RESUMEN
BACKGROUND: Egg is a common cause of food-allergic reactions, especially among young children. Some egg-allergic patients do, however, tolerate heated egg products and component-resolved diagnostics (CRD) may facilitate prediction of different disease manifestations. Commercially available preparations of the egg-white allergens, ovomucoid, ovalbumin, conalbumin and lysozyme, have been reported to contain impurities which interfere with accurate CRD. METHODS: Commercial preparations of the 4 egg-white allergens were characterized using allergen-specific monoclonal chimeric human/mouse IgE antibodies in experimental ImmunoCAP® tests. Further purification of commercial ovomucoid, ovalbumin and conalbumin preparations was performed by chromatography based on affinity to monoclonal antibodies. Purity was monitored by size exclusion chromatography, SDS-PAGE, Western blotting and experimental ImmunoCAP tests using allergen-specific chimeric IgE antibodies. IgE reactivity to the highly purified egg components was analyzed in 83 samples from egg white-sensitized individuals. RESULTS: Preparations of commercially available ovomucoid, ovalbumin and conalbumin were found to contain other egg allergens which were removed by chromatographic purification. No impurities were detected in the commercial lysozyme preparation. Previously unknown complexes between the target allergens and contaminating allergens were detected and removed by affinity chromatography. IgE reactivity to ovalbumin was most common in the analyzed samples (87%), followed by ovomucoid (72%), conalbumin (69%) and lysozyme (58%). CONCLUSIONS: In this study we demonstrate the advantage of using monoclonal antibodies for purification, and monoclonal chimeric IgE antibodies for characterization, of egg allergens intended for CRD. Our study also established that ovalbumin, ovomucoid, conalbumin and lysozyme are all major allergens.
Asunto(s)
Alérgenos/aislamiento & purificación , Cromatografía de Afinidad/métodos , Conalbúmina/aislamiento & purificación , Hipersensibilidad al Huevo/diagnóstico , Clara de Huevo/química , Ovalbúmina/aislamiento & purificación , Ovomucina/aislamiento & purificación , Alérgenos/inmunología , Anticuerpos Monoclonales/inmunología , Conalbúmina/inmunología , Humanos , Inmunoglobulina E/inmunología , Pruebas Inmunológicas , Ovalbúmina/inmunología , Ovomucina/inmunologíaRESUMEN
BACKGROUND: Dog dander is an important cause of respiratory allergy, but the spectrum of known dog allergens appears incomplete. Two lipocalins, Can f 1 and Can f 2, and serum albumin, Can f 3, have been characterized in detail but do not fully account for the IgE antibody-binding activity of dog dander extract. Allergen activity has previously been detected in dog urine but not further characterized. OBJECTIVE: We sought to identify, characterize, and assess the importance of allergen components in dog urine. METHODS: Dog urine was fractionated by means of size exclusion chromatography and examined for IgE antibody binding. A protein present in one fraction displaying IgE antibody-binding activity was identified by means of N-terminal sequencing and mass spectrometry. A recombinant form of the protein was produced in Pichia pastoris. IgE antibody binding to dog allergen components among sera of 37 subjects with dog allergy was determined by means of ImmunoCAP analysis. RESULTS: An IgE antibody-binding protein was isolated from dog urine and identified as prostatic kallikrein. A closely related or identical protein was detected in dog dander. The recombinant prostatic kallikrein displayed immunologic and biochemical properties similar to those of the natural protein and bound IgE antibodies from 26 (70%) of 37 sera of subjects with dog allergy, 14 of which reacted to none of Can f 1, Can f 2, or Can f 3. The dog allergen identified here was found to cross-react with human prostate-specific antigen, a key culprit in IgE-mediated vaginal reactions to semen. CONCLUSION: Prostatic kallikrein is a new major dog allergen.
Asunto(s)
Alérgenos/inmunología , Perros/inmunología , Inmunoglobulina E/inmunología , Hipersensibilidad Respiratoria/inmunología , Calicreínas de Tejido/inmunología , Adulto , Alérgenos/orina , Animales , Reacciones Cruzadas/inmunología , Humanos , Masculino , Persona de Mediana Edad , Próstata/inmunología , Antígeno Prostático Específico/inmunología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Calicreínas de Tejido/aislamiento & purificación , Calicreínas de Tejido/orinaRESUMEN
Identification and characterization of membrane proteins is of increasing importance in modern proteomic studies. It is of central interest to have access to methods that combine efficient solubilization with enrichment of proteins and intact protein complexes. Separation methods have been developed based on nondenaturing detergent extraction of yeast mitochondrial membrane proteins followed by enrichment of hydrophobic proteins in aqueous two-phase system. Combining the zwitterionic detergent Zwittergent 3-10 and the nonionic detergent Triton X-114 results in a complementary solubilization of proteins, which is similar to that of the anionic detergent sodium dodecyl sulfate (SDS) but with the important advantage of being nondenaturing. Detergent/polymer two-phase system partitioning offers removal of soluble proteins, which can be further improved by manipulation of the driving forces governing protein distribution between the phases. Integral and peripheral membrane protein subunits from intact membrane protein complexes partition to the detergent phase while soluble proteins are found in the polymer phase. A protocol is presented which combines nondenaturing solubilization of membrane proteins with extraction in detergent/polymer two-phase system for application in proteomic studies as a mild and efficient method for enrichment of membrane proteins and membrane protein complexes.
