| 1. |
- Stella, Roberto, et al.
(författare)
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Relative Quantification of Membrane Proteins in Wild-Type and Prion Protein (PrP)-Knockout Cerebellar Granule Neurons
- 2012
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 11:2, s. 523-536
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Tidskriftsartikel (refereegranskat)abstract
- Approximately 25% of eukaryotic proteins possessing homology to at least two trans membrane domains are predicted to be embedded in biological membranes. Nevertheless, this group of proteins is not usually well represented in proteome-wide experiments due to their refractory nature. Here we present a quantitative mass spectrometry-based comparison of membrane protein expression in cerebellar granule neurons grown in primary culture that were isolated from wild-type mice and mice lacking the cellular prion protein. This protein is a cell-surface glycoprotein that is mainly expressed in the central nervous system and is involved in several neurodegenerative disorders, though its physiological role is unclear. We used a low specificity enzyme a-chymotrypsin to digest membrane proteins preparations that had been separated by SDS-PAGE. The resulting peptides were labeled with tandem mass tags and analyzed by MS. The differentially expressed proteins identified using this approach were further analyzed by multiple reaction monitoring to confirm the expression level changes.
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| 2. |
- Cifani, Paolo, et al.
(författare)
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Hunting for Protein Markers of Hypoxia by Combining Plasma Membrane Enrichment with a New Approach to Membrane Protein Analysis
- 2011
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 10:4, s. 1645-1656
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Tidskriftsartikel (refereegranskat)abstract
- Nontransient hypoxia is strongly associated with malignant lesions, resulting in aggressive behavior and resistance to treatment. We present an analysis of mRNA and protein expression changes in neuroblastoma cell lines occurring upon the transition from normoxia to hypoxia. The correlation between mRNA and protein level changes was poor, although some known hypoxia-driven genes and proteins correlated well. We present previously undescribed membrane proteins expressed under hypoxic conditions that are candidates for evaluation as biomarkers.
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| 3. |
- Bruzelius, Katharina, et al.
(författare)
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Biosynthesis of selenoproteins in cultured bovine mammary cells.
- 2008
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Ingår i: Journal of Trace Elements in Medicine and Biology. - : Elsevier BV. - 1878-3252 .- 0946-672X. ; 22:3, s. 224-233
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Tidskriftsartikel (refereegranskat)abstract
- The biosynthesis of selenoproteins was studied in relation to milk formation and mammary cell biology by incubating the bovine mammary cell line MAC-T with (75Se)selenite. Intracellular proteins and proteins secreted into the cell culture medium were separated by 2D electrophoresis, the selenoproteins were detected by autoradiography, and the proteins were identified by MALDI-TOF. Approximately 35 75Se-containing spots were found in the cell proteins from MAC-T cells. Among them, one-third showed high intensity. The strongest spot was identified as glutathione peroxidase 1. About 20 spots were observed in protein precipitated from cell culture medium, one-third of them being distinctly visible. In an attempt to study a perturbation of the system, the effect of retinoic acid (RA) on the formation of selenoproteins was investigated. The concentration of 75Se in total cell protein was reduced by about 35% in cells cultured with RA compared with control cells, while the opposite effect was observed in protein precipitated from cell culture medium, which contained 60% more 75Se in RA-treated samples than in controls. There were also indications that RA might affect different selenoproteins in different ways. The methods described provide a promising approach for further studies of the regulation of selenoprotein formation in the mammary gland.
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| 4. |
- Levander, Fredrik, et al.
(författare)
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Automated methods for improved protein identification by peptide mass fingerprinting
- 2004
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Ingår i: Proteomics. - Weinheim : Wiley. - 1615-9861 .- 1615-9853. ; 4:9, s. 2594-2601
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Tidskriftsartikel (refereegranskat)abstract
- In order to maximize protein identification by peptide mass fingerprinting noise peaks must be removed from spectra and recalibration is often required. The preprocessing of the spectra before database searching is essential but is time-consuming. Nevertheless, the optimal database search parameters often vary over a batch of samples. For high-throughput protein identification, these factors should be set automatically, with no or little human intervention. In the present work automated batch filtering and recalibration using a statistical filter is described. The filter is combined with multiple data searches that are performed automatically. We show that, using several hundred protein digests, protein identification rates could be more than doubled, compared to standard database searching. Furthermore, automated large-scale in-gel digestion of proteins with endoproteinase LysC, and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis, followed by subsequent trypsin digestion and MALDI-TOF analysis were performed. Several proteins could be identified only after digestion with one of the enzymes, and some less significant protein identifications were confirmed after digestion with the other enzyme. The results indicate that identification of especially small and low-abundance proteins could be significantly improved after sequential digestions with two enzymes.
