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Proteomic Strategy (proteomic + strategy)
Selected AbstractsProteomic strategies in multiple sclerosis and its animal modelsPROTEOMICS - CLINICAL APPLICATIONS, Issue 11 2007Stella Elkabes Dr. Abstract The early and precise diagnosis, the prognosis, and the clinical management of multiple sclerosis, remain a considerable challenge. In recent years, the development of novel and powerful proteomic techniques prompted the use of these approaches for the search of unique biomarkers in the cerebrospinal fluid of multiple sclerosis patients. A few studies have also utilized proteomics to delineate the profile of differentially expressed proteins in animal models of the human disease in order to gain global insights into affected pathways. The identification of differentially expressed proteins may be an initial step in the discovery of novel targets and mechanisms that play critical roles in the pathology of multiple sclerosis. Based on these findings, future investigations may elucidate the events leading to demyelination, axonal damage, and neurodegeneration, providing better insights into mechanisms governing the onset and progression of the disease. Although these proteomic studies provide valuable information, they are also faced with a number of challenges. The present review discusses some of the strengths and limitations of proteomic investigations as applied to multiple sclerosis. [source] Proteomic strategies to elucidate pathogenic mechanisms of spirochetesPROTEOMICS - CLINICAL APPLICATIONS, Issue 9 2007Jarlath E. Nally Dr. Abstract Spirochetes are a unique group of bacteria that include several motile and highly invasive pathogens that cause a multitude of acute and chronic disease processes. Nine genomes of spirochetes have been completed, which provide significant insights into pathogenic mechanisms of disease and reflect an often complex lifestyle associated with a wide range of environmental and host factors encountered during disease transmission and infection. Characterization of the outer membrane of spirochetes is of particular interest since it interacts directly with the host and environs during disease and likely contains candidate vaccinogens and diagnostics. In concert with appropriate fractionation techniques, the tools of proteomics have rapidly evolved to characterize the proteome of spirochetes. Of greater significance, studies have confirmed the differential expression of many proteins, including those of the outer membrane, in response to environmental signals encountered during disease transmission and infection. Characterization of the proteome in response to such signals provides novel insights to understand pathogenic mechanisms of spirochetes. [source] Mass spectrometry in aging researchMASS SPECTROMETRY REVIEWS, Issue 5 2005Christian Schöneich Abstract This review covers the application of mass spectrometric techniques to aging research. Modern proteomic strategies will be discussed as well as the targeted analysis of specific proteins for the correlation of post-translational modifications with protein function. Selected examples will show both the power and also current limitations of the respective techniques. Experimental results and strategies are discussed in view of current theories of the aging process. © 2004 Wiley Periodicals, Inc., Mass Spec Rev 24:701,718, 2005 [source] High-resolution biomarker discovery: Moving from large-scale proteome profiling to quantitative validation of lead candidatesPROTEOMICS - CLINICAL APPLICATIONS, Issue 10-11 2008Johannes A. Hewel Abstract Diverse proteomic techniques based on protein MS have been introduced to systematically characterize protein perturbations associated with disease. Progress in clinical proteomics is essential for personalized medicine, wherein treatments will be tailored to individual needs based on patient stratification using noninvasive disease monitoring procedures to reveal the most appropriate therapeutic targets. However, breakthroughs await the successful development and application of a robust proteomic pipeline capable of identifying and rigorously assessing the relevance of multiple candidate proteins as informative diagnostic and prognostic indicators or suitable drug targets involved in a pathological process. While steady progress has been made toward more comprehensive proteome profiling, the emphasis must now shift from in depth screening of reference samples to stringent quantitative validation of selected lead candidates in a broader clinical context. Here, we present an overview of the emerging proteomic strategies for high-throughput protein detection focused primarily on targeted MS/MS as the basis for biomarker verification in large clinical cohorts. We discuss the conceptual promise and practical pitfalls of these methods in terms of achieving higher dynamic range, higher throughput, and more reliable quantification, highlighting research avenues that merit additional inquiry. [source] A novel approach to tag and identify