| 1. |
- Apweiler, Rolf, et al.
(författare)
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Approaching clinical proteomics : current state and future fields of application in cellular proteomics
- 2009
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Ingår i: Cytometry. Part A : the journal of the International Society for Analytical Cytology. - : Wiley. - 1552-4922. ; 75A:10, s. 816-832
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Forskningsöversikt (refereegranskat)abstract
- Recent developments in proteomics technology offer new opportunities for clinical applications in hospital or specialized laboratories including the identification of novel biomarkers, monitoring of disease, detecting adverse effects of drugs, and environmental hazards. Advanced spectrometry technologies and the development of new protein array formats have brought these analyses to a standard, which now has the potential to be used in clinical diagnostics. Besides standardization of methodologies and distribution of proteomic data into public databases, the nature of the human body fluid proteome with its high dynamic range in protein concentrations, its quantitation problems, and its extreme complexity present enormous challenges. Molecular cell biology (cytomics) with its link to proteomics is a new fast moving scientific field, which addresses functional cell analysis and bioinformatic approaches to search for novel cellular proteomic biomarkers or their release products into body fluids that provide better insight into the enormous biocomplexity of disease processes and are suitable for patient stratification, therapeutic monitoring, and prediction of prognosis. Experience from studies of in vitro diagnostics and especially in clinical chemistry showed that the majority of errors occurs in the preanalytical phase and the setup of the diagnostic strategy. This is also true for clinical proteomics where similar preanalytical variables such as inter- and intra-assay variability due to biological variations or proteolytical activities in the sample will most likely also influence the results of proteomics studies. However, before complex proteomic analysis can be introduced at a broader level into the clinic, standardization of the preanalytical phase including patient preparation, sample collection, sample preparation, sample storage, measurement, and data analysis is another issue which has to be improved. In this report, we discuss the recent advances and applications that fulfill the criteria for clinical proteomics with the focus on cellular proteomics (cytoproteomics) as related to preanalytical and analytical standardization and to quality control measures required for effective implementation of these technologies and analytes into routine laboratory testing to generate novel actionable health information. It will then be crucial to design and carry out clinical studies that can eventually identify novel clinical diagnostic strategies based on these techniques and validate their impact on clinical decision making.
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| 2. |
- Apweiler, Rolf, et al.
(författare)
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Approaching clinical proteomics : current state and future fields of application in fluid proteomics
- 2009
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Ingår i: Clinical Chemistry and Laboratory Medicine. - 1434-6621 .- 1437-4331. ; 47:6, s. 724-744
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Forskningsöversikt (refereegranskat)abstract
- The field of clinical proteomics offers opportunities to identify new disease biomarkers in body fluids, cells and tissues. These biomarkers can be used in clinical applications for diagnosis, stratification of patients for specific treatment, or therapy monitoring. New protein array formats and improved spectrometry technologies have brought these analyses to a level with potential for use in clinical diagnostics. The nature of the human body fluid proteome with its large dynamic range of protein concentrations presents problems with quantitation. The extreme complexity of the proteome in body fluids presents enormous challenges and requires the establishment of standard operating procedures for handling of specimens, increasing sensitivity for detection and bioinformatical tools for distribution of proteomic data into the public domain. From studies of in vitro diagnostics, especially in clinical chemistry, it is evident that most errors occur in the preanalytical phase and during implementation of the diagnostic strategy. This is also true for clinical proteomics, and especially for fluid proteomics because of the multiple pretreatment processes. These processes include depletion of high-abundance proteins from plasma or enrichment processes for urine where biological variation or differences in proteolytic activities in the sample along with preanalytical variables such as inter- and intra-assay variability will likely influence the results of proteomics studies. However, before proteomic analysis can be introduced at a broader level into the clinical setting, standardization of the preanalytical phase including patient preparation, sample collection, sample preparation, sample storage, measurement and data analysis needs to be improved. In this review, we discuss the recent technological advances and applications that fulfil the criteria for clinical proteomics, with the focus on fluid proteomics. These advances relate to preanalytical factors, analytical standardization and quality-control measures required for effective implementation into routine laboratory testing in order to generate clinically useful information. With new disease biomarker candidates, it will be crucial to design and perform clinical studies that can identify novel diagnostic strategies based on these techniques, and to validate their impact on clinical decision-making.
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| 3. |
- Hauck, Stefanie M, et al.
(författare)
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Differential modification of phosducin protein in degenerating rd1 ret is associated with constitutively active CaMKII in rod outer segments.