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| 5. |
- Scholz, Birger, et al.
(författare)
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Impact of Temperature Dependent Sampling Procedures in Proteomics and Peptidomics : A Characterization of the Liver and Pancreas Post Mortem Degradome
- 2011
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 10:3, s. M900229MCP200-
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Tidskriftsartikel (refereegranskat)abstract
- Little is known about the nature of post mortem degradation of proteins and peptides on a global level, the so-called degradome. This is especially true for nonneural tissues. Degradome properties in relation to sampling procedures on different tissues are of great importance for the studies of, for instance, post translational modifications and/or the establishment of clinical biobanks. Here, snap freezing of fresh(< 2 min post mortem time) mouse liver and pancreas tissue is compared with rapid heat stabilization with regard to effects on the proteome (using two-dimensional differential in-gel electrophoresis) and peptidome (using label free liquid chromatography). We report several proteins and peptides that exhibit heightened degradation sensitivity, for instance superoxide dismutase in liver, and peptidyl-prolyl cis-trans isomerase and insulin C-peptides in pancreas. Tissue sampling based on snap freezing produces a greater amount of degradation products and lower levels of endogenous peptides than rapid heat stabilization. We also demonstrate that solely snap freezing related degradation can be attenuated by subsequent heat stabilization. We conclude that tissue sampling involving a rapid heat stabilization step is preferable to freezing with regard to proteomic and peptidomic sample quality.
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| 6. |
- Wåhlander, Åsa, et al.
(författare)
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Parallel post-source decay for increasing protein identification confidence levels from 2-D gels
- 2008
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Ingår i: Proteomics. - : Wiley. - 1615-9853 .- 1615-9861. ; 8:9, s. 1771-1779
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Tidskriftsartikel (refereegranskat)abstract
- Peptide mass fingerprinting (PMF) has over the years become one of the most commonly used tools for high-throughput analysis and identification of proteins. This method is applicable when relatively simple samples have to be analysed and it is commonly used for analysing proteins previously separated by 2-DE. The most common type of instrument used for this approach is the MALDI-TOF that has proved to be particularly suitable for the PMF analysis because of its characteristics of speed, robustness, sensitivity and automation. We have used a MALDI-TOF equipped with a novel parallel PSD capability (MALDI micro MX), to perform the analysis of two sets of different biological samples isolated by 2-DE. By using a method that integrates the data obtained by PMF analysis with the PSD data obtained in the same experiment, we show that the new multiplexed PSD solution increases the protein identification rate compared to the normal PMF approach. We also investigated the use of a charge-directed fragmentation modification reagent to improve the identification rate and confidence levels.
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| 7. |
- Bengtsson, Sofia, et al.
(författare)
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Large-scale proteomics analysis of human ovarian cancer for biomarkers
- 2007
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 6:4, s. 1440-1450
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Tidskriftsartikel (refereegranskat)abstract
- Ovarian cancer is usually found at a late stage when the prognosis is often bad. Relative survival rates decrease with tumor stage or grade, and the 5-year survival rate for women with carcinoma is only 38%. Thus, there is a great need to find biomarkers that can be used to carry out routine screening, especially in high-risk patient groups. Here, we present a large-scale study of 64 tissue samples taken from patients at all stages and show that we can identify statistically valid markers using nonsupervised methods that distinguish between normal, benign, borderline, and malignant tissue. We have identified 217 of the significantly changing protein spots. We are expressing and raising antibodies to 35 of these. Currently, we have validated 5 of these antibodies for use in immunohistochemical analysis using tissue microarrays of healthy and diseased ovarian, as well as other, human tissues.
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| 8. |
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| 9. |
- Bäck, Peter, et al.
(författare)
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Automating gel image acquisition
- 2003
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 2:6, s. 662-664
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Tidskriftsartikel (refereegranskat)abstract
- We describe the design and implementation of a robotic solution to automate the acquisition of gel images. The soft- and hardware aspects are outlined together with the various safety aspects that need to be addressed.
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| 10. |
- Corthals, Garry L., et al.
(författare)
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The transition of the European Proteomics Association into the future
- 2011
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Ingår i: Journal of Proteomics. - : Elsevier BV. - 1874-3919. ; 75:1, s. 18-22
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The following report provides an overview of the discussions and outcome of the EuPA General Council meeting that took place in Estoril 20-21 October 2010. During the annual meeting future policy and action plans in a variety of areas are decided. Several important points were decided upon during this meeting including the expansion of the EuPA Executive Committee by introducing a new EuPA committee - EuPA Developments - that will initially spearhead activities in standardisation, imaging ms and biobanking. The EuPA General Council also invited Russia as its 17th member. More details about these and additional activities are presented in the article. (C) 2011 Published by Elsevier B.V.
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