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Low-abundance Proteins (low-abundance + protein)
Selected AbstractsBody 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] Cover Picture: Electrophoresis 16'2010ELECTROPHORESIS, Issue 16 2010Article first published online: 19 AUG 2010 Issue no. 16 is a regular issue with an Emphasis on "Proteins and Proteomics" comprising 20 manuscripts distributed over 4 separate parts. Part I has 7 research articles on various aspects of proteins and proteomics including combinatorial peptide ligand library for accessing low abundance proteins, analysis of membrane proteins, proteomic profiling of human colon cancer cells, quantitative determinations of biomarkers in clinical diagnostics, recombinant factor VIII, analysis of E. coli soluble proteins, and a weakly basic amino-reactive fluorescent label for IEF of proteins and chip electrophoresis. Part II has 2 research articles dealing with the CE analysis of magnetic nanoparticles and a microfluidic magnetic bead impact for cell stimulation. Part III consists of 2 research articles dealing with on-line preconcentration in CE. Instrumentation, devices and various methodologies are described in 9 research articles, which make the content of Part IV. Featured articles include: Combinatorial peptide ligand library plasma treatment: Advantages for accessing low-abundance proteins ((doi: 10.1002/elps.201000188)) Precautions to improve the accuracy of quantitative determinations of biomarkers in clinical diagnostics ((doi: 10.1002/elps.201000243)) Rapid identification of Candida albicans in blood by combined capillary electrophoresis and fluorescence in situ hybridization ((doi: 10.1002/elps.201000138)) [source] Proteomics in globe artichoke: Protein extraction and sample complexity reduction by PEG fractionationELECTROPHORESIS, Issue 9 2009Alberto Acquadro Abstract Here, we report the first leaf proteome analysis for globe artichoke. Three protein extraction protocols were tested and a reproducible Mg/NP-40-based method was established. Ribulose-1,5-biphosphate carboxylase-oxygenase (RuBisCO) is a highly abundant leaf protein, and its presence masks co-localizing, less abundant proteins. To remove RuBisCO from the sample, and thereby improve spot resolution, a PEG fractionation approach was elaborated. 2-DE profiles of various PEG fractions showed that the fractionation procedure was successful in excluding most of the RuBisCO, allowing for the detection of many low-abundance proteins. Western blot analysis was able to confirm the reduction in RuBisCO content achieved by PEG fractionation. In all, 841 distinct protein spots were detected, and 40 of these, selected from the RuBisCO region of the 2-DE profile, were successfully identified by MS. A number of homologues of these proteins also co-localize with RuBisCO in Arabidopsis thaliana. [source] Micro-high-performance liquid chromatography platform for the depletion of high-abundance proteins and subsequent on-line concentration/capturing of medium and low-abundance proteins from serum.ELECTROPHORESIS, Issue 16 2008Application to profiling of protein expression in healthy, osteoarthritis sera by 2-D gel electrophoresis No abstract is available for this article. [source] Sample complexity reduction for two-dimensional electrophoresis using solution isoelectric focusing prefractionationELECTROPHORESIS, Issue 12 2008Matthew R. Richardson Abstract Despite its excellent resolving power, 2-DE is of limited use when analyzing cellular proteomes, especially in differential expression studies. Frequently, fewer than 2000 protein spots are detected on a single 2-D gel (a fraction of the total proteome) regardless of the gel platform, sample, or detection method used. This is due to the vast number of proteins expressed and their equally vast dynamic range. To exploit 2-DE unique ability as both an analytical and a preparative tool, the significant sample prefractionation is necessary. We have used solution isoelectric focusing (sIEF) via the ZOOMŪ IEF Fractionator (Invitrogen) to generate sample fractions from complex bacterial lysates, followed by parallel 2-DE, using narrow-range IPG strips that bracket the sIEF fractions. The net result of this process is a significant enrichment of the bacterial proteome resolved on multiple 2-D gels. After prefractionation, we detected 5525 spots, an approximate 3.5-fold increase over the 1577 spots detected in an unfractionated gel. We concluded that sIEF is an effective means of prefractionation to increase depth of field and improve the analysis of low-abundance proteins. [source] Comprehensive proteome analysis of mouse liver by ampholyte-free liquid-phase isoelectric focusingELECTROPHORESIS, Issue 