geranylgeranylated proteinsELECTROPHORESIS, Issue 20 2009Lai N. Chan Abstract A recently developed proteomic strategy, the "GG-azide"-labeling approach, is described for the detection and proteomic analysis of geranylgeranylated proteins. This approach involves metabolic incorporation of a synthetic azido-geranylgeranyl analog and chemoselective derivatization of azido-geranylgeranyl-modified proteins by the "click" chemistry, using a tetramethylrhodamine-alkyne. The resulting conjugated proteins can be separated by 1-D or 2-D and pH fractionation, and detected by fluorescence imaging. This method is compatible with downstream LC-MS/MS analysis. Proteomic analysis of conjugated proteins by this approach identified several known geranylgeranylated proteins as well as Rap2c, a novel member of the Ras family. Furthermore, prenylation of progerin in mouse embryonic fibroblast cells was examined using this approach, demonstrating that this strategy can be used to study prenylation of specific proteins. The "GG-azide"-labeling approach provides a new tool for the detection and proteomic analysis of geranylgeranylated proteins, and it can readily be extended to other post-translational modifications. [source] Proteomic analysis identifies in vivo candidate matrix metalloproteinase-9 substrates in the left ventricle post-myocardial infarctionPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 11 2010Rogelio Zamilpa Abstract Matrix metalloproteinase-9 (MMP-9) deletion has been shown to improve remodeling of the left ventricle post-myocardial infarction (MI), but the mechanisms to explain this improvement have not been fully elucidated. MMP-9 has a broad range of in vitro substrates, but relevant in vivo substrates are incompletely defined. Accordingly, we evaluated the infarct regions of wild-type (wt) and MMP-9 null (null) mice using a proteomic strategy. Wt and null groups showed similar infarct sizes (48±3 in wt and 45±3% in null), indicating that both groups received an equal injury stimulus. Left ventricle infarct tissue was homogenized and analyzed by 2-DE and MS. Of 31 spot intensity differences, the intensities of 9 spots were higher and 22 spots were lower in null mice compared to wt (all p<0.05). Several extracellular matrix proteins were identified in these spots by MS, including fibronectin, tenascin-C, thrombospondin-1, and laminin. Fibronectin was observed on the gels at a lower than expected molecular weight in the wt group, which suggested substrate cleavage, and the lower molecular weight spot was observed at lower intensity in the MMP-9 null group, which suggested cleavage by MMP-9. Immunoblotting confirmed the presence of fibronectin cleavage products in the wt samples and lower levels in the absence of MMP-9. In conclusion, examining infarct tissue from wt and MMP-9 null mice by proteomic analysis provides a powerful and unique method to identify in vivo candidate MMP substrates. [source] Proteomic analysis of fast and slow muscles from normal and kyphoscoliotic mice using protein arrays, 2-DE and MSPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 16 2006Marie-Catherine Le Bihan Abstract A proteomic strategy based upon the integrated use of SELDI-TOF/MS, 2-DE and MALDI-TOF/MS has been used to identify a panel of fast muscle protein markers: MLC1F, MLC3F, fast troponin,C (STNC) and slow muscle markers: MLC1SB and MLC2v. MLC3F, MLC1F and STNC were virtually absent in the physiologically ,pure slow' soleus muscle of kyphoscoliotic mutant mice compared to control BDmice, whereas MLC2v increased threefold. A SELDI-TOF/MS peak at 18,012,Da in spectra from strong anionic exchange protein array fractions of fast vastus muscle was confirmed as STNC by its specific depletion from crude extracts of vastus muscle using an anti-TNC mAb. SELDI-TOF/MS also identified MLC2F phosphorylation in crude muscle extracts after treatment with alkaline phosphatase. High probability protein identifications were achieved by SELDI-TOF/MS PMF based upon the resolution of large peptides formed by partial cleavage and high peptide coverage. When the pI from 2-D gels and molecular weight estimations from SELDI-TOF/MS were entered into the TagIdent algorithm, high probability protein identity predictions were obtained that were confirmed later by PMF. We confirm that SELDI-TOF/MS can be integrated with other proteomics techniques for the efficient analysis of protein expression changes and PTMs associated with physiological changes in skeletal muscle. [source] Power and limitations of electrophoretic separations in proteomics strategiesMASS SPECTROMETRY REVIEWS, Issue 5 2009Thierry Rabilloud Abstract Proteomics can be defined as the large-scale analysis of proteins. Due to the complexity of biological systems, it is required to concatenate various separation techniques prior to mass spectrometry. These techniques, dealing with proteins or peptides, can rely on chromatography or electrophoresis. In this review, the electrophoretic techniques are under scrutiny. Their principles are recalled, and their applications