- 2006
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Ingår i: Molecular & Cellular Proteomics. - 1535-9484. ; 5:2, s. 324-336
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Tidskriftsartikel (refereegranskat)abstract
- Retinitis pigmentosa comprises a heterogeneous group of incurable progressive blinding diseases with unknown pathogenic mechanisms. The retinal degeneration 1 (rd1) mouse is a retinitis pigmentosa model that carries a mutation in a rod photoreceptor-specific phosphodiesterase gene, leading to rapid degeneration of these cells. Elucidation of the molecular differences between rd1 and healthy retinae is crucial for explaining this degeneration and could assist in suggesting novel therapies. Here we used high resolution proteomics to compare the proteomes of the rd1 mouse retina and its congenic, wildtype counterpart at postnatal day 11 when photoreceptor death is profound. Over 3000 protein spots were consistently resolved by two-dimensional gel electrophoresis and subjected to a rigorous filtering procedure involving computer-based spot analyses. Five proteins were accepted as being differentially expressed in the rd1 model and subsequently identified by mass spectrometry. The difference in one such protein, phosducin, related to an altered modification pattern in the rd1 retina rather than to changed expression levels. Additional experiments showed phosducin in healthy retinae to be highly phosphorylated in the dark- but not in the light-adapted phase. In contrast, rd1 phosducin was highly phosphorylated irrespective of light status, indicating a dysfunctional rd1 light/dark response. The increased rd1 phosducin phosphorylation coincided with increased activation of calcium/calmodulin-activated protein kinase II, which is known to utilize phosducin as a substrate. Given the increased rod calcium levels present in the rd1 mutation, calcium-evoked overactivation of this kinase may be an early and long sought for step in events leading to photoreceptor degeneration in the rd1 mouse.
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| 4. |
- Hauck, Stefanie M., et al.
(författare)
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Identification of paracrine neuroprotective candidate proteins by a functional assay-driven proteomics approach
- 2008
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Ingår i: Molecular & Cellular Proteomics. - 1535-9484. ; 7:7, s. 1349-1361
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Tidskriftsartikel (refereegranskat)abstract
- Glial cells support neuronal survival and function by secreting neurotrophic cytokines. Retinal Mueller glial cells (RMGs) support retinal neurons, especially photoreceptors. These highly light-sensitive sensory neurons receive vision, and their death results in blinding diseases. It has been proposed that RMGs release factors that support photoreceptor survival, but the nature of these factors remains to be elucidated. To discover such neurotrophic factors, we developed an integrated work flow toward systematic identification of neuroprotective proteins, which are, like most cytokines, expressed only in minute amounts. This strategy can be generally applied to identify secreted bioactive molecules from any body fluid once a recipient cell for this activity is known. Toward this goal we first isolated conditioned medium (CM) from primary porcine RMGs cultured in vitro and tested for survival-promoting activity using primary photoreceptors. We then developed a large scale, microplate-based cellular high content assay that allows rapid assessment of primary photoreceptor survival concomitant with biological activity in vitro. The enrichment strategy of bioactive proteins toward their identification consists of several fractionation steps combined with tests for biological function. Here we combined 1) size fractionation, 2) ion exchange chromatography, 3) reverse phase liquid chromatography, and 4) mass spectrometry (Q-TOF MS/MS or MALDI MS/MS) for protein identification. As a result of this integrated work flow, the insulin-like growth factor-binding proteins IGFBP5 and IGFBP7 and connective tissue growth factor (CTGF) were identified as likely candidates. Cloning and stable expression of these three candidate factors in HEK293 cells produced conditioned medium enriched for either one of the factors. IGFBP5 and CTGF, but not IGFBP7, significantly increased photoreceptor survival when secreted from HEK293 cells and when added to the original RMG-CM. This indicates that the survival-promoting activity in RMG-CM is multifactorial with IGFBP5 and CTGF as an integral part of this activity.
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| 5. |
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| 6. |
- Paquet-Durand, Francois, et al.
(författare)
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PKG activity causes photoreceptor cell death in two retinitis pigmentosa models
- 2009
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Ingår i: Journal of Neurochemistry. - : Wiley. - 1471-4159 .- 0022-3042. ; 108:3, s. 796-810
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Tidskriftsartikel (refereegranskat)abstract
- Photoreceptor degeneration in retinitis pigmentosa is one of the leading causes of hereditary blindness in the developed world. Although causative genetic mutations have been elucidated in many cases, the underlying neuronal degeneration mechanisms are still unknown. Here, we show that activation of cGMP-dependent protein kinase (PKG) hallmarks photoreceptor degeneration in rd1 and rd2 human homologous mouse models. When induced in wild-type retinae, PKG activity was both necessary and sufficient to trigger cGMP-mediated photoreceptor cell death. Target-specific, pharmacological inhibition of PKG activity in both rd1 and rd2 retinae strongly reduced photoreceptor cell death in organotypic retinal explants. Likewise, inhibition of PKG in vivo, using three different application paradigms, resulted in robust photoreceptor protection in the rd1 retina. These findings suggest a pivotal role for PKG activity in cGMP-mediated photoreceptor degeneration mechanisms and highlight the importance of PKG as a novel target for the pharmacological intervention in RP.
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| 7. |
- Taussig, Michael J., et al.
(författare)
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ProteomeBinders : planning a European resource of affinity reagents for analysis of the human proteome
- 2007
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Ingår i: Nature Methods. - : Springer Science and Business Media LLC. - 1548-7091 .- 1548-7105. ; 4:1, s. 13-17
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Tidskriftsartikel (refereegranskat)abstract
- ProteomeBinders is a new European consortium aiming to establish a comprehensive resource of well-characterized affinity reagents, including but not limited to antibodies, for analysis of the human proteome. Given the huge diversity of the proteome, the scale of the project is potentially immense but nevertheless feasible in the context of a pan-European or even worldwide coordination.
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