11 2008Hua Zhong Abstract In this study, ampholyte-free liquid-phase IEF (LIEF) was combined with narrow pH range 2-DE and SDS-PAGE RP-HPLC for comprehensive analysis of mouse liver proteome. Because LIEF prefractionation was able to reduce the complexity of the sample and enhance the loading capacity of IEF strips, the number of visible protein spots on subsequent 2-DE gels was significantly increased. A total of 6271 protein spots were detected after integrating five narrow pH range 2-DE gels following LIEF prefractionation into a single virtual 2-DE gel. Furthermore, the pH,3,5 LIEF fraction and the unfractionated sample were separated by pH,3,6 2-DE and identified by MALDI-TOF/TOF MS, respectively. In parallel, the pH 3,5 LIEF fraction was also analyzed by SDS-PAGE RP-HPLC MS/MS. LIEF-2-DE and LIEF-HPLC could obviously improve the separation efficiency and the confidence of protein identification, which identified a higher number of low-abundance proteins and proteins with extreme physicochemical characteristics or post-translational modifications compared to conventional 2-DE method. Furthermore, there were 207 proteins newly identified in mouse liver in comparison with previously reported large-scale datasets. It was observed that the combination of LIEF-2-DE and LIEF-HPLC was effective in promoting MS-based liver proteome profiling and could be applied on similar complex tissue samples. [source] Removal of high-abundance proteins for nuclear subproteome studies in rice (Oryza sativa) endospermELECTROPHORESIS, Issue 3 2008Guosheng Li Abstract Endosperm is a highly specialized storage organ with three sets of genomes. It is one of the most economically important organs in plants. Endosperm development involves parental imprinting and endoreduplication. A thorough study of the endosperm proteome, particularly the nuclear proteome, may provide critical insight into the regulation of seed development. Unfortunately, endosperm is extremely rich in starch grains and protein bodies of different sizes, making proteome studies on nonstorage proteins, particularly the low-abundance proteins, very challenging. Here we have developed a chromatographic method to remove large starch grains and an electrophoresis method to recover low-abundance proteins, respectively. Using these methods, we have identified 468 proteins from the nuclear enriched fraction of rice endosperm, including transcription factors, histone modification proteins, kinetochore proteins, centromere/microtubule binding proteins, and transposon proteins. Among the 468 proteins, 208 (44%) are hypothetical proteins, indicating that the endosperm proteome is poorly explored. In addition, analyses of the MS/MS data using BioWorks 3.1 have identified 59 putative acetylated proteins and 40 putative methylated proteins. Our studies have developed a method to remove starch grains and recover low-abundance proteins, respectively. The methods should be applicable to other organisms. [source] Biomarker discovery in breast cancer serum using 2-D differential gel electrophoresis/ MALDI-TOF/TOF and data validation by routine clinical assaysELECTROPHORESIS, Issue 8 2006Hong-Lei Huang Abstract In the present study, we used 2-D differential gel electrophoresis (2-D DIGE) and MS to screen biomarker candidates in serum samples obtained from 39,patients with breast cancer and 35,controls. First, we pooled the serum samples matched with age and menopausal status. Then, we depleted the two most abundant proteins albumin and IgG by immunoaffinity chromatography under partly denaturing conditions in order to enrich low-abundance proteins and proteins with low molecular weight. Concentrated and desalted samples were labeled with three different CyDyes including one internal standard, pooled from all the samples, and separated with 2-D DIGE in triplicate experiments. Biological variations of the protein expression level were analyzed with DeCyder software and evaluated for reproducibility and statistical significance. The profile of differentially expressed protein spots between patients and controls revealed proapolipoprotein A-I, transferrin, and hemoglobin as up-regulated and three spots, apolipoprotein,A-I, apolipoprotein,C-III, and haptoglobin,,2 as down-regulated in patients. Finally, routine clinical immunochemical reactions were used to validate selected candidate biomarkers by quantitative determination of specific proteins in all individual serum samples. The serum level of transferrin correlated well with the 2-D-DIGE results. However, the serum levels of apolipoprotein A-I and haptoglobin could not be detected with the clinical routine diagnostic tests. This demonstrated an advantage 2-D DIGE still has over other techniques. 