Asunto(s)
Fraccionamiento Químico/métodos , Proteínas Mitocondriales/aislamiento & purificación , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Saccharomyces cerevisiae/química , Detergentes/química , Electroforesis en Gel Bidimensional , Interacciones Hidrofóbicas e Hidrofílicas , Octoxinol , Polietilenglicoles/química , Polímeros/química , Compuestos de Amonio Cuaternario/química , Dodecil Sulfato de Sodio/químicaRESUMEN
Methods that combine efficient solubilization with enrichment of proteins and intact protein complexes are of central interest in current membrane proteomics. We have developed methods based on nondenaturing detergent extraction of yeast mitochondrial membrane proteins followed by enrichment of hydrophobic proteins in aqueous two-phase system. Combining the zwitterionic detergent Zwittergent 3-10 and the nonionic detergent Triton X-114 results in a complementary solubilization of proteins, which is similar to that of the anionic detergent sodium dodecyl sulfate (SDS) but with the important advantage of being nondenaturing. Detergent/polymer two-phase system partitioning offers removal of soluble proteins that can be further improved by manipulation of the driving forces governing protein distribution between the phases. Integral and peripheral membrane protein subunits from intact membrane protein complexes partition to the detergent phase while soluble proteins are found in the polymer phase. An optimized solubilization protocol is presented in combination with detergent/polymer two-phase partitioning as a mild and efficient method for initial enrichment of membrane proteins and membrane protein complexes in proteomic studies.
Asunto(s)
Proteínas de la Membrana/aislamiento & purificación , Polímeros/química , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Saccharomyces cerevisiae/química , Detergentes , Proteínas de la Membrana/química , Transición de Fase , Proteínas de Saccharomyces cerevisiae/química , SolubilidadRESUMEN
BACKGROUND: The aim of this study was to purify the major oak pollen allergen, Que a 1, to perform biochemical and immunological characterization of the allergen and to develop an experimental native (n) Que a 1 ImmunoCAP(R). METHODS: Que a 1 was purified from oak pollen extract using affinity chromatography and characterized by SDS-PAGE, two-dimensional (2D) PAGE, mass spectrometry (MS), N-terminal sequencing and specific IgE inhibition on ImmunoCAP. Samples from 16 subjects sensitized to oak pollen were analyzed by ImmunoCAP for IgE reactivity to nQue a 1, and recombinant (r)Bet v 1 and 2 (profilin). They were also studied in IgE immunoblotting. RESULTS: The purity of nQue a 1 was >95%, since a single band was observed on silver-stained SDS-PAGE. The identity was verified by MS analysis, and 2D-PAGE revealed several isoforms. The obtained N-terminal sequence of 50-amino-acid residues from nQue a 1 showed a 58-74% sequence identity with other pathogenesis-related class 10 allergens. Specific IgE inhibition verified a preserved immunoreactivity (70-92% inhibition). All subjects were sensitized to Que a 1 and Bet v 1, and two to profilin. The IgE antibody levels to nQue a 1 were generally lower than to rBet v 1. The obtained results correlated well with IgE immunoblotting. CONCLUSIONS: We present a highly purified and extensively characterized preparation of nQue a 1. Que a 1 seems to be an allergen of equal importance in oak pollen as Bet v 1 in birch pollen. An nQue a 1 ImmunoCAP will be useful in component-resolved diagnostics.
Asunto(s)
Alérgenos/aislamiento & purificación , Inmunoglobulina E/inmunología , Proteínas de Plantas/aislamiento & purificación , Polen/química , Quercus/inmunología , Alérgenos/análisis , Alérgenos/inmunología , Secuencia de Aminoácidos , Antígenos de Plantas , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Humanos , Immunoblotting , Datos de Secuencia Molecular , Proyectos Piloto , Proteínas de Plantas/análisis , Proteínas de Plantas/inmunología , Polen/inmunología , Quercus/química , Rinitis Alérgica Estacional/sangre , Rinitis Alérgica Estacional/inmunología , Alineación de Secuencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
It is of central interest in membrane proteomics to establish methods that combine efficient solubilization with enrichment of proteins and intact protein complexes. We have investigated the quantitative and qualitative solubilization efficiency of five commercially available detergents using mitochondria from the yeast Saccharomyces cerevisiae as model system. Combining the zwitterionic detergent Zwittergent 3-10 and the non-ionic detergent Triton X-114 resulted in a complementary solubilization of proteins, which was similar to that of the anionic detergent sodium dodecyl sulfate (SDS). The subsequent removal of soluble proteins by detergent/polymer two-phase system partitioning was further enhanced by addition of SDS and increasing pH. A large number of both integral and peripheral membrane protein subunits from mitochondrial membrane protein complexes were identified in the detergent phase. We suggest that the optimized solubilization protocol in combination with detergent/polymer two-phase partitioning is a mild and efficient method for initial enrichment of membrane proteins and membrane protein complexes in proteomic studies.