for peptide and protein separations are presented and critically discussed. In addition, the features that are specific to gel electrophoresis and that interplay with mass spectrometry (i.e., protein detection after electrophoresis, and the process leading from a gel piece to a solution of peptides) are also discussed. © 2008 Wiley Periodicals, Inc., Mass Spec Rev 28:816,843, 2009 [source] Body fluid proteomics: Prospects for biomarker discoveryPROTEOMICS - CLINICAL APPLICATIONS, Issue 9 2007Sung-Min Ahn Abstract Many diseases are caused by perturbations of cellular signaling pathways and related pathway networks as a result of genetic aberrations. These perturbations are manifested by altered cellular protein profiles in the fluids bathing tissue/organs (i.e., the tissue interstitial fluid, TIF). A major challenge of clinical chemistry is to quantitatively map these perturbed protein profiles , the so-called "signatures of disease" , using modern proteomic technologies. This information can be utilized to design protein biomarkers for the early detection of disease, monitoring disease progression and efficacy of drug action. Here, we discuss the use of body fluids in the context of prospective biomarker discovery, and the marked 1000,1500-fold dilution of body fluid proteins, during their passage from TIF to the circulatory system. Further, we discuss proteomics strategies aimed at depleting major serum proteins, especially albumin, in order to focus on low-abundance protein/peptides in plasma. A major limitation of depletion strategies is the removal of low-molecular weight protein/peptides which specifically bind major plasma proteins. We present a prototype model, using albumin, for understanding the multifaceted nature of biomarker research, highlighting the involvement of albumin in Alzheimer's disease. This model underscores the need for a system-level understanding for biomarker research and personalized medicine. [source] Bioaffinity magnetic reactor for peptide digestion followed by analysis using bottom-up shotgun proteomics strategyJOURNAL OF SEPARATION SCIENCE, JSS, Issue 3 2008Lucie Korecká Abstract We report an efficient and streamlined way to improve the analysis and identification of peptides and proteins in complex mixtures of soluble proteins, cell lysates, etc. By using the shotgun proteomics methodology combined with bioaffinity purification we can remove or minimize the interference contamination of a complex tryptic digest and so avoid the time-consuming separation steps before the final MS analysis. We have proved that by means of enzymatic fragmentation (endoproteinases with Arg-C or/and Lys-C specificity) connected with the isolation of specific peptides we can obtain a simplified peptide mixture for easier identification of the entire protein. A new bioaffinity sorbent was developed for this purpose. Anhydrotrypsin (AHT), an inactive form of trypsin with an affinity for peptides with arginine (Arg) or lysine (Lys) at the C-terminus, was immobilized onto micro/nanoparticles with superparamagnetic properties (silica magnetite particles (SiMAG),Carboxyl, Chemicell, Germany). This AHT carrier with a determined binding capacity (26.8 nmol/mg of carrier) was tested with a model peptide, human neurotensin, and the resulting MS spectra confirmed the validity of this approach. [source] A proof-of-principle gel-free proteomics strategy for the identification of predictive biomarkers for the onset of pre-eclampsiaBJOG : AN INTERNATIONAL JOURNAL OF OBSTETRICS & GYNAECOLOGY, Issue 11 2009RT Blankley Objective, Progress in the prevention and treatment of women at risk of pre-eclampsia (PE) still remains hindered by the lack of clinical screening tools that can accurately predict which mothers are at risk. The identification and validation of predictive biomarkers is therefore seen as a critical milestone towards improved healthcare provision and the clinical testing of new therapeutic strategies. Gel-free proteomic technologies offer the capability of analysing hundreds of plasma proteins simultaneously, but as yet these methods have not been applied to pregnancy complications. To assess the feasibility of such an approach to plasma biomarker research in pregnancy we have applied the technique to samples from women with PE to gestation-matched controls. Sample, Pooled plasma samples taken at time of disease from women with PE (n = 23) and gestation-matched controls (n = 23). Methods, Proteomics strategy for relative quantification of proteins using mass spectrometry. Results, We identified several differences, including elevated levels of endoglin, PAPP-A and PSG1 in PE plasma. Increased levels of endoglin were validated using immunoassay analysis of individual plasma samples. Conclusions, Although at a relatively early stage, this mass spectrometry-based approach shows promise as a tool to identify global protein changes in plasma. The application of these methods to pre-disease samples is the next step in the identification of clinically useful biomarkers. [source] | |||||||||||||