2-D DIGE can distinguish between isoforms of proteins, where the overall immunochemical quantification does fail due to a lack of isoform-special antibodies. [source] Detergent addition to tryptic digests and ion mobility separation prior to MS/MS improves peptide yield and protein identification for in situ proteomic investigation of frozen and formalin-fixed paraffin-embedded adenocarcinoma tissue sectionsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 10 2009Marie-Claude Djidja Abstract The identification of proteins involved in tumour progression or which permit enhanced or novel therapeutic targeting is essential for cancer research. Direct MALDI analysis of tissue sections is rapidly demonstrating its potential for protein imaging and profiling in the investigation of a range of disease states including cancer. MALDI-mass spectrometry imaging (MALDI-MSI) has been used here for direct visualisation and in situ characterisation of proteins in breast tumour tissue section samples. Frozen MCF7 breast tumour xenograft and human formalin-fixed paraffin-embedded breast cancer tissue sections were used. An improved protocol for on-tissue trypsin digestion is described incorporating the use of a detergent, which increases the yield of tryptic peptides for both fresh frozen and formalin-fixed paraffin-embedded tumour tissue sections. A novel approach combining MALDI-MSI and ion mobility separation MALDI-tandem mass spectrometry imaging for improving the detection of low-abundance proteins that are difficult to detect by direct MALDI-MSI analysis is described. In situ protein identification was carried out directly from the tissue section by MALDI-MSI. Numerous protein signals were detected and some proteins including histone H3, H4 and Grp75 that were abundant in the tumour region were identified. [source] Large scale depletion of the high-abundance proteins and analysis of middle- and low-abundance proteins in human liver proteome by multidimensional liquid chromatographyPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 5 2008Mingxia Gao Abstract An unbiased method for large-scale depletion of high-abundance proteins and identification of middle- or low-abundance proteins by multidimensional LC (MDLC) was demonstrated in this paper. At the protein level, the MDLC system, coupling the first dimensional strong cation exchange (SCX) chromatography with the second dimensional RP-HPLC, instead of immunoaffinity technology, was used to deplete high-abundance proteins. Sixty-two fractions from SCX were separated further by RPLC. UV absorption spectra were observed to differentiate high-abundance proteins from middle- or low-abundance proteins. After the depletion of high-abundance proteins, middle- or low-abundance proteins were enriched, digested, and separated by online 2D-micro-SCX/cRPLC. The eluted peptides were deposited on the MALDI target and detected by MALDI-TOF/TOF MS. This depletion strategy was applied to the proteome of the normal human liver (NHL) provided by the China Human Liver Proteome Project (CHLPP). In total, 58 high-abundance proteins were depleted in one experiment. The strategy increases greatly the number of identified proteins and around 1213 proteins were identified, which was about 2.7 times as that of the nondepletion method. [source] A novel approach and protocol for discovering extremely low-abundance proteins in serumPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 17 2006Yoshinori Tanaka Abstract The proteomic analysis of serum (plasma) has been a major approach to determining biomarkers essential for early disease diagnoses and drug discoveries. The determination of these biomarkers, however, is analytically challenging since the dynamic concentration range of serum proteins/peptides is extremely wide (more than 10,orders of magnitude). Thus, the reduction in sample complexity prior to proteomic analyses is essential, particularly in analyzing low-abundance protein biomarkers. Here, we demonstrate a novel approach to the proteomic analyses of human serum that uses an originally developed serum protein separation device and a sequentially linked 3-D-LC-MS/MS system. Our hollow-fiber-membrane-based serum pretreatment device can efficiently deplete high-molecular weight proteins and concentrate low-molecular weight proteins/peptides automatically within 1,h. Four independent analyses of healthy human sera pretreated using this unique device, followed by the 3-D-LC-MS/MS successfully produced 12,000,13,000 MS/MS spectra and hit around 1800,proteins (>95% reliability) and 2300,proteins (>80% reliability). We believe that the unique serum pretreatment device and proteomic analysis protocol reported here could be