Asunto(s)
Membrana Celular/química , Detergentes/química , Proteínas de la Membrana/análisis , Polímeros/química , Proteoma/análisis , Fraccionamiento Químico/métodos , Electroforesis en Gel de Poliacrilamida , Proteínas de la Membrana/química , Proteínas Mitocondriales/análisis , Proteínas Mitocondriales/química , Estructura Molecular , Reproducibilidad de los Resultados , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/análisis , Proteínas de Saccharomyces cerevisiae/química , Solubilidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Agua/químicaRESUMEN
For proteomic monitoring of processes such as allergy or inflammation an efficient pre-fractionation strategy is required. We isolated plasma membranes from human peripheral blood mononuclear (PBM) cells by aqueous two-phase partitioning. After 1DE combined with LC-MS/MS, several cell surface marker proteins and in total 60 different plasma membrane proteins (out of 84 identified proteins, i.e., 72%) were detected. Plasma membranes obtained were from only one human donor, the procedure is therefore applicable for individual patient screening.
Asunto(s)
Biomarcadores/análisis , Proteínas Sanguíneas/análisis , Membrana Celular/metabolismo , Proteómica/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Antígenos CD/análisis , Antígenos CD/metabolismo , Biomarcadores/sangre , Biomarcadores/metabolismo , Proteínas Sanguíneas/metabolismo , Cromatografía Liquida/métodos , HumanosRESUMEN
As reduction of sample complexity is a central issue in membrane proteomic research, the need for new pre-fractionation methods is significant. Here we present a method for fast and efficient enrichment of Escherichia coli inner membranes expressing a His-tagged integral membrane L-fucose-proton symporter (FucP). An enriched inner membrane fraction was obtained from a crude membrane mixture using affinity two-phase partitioning in combination with nickel-nitrilotriacetic acid (Ni-NTA) immobilized on agarose beads. Due to interaction between the beads and FucP, inner membranes were selectively partitioned to the bottom phase of a polymer/polymer aqueous two-phase system consisting of poly(ethylene glycol) (PEG) and dextran. The partitioning of membranes was monitored by assaying the activity of an inner membrane marker protein and measuring the total protein content in both phases. The enrichment of inner membrane proteins in the dextran phase was also investigated by proteomic methodology, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), trypsin digestion and liquid chromatography in combination with tandem mass spectrometry (LC-MS/MS). Using a high level of significance (99.95%) in the subsequent database search, 36 proteins assigned to the inner membrane were identified in the bottom phase, compared to 29 when using the standard sucrose gradient centrifugation method for inner membrane isolation. Furthermore, metal affinity two-phase partitioning was up to 10 times faster than sucrose gradient centrifugation. The separation conditions in these model experiments provide a basis for the selective isolation of E. coli membranes expressing His-tagged proteins and can therefore facilitate research on such membrane proteomes.
Asunto(s)
Membrana Celular , Cromatografía de Afinidad/métodos , Escherichia coli/química , Western Blotting , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Espectrometría de MasasRESUMEN
Pre-fractionation of a complex mixture of proteins increases the resolution in analytical separations of proteins from cells, tissues or organisms. Here we demonstrate a novel method for pre-fractionation of membrane proteins by a detergent-based aqueous two-phase system. Membrane proteins are strongly under-represented in proteomic studies based on two-dimensional electrophoresis (2-DE). As a model system, we have isolated mitochondria from the yeast Saccharomyces cerevisiae. Mitochondrial proteins were fractionated in an aqueous two-phase system consisting of the polymer poly(ethylene glycol) and either of two commonly used non-ionic detergents, Triton X-114 or dodecyl maltoside (DDM). Soluble proteins partitioned mainly to the polymer phase while membrane proteins were enriched in the detergent phase, as identified from one-dimensional electrophoresis (1-DE) and/or 2-DE followed by mass spectrometric analysis. Pre-fractionation was further enhanced by addition of an anionic detergent, sodium dodecyl sulfate, or a chaotropic salt, NaClO4, and by raising the pH in the system. The two-phase system pre-fractionation was furthermore combined with an alternative two-dimensional high-resolution separation method, namely ion-exchange chromatography and 1-DE. By this approach a larger number of membrane proteins could be identified compared to separation with conventional 2-DE. Thus, pre-fractionation of complex protein mixtures using the aqueous two-phase systems developed here will help to disclose larger proportions of membrane proteins in different proteomes.