| 1. |
|
|
| 2. |
- Kaplan, Anders, et al.
(författare)
-
An Automated Method for Scanning LC−MS Data Sets for Significant Peptides and Proteins, Including Quantitative Profiling and Interactive Confirmation : An Automated Method for Scanning LC−MS Data Sets for Significant Peptides and Proteins, Including Quantitative Profiling and Interactive Confirmation
- 2007
-
Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 6:7, s. 2888-2895
-
Tidskriftsartikel (refereegranskat)abstract
- Differential quantification of proteins and peptides by LC-MS is a promising method to acquire knowledge about biological processes, and for finding drug targets and biomarkers. However, differential protein analysis using LC-MS has been held back by the lack of suitable software tools. Large amounts of experimental data are easily generated in protein and peptide profiling experiments, but data analysis is time-consuming and labor-intensive. Here, we present a fully automated method for scanning LC-MS/MS data for biologically significant peptides and proteins, including support for interactive confirmation and further profiling. By studying peptide mixtures of known composition, we demonstrate that peptides present in different amounts in different groups of samples can be automatically screened for using statistical tests. A linear response can be obtained over almost 3 orders of magnitude, facilitating further profiling of peptides and proteins of interest. Furthermore, we apply the method to study the changes of endogenous peptide levels in mouse brain striatum after administration of reserpine, a classical model drug for inducing Parkinson disease symptoms.
|
|
| 3. |
|
|
| 4. |
- Nilsson, Anna, et al.
(författare)
-
Increased striatal mRNA and protein levels of the immunophilin FKBP-12 in experimental Parkinson’s Disease and identification of FKBP-12-binding proteins
- 2007
-
Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 6:10, s. 3952-3961
-
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.
|
|
| 5. |
|
|
| 6. |
- Sköld, Karl, et al.
(författare)
-
The significance of biochemical and molecular sample integrity in brain proteomics and peptidomics: Stathmin (2-20) and peptides as sample quality indicators.
- 2007
-
Ingår i: Proteomics. - : Wiley. - 1615-9853 .- 1615-9861. ; 7:24, s. 4445-4456
-
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.
|
|
| 7. |
|
|
| 8. |
- Svensson, Marcus, et al.
(författare)
-
Heat stabilization of the tissue proteome : a new technology for improved proteomics
- 2009
-
Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 8:2, s. 974-981
-
Tidskriftsartikel (refereegranskat)abstract
- After tissue or body fluid sampling, proteases and other protein-modifying enzymes can rapidly change composition of the proteome. As a direct consequence, analytical results will reflect a mix of in vivo proteome and ex vivo degradation products. Vital information about the presampling state may be destroyed or distorted, leading to variation between samples and incorrect conclusions. Sample stabilization and standardization of sample handling can reduce or eliminate this problem. Here, a novel tissue stabilization system which utilizes a combination of heat and pressure under vacuum was used to stop degradation in mouse brain tissue immediately after sampling. It was found by biochemical assays that enzymatic activity was reduced to background levels in stabilized samples. Western blot analysis confirmed that post-translational phosphorylations of analyzed proteins were stable and conserved for up to 2 h at room temperature and that peptide extracts were devoid of abundant protein degradation fragments. The combination of reduced complexity and proteolytic inactivation enabled mass spectrometric identification of several neuropeptides and endogenous peptides including modified species at higher levels compared to nonstabilized samples. The tissue stabilizing system ensures reproducible and rapid inactivation of enzymes. Therefore, the system provides a powerful improvement to proteomics by greatly reducing the complexity and dynamic range of the proteome in tissue samples and enables enhanced possibilities for discovery and analysis of clinically relevant protein/peptide biomarkers.
|
|
| 9. |
- Svensson, Marcus, et al.
(författare)
-
Neuropeptidomics : MS applied to the discovery of novel peptides from the brain
- 2007
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 79:1, s. 14-21
-
Tidskriftsartikel (refereegranskat)abstract
- Peptidomics involves the comprehensive analysis of the peptide content of a certain cell, organ, body fluid, or organism. Per E. Andrén and colleagues at Uppsala University and the Karolinska Institutet (both in Sweden) describe neuropeptidomics approaches in brain lysates and tissue sections to study peptide expression in disease models and to identify potentially biologically active neuropeptides.
|
|
| 10. |
- Svensson, Marcus, 1972-
(författare)
-
Neuropeptidomics – Expanding Proteomics Downwards
- 2007
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Biological function is mainly carried out by a dynamic population of proteins which may be used as markers for disease diagnosis, prognosis, and as a guide for effective treatment. In analogy to genomics, the study of proteins is called proteomics and it is generally performed by two-dimensional gel electrophoresis and mass spectrometric methods. However, gel based proteomics is methodologically restricted from the low mass region which includes important endogenous peptides. Furthermore, the study of endogenous peptides, peptidomics, is compromised by protein fragments produced post mortem during conventional sample handling. In this thesis nanoflow liquid chromatography and mass spectrometry have been used together with improved methods for sample preparation to semi-quantitatively monitor peptides in brain tissue. The proteolysis of proteins and rise of fragments in the low mass region was studied in a time-course study up to ten minutes, where a potential marker for sample quality was found. When rapidly denatured brain tissue was analyzed, the methods enabled detection of hundreds of peptides and identifications of several endogenous peptides not previously described in the literature. The identification process of endogenous peptides has been improved by creating small targeted sequence collections from existing databases. In applications of the MPTP model for Parkinson’s disease the protein and peptide expressions were compared to controls. Several proteins were significantly changed belonging to groups of mitochondrial, cytoskeletal, and vesicle associated proteins. In the peptidomic study, the levels of the small protein PEP-19 was found to be significantly decreased in the striatum of MPTP administered animals. Using imaging mass spectrometry the spatial distribution of PEP-19 was found to be predominant in the striatum and the levels were concordantly decreased in the parkinsonian tissue as verified by immunoblotting.
|
|
