| 1. |
- Madeira, Alexandra, et al.
(författare)
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Coupling surface plasmon resonance to mass spectrometry to discover novel protein-protein interactions
- 2009
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Ingår i: Nature Protocols. - : Springer Science and Business Media LLC. - 1754-2189 .- 1750-2799. ; 4:7, s. 1023-1037
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Tidskriftsartikel (refereegranskat)abstract
- The elucidation of protein-protein interaction networks is a crucial task in the postgenomic era. In this protocol, we describe our approach to discover protein-protein interactions using the surface plasmon resonance technique coupled to mass spectrometry ( MS). A peptide or a protein is immobilized on a sensor chip and then exposed to brain extracts injected through the surface of the chip by a microfluidic system. The interactions between the immobilized ligand and the extracts can be monitored in real time. Proteins interacting with the peptide/protein are recovered, trypsinated and identified using MS. The data obtained are searched against a sequence database using the Mascot 2.1 software. Control experiments using blank sensor chips and/or randomized peptides are carried out to exclude nonspecific interactors. The protocol can be carried out in < 3 days. Other methods, such as yeast two-hybrid systems or pull-down approaches followed by MS, are widely used to screen protein-protein interactions. However, as the yeast two-hybrid system requires protein interactions in the nucleus of yeast, proteins that are abundant in other compartments may not be detected. Pull-down approaches based on immunoprecipitation can be used to study endogenous proteins but they require specific antibodies. The protocol presented here does not require the specific labeling or modification of proteins.
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| 2. |
- Madeira, Alexandra, et al.
(författare)
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Identification of protein-protein interactions by surface plasmon resonance followed by mass spectrometry
- 2011
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Ingår i: Current protocols in protein science. - Hoboken, NJ, USA : John Wiley & Sons, Inc.. - 1934-3663. ; Suppl. 65, s. 19.21.1-19.21.9
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Tidskriftsartikel (refereegranskat)abstract
- Elucidation of the function and meaning of the protein networks can be useful in the understanding of many pathological processes and the identification of new therapeutic targets. This unit describes an approach to discover protein-protein interactions by coupling surface plasmon resonance to mass spectrometry. Briefly, a protein is covalently bound to a sensor chip, which is then exposed to brain extracts injected over the surface via a microfluidic system. This allows the monitoring in real-time of the interactions between the immobilized ligand and the extracts. Interacting proteins from the extracts are then recovered, trypsinized, and identified using mass spectrometry. The data obtained are searched against a sequence database using the Mascot software. To exclude nonspecific interactors, control experiments using blank sensor chips, and/or randomized peptides, are performed. The protocol presented here does not require specific labeling or modification of proteins and can be performed in <4 days.
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| 3. |
- Nilsson, Anna, et al.
(författare)
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Increased striatal mRNA and protein levels of the immunophilin FKBP-12 in experimental Parkinson’s Disease and identification of FKBP-12-binding proteins
- 2007
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 6:10, s. 3952-3961
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Tidskriftsartikel (refereegranskat)abstract
- FKBP-12, a 12 kDa FK506-binding protein (neuroimmunophilin), acts as a receptor for the immunosuppressant drug FK506. Neuroimmunophilins, including FKBP-12, are abundant in the brain and have been shown to be involved in reversing neuronal degeneration and preventing cell death. In this report, we have utilized several analytical techniques, such as in situ hybridization, Western blotting, two-dimensional gel electrophoresis, and liquid chromatography electrospray tandem mass spectrometry to study the transcriptional expression as well as protein levels of FKBP-12 in the unilateral 6-hydroxydopamine (6-OHDA) rat model of Parkinson's disease. The FKBP-12 protein was also detected directly on brain tissue sections using mass spectrometry profiling. We found increased levels of FKBP-12 mRNA and protein in the dorsal and middle part of the 6-OHDA lesioned striatum. Thus, these studies clearly demonstrate that FKBP-12 is increased in the brain of a common animal model of Parkinson's disease (PD). Additionally, we have identified potential binding partners to FKBP-12 that may be implicated in the pathophysiology of Parkinson's disease, such as alpha-enolase, 14-3-3 zeta/delta, pyruvate kinase isozymes, and heat shock protein 70, using surface plasmon resonance sensor technology in combination with mass spectrometry. In conclusion, these data strongly suggests that FKBP-12 is altered in an experimental model of PD.
