with the proteome data suggest that Regent presents a strict balance between ROS control and signaling leading to plant cell death activation. Our data reveals the genotype-dependent modulation of plant metabolism and defense responses providing new insights into underlying molecular processes of grapevine resistance against the downy mildew fungus.
Project description:The age of mosquitoes is a crucial determinant of their susceptibility to infection, probability of survival to transmit pathogens and tolerance to insecticides. We investigated changes to the abundance of proteins found in heads and thoraces of Anopheles gambiae and Anopheles stephensi as they aged. Protein expression changes were assessed using two-dimensional difference gel electrophoresis and the identity of differentially expressed proteins was determined by using either matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry or capillary high-pressure liquid chromatography coupled with a linear ion-trap (LTQ)-Orbitrap XL hybrid mass spectrometer. Protein biomarkers were validated by quantitative Western blot analysis.
Project description:Muscle atrophy is one of the main deleterious consequences of ageing and physical inactivity. Although basic knowledge regarding the underlying mechanisms of muscle atrophy is continuously growing, there are still no efficient therapeutic strategies for its prevention and treatment. Hibernating bears exhibit a strong and unique ability to preserve muscle mass in conditions where muscle atrophy is observed in humans. However, underlying mechanisms have not been understood yet. To fill this gap, the aim of this study was to characterize changes in the bear muscle proteome during hibernation versus the active period. Muscle biopsies were obtained from Ursus arctos bears.