| 1. |
- Everberg, Henrik, et al.
(författare)
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Affinity Purification of Egg-White Allergens for Improved Component-Resolved Diagnostics
- 2011
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Ingår i: International Archives of Allergy and Immunology. - : S. Karger AG. - 1018-2438 .- 1423-0097. ; 154:1, s. 33-41
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Tidskriftsartikel (refereegranskat)abstract
- 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 (R) 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.
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| 2. |
- Everberg, Henrik, et al.
(författare)
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Aqueous two-phase partitioning for proteomic monitoring of cell surface biomarkers in human peripheral blood mononuclear cells
- 2006
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 5:5, s. 1168-75.
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- Everberg, Henrik, et al.
(författare)
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Efficient and non-denaturing membrane solubilization combined with enrichment of membrane protein complexes by detergent/polymer aqueous two-phase partitioning for proteome analysis
- 2006
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1122:1-2, s. 35-46
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Tidskriftsartikel (refereegranskat)abstract
- 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. (c) 2006 Elsevier B.V. All rights reserved.
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| 4. |
- Everberg, Henrik, et al.
(författare)
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Enrichment of membrane proteins by partitioning in detergent/polymer aqueous two-phase systems.
- 2008
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1940-6029. ; 424, s. 403-412
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Everberg, Henrik, et al.
(författare)
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Extraction of yeast mitochondrial membrane proteins by solubilization and detergent/polymer aqueous two-phase partitioning.
- 2009
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1940-6029. ; 528, s. 72-81
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
- Everberg, Henrik, et al.
(författare)
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Isolation of Escherichia coli inner membranes by metal affinity two-phase partitioning
- 2006
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1118:2, s. 244-252
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Tidskriftsartikel (refereegranskat)abstract
- 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-nitrilotri acetic 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. (c) 2006 Elsevier B.V. All rights reserved.
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| 7. |
- Everberg, Henrik, et al.
(författare)
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Protein pre-fractionation in detergent-polymer aqueous two-phase systems for facilitated proteomic studies of membrane proteins
- 2004
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1029:1-2, s. 113-124
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Tidskriftsartikel (refereegranskat)abstract
- 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 (I-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. (C) 2004 Elsevier B.V. All rights reserved.
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| 8. |
- Everberg, Henrik
(författare)
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Separation Methods in Membrane Proteomics -Integration of Aqueous Two-Phase Partitioning for Enrichment and Pre-Fractionation
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The study of the protein complement of a genome is called proteomics. Proteomics can be defined as identification, quantification and characterization of a total set of proteins expressed at different time-points or under different cellular states to provide global information about biological processes. The study of membrane proteins is a challenging area in the development of proteomic research. Despite their importance in biology and medicine, membrane proteins are remarkably underrepresented in the majority of current proteomic studies, which is largely due to poor solubility in aqueous solutions and thus incompatibility with the traditional separation method, two-dimensional gel electrophoresis. This stresses the need for finding efficient strategies for enrichment and pre-fractionation, and development of novel high-resolution separation techniques for progress of membrane proteomics. In this thesis, aqueous two-phase partitioning has been studied for enrichment and pre-fractionation of proteins to facilitate membrane proteomes analysis. Aqueous two-phase partitioning is a method used in different bioseparation applications for initial recovery of biomolecules mainly based on their surface properties. A number of different aqueous two-phase systems have been used and evaluated with a wide variety of proteomic separation and identification techniques. Detergent/polymer aqueous two-phase partitioning was shown to be efficient for pre-fractionation of yeast mitochondrial proteins. Furthermore, the structural integrity of multi-subunit membrane protein complexes was maintained in the detergent/polymer two-phase partitioning experiments. The importance of screening experimental conditions for optimized extraction of membrane proteins was demonstrated by the efficient and non-denaturing solubilization obtained by the combination of zwitterionic and non-ionic detergents. Metal affinity two-phase partitioning was investigated for selective enrichment of inner membrane vesicles from E. coli and provided a rapid enrichment compared to traditional sucrose gradient centrifugation. PEG/dextran two-phase partitioning was employed for rapid and efficient enrichment of plasma membranes from human peripheral blood mononuclear cells. The identification of a number of unique signature peptides derived from nine cell surface-specific CD-antigens showed that the proposed strategy can potentially be used for proteomic monitoring of disease reactions and therapeutic treatment on membrane bound cell surface proteins. In conclusion, reduction of sample complexity is a key issue in all proteomic studies but especially so in membrane proteomics. The use of aqueous two-phase partitioning for enrichment and pre-fractionation combined with efficient high-resolution separation techniques will provide a useful approach for membrane proteins and membrane protein complexes in future proteomic studies.
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| 9. |
- Movérare, Robert, et al.
(författare)
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Purification and characterization of the major oak pollen allergen Que a 1 for component-resolved diagnostics using ImmunoCAP
- 2008
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Ingår i: International Archives of Allergy and Immunology. - : S. Karger AG. - 1018-2438 .- 1423-0097. ; 146:3, s. 203-211
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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