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| 4. |
- Sjögren, Benita
(författare)
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Involvement of lipid rafts in G protein-coupled monoamine receptor trafficking and signaling : a pharmacological approach
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The present work focused on lipid raft-mediated modulation of signaling and trafficking of serotonin (5-HT) and dopamine receptors. The 5-HT system is one of the most complex neurotransmitter systems, with a wide distribution both in the CNS and in the periphery. It is involved in the regulation of many biological functions as diverse as mood, metabolism and cardiovascular tone. 5-HT acts via at least 14 receptors, divided into seven subgroups according to sequence homology and mode of signal transduction. All of them are G protein-coupled receptors (GPCRs), except for the 5-HT3 receptor, which is a ligand-gated ion channel. The dopamine system is involved in the motor, cognitive and reward systems of the brain. Dopamine acts via five receptors, all GPCRs, divided into D1-like and D2-like receptors according to their mode of signal transduction. Dysregulation of the dopamine system is the underlying cause of addiction, schizophrenia and Parkinson s disease (PD). The Singer-Nicholson fluidic mosaic model has for many years been the general model for the structure of biological membranes. In recent years this view has been revised with the discovery of liquid ordered microdomains, lipid rafts, enriched in cholesterol and sphingolipids. These microdomains play important roles in regulating signal transduction and trafficking events. A subgroup of lipid rafts, caveolae, is flask-shaped invaginations of the plasma membrane. They contain caveolin (Cav) proteins that can interact with other proteins, including GPCRs. Recently it was discovered that lipid rafts and caveolae participate in endocytosis and receptor trafficking. Although the exact mechanism for these processes is not yet fully understood, it seems clear that there can be alternative routes of receptor internalization than the classical endosomatic pathway via clathrin coated vesicles. In the present work we found that decreases of cholesterol, gangliosides, sphingomyelin or Cav-1 (by RNA interference) levels, attenuated maximum agonist and antagonist binding to 5-HT7 receptors. This could not be explained by a mere reduction in total receptor protein levels. Furthermore, signaling via 5-HT7 receptors was attenuated by cholesterol depletion with HMG-CoA reductase inhibitors, statins, which are widely used in the treatment of atherosclerosis. Reduction of cholesterol levels also attenuated signaling via 5-HT1A receptors in primary neuronal cultures. Furthermore, we could detect dopamine D1 receptors in low density regions in a sucrose gradient, suggesting lipid raft localization. This was further confirmed by the finding that reduction of cholesterol levels inhibited signaling via dopamine D1 receptors in mouse striatal slices. These results give further indications that lipid raft play important roles in regulating monoamine GPCRs in vivo. We have also found that Cav-1, a key protein in caveolae, regulates cell surface expression of 5-HT7 receptors. Biochemical and cellular assays showed that 5-HT7 receptors are located in low density regions in a sucrose gradient together with Cav-1, indicating lipid raft localization. Furthermore, it was found that 5-HT7 receptors undergo agonist induced internalization and that this could be inhibited by reducing Cav-1 expression with RNA interference. The internalization could also be inhibited with genistein, known to block lipid raft-mediated internalization. In contrast, hypertonic sucrose solution could not inhibit internalization. Altogether this suggests that 5-HT7 receptors are internalized through a clathrin-independent mechanism that is dependent on Cav-1.
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| 5. |
- Sköld, Karl, et al.
(författare)
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The significance of biochemical and molecular sample integrity in brain proteomics and peptidomics: Stathmin (2-20) and peptides as sample quality indicators.
- 2007
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Ingår i: Proteomics. - : Wiley. - 1615-9853 .- 1615-9861. ; 7:24, s. 4445-4456
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Tidskriftsartikel (refereegranskat)abstract
- Comparisons of transcriptional and translational expression in normal and abnormal states are important to reach an understanding of pathogenesis and pathophysiology. Maintaining the biochemical, molecular, and structural sample integrity is essential for correct sample comparisons. We demonstrate that both proteins and neuropeptides, including their PTMs, are subjected to massive degradation in the brain already 1 min postmortem. Further, markers for determining the integrity and status of a biological sample were identified. The protein fragment stathmin 2-20 correlated well with the general level of postmortem degradation and may serve as a sample quality indicator for future work, both in animal and human postmortem brains. Finally, a novel method for preventing degradation of proteins and peptides in postmortem tissue is presented using rapid and uniform conductive heat transfer on tissue prior to the actual sample preparation procedures, which enables the relatively low-abundant neuropeptides to remain intact, minimizes degradation of proteins by proteolysis, and conserves the PTMs of the neuropeptides.
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| 6. |
- Öhman, Elisabet, et al.
(författare)
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Use of Surface Plasmon Resonance Coupled with Mass Spectrometry Reveals an Interaction between the Voltage-Gated Sodium Channel Type X alpha-Subunit and Caveolin-1
- 2008
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 7:12, s. 5333-5338
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Tidskriftsartikel (refereegranskat)abstract
- The combination of surface plasmon resonance and mass spectrometry is emerging as a sensitive tool for the elucidation of protein-protein interactions. With the use of surface plasmon resonance-mass spectrometry, peptides, and brain extracts, we now report a novel interaction between the voltage-gated sodium channel type X alpha-subunit and caveolin-1, the central protein controlling caveolae formation. Surface plasmon resonance binding analyses show that this interaction involves amino acids 85-103 of voltage-gated sodium channel type X alpha-subunit and amino acids 81-100 of caveolin-1, a known scaffolding domain of caveolin-1. It is anticipated that the surface plasmon resonance-mass spectrometry approach utilized in this study will be important for the elucidation of protein-protein network analysis in native tissues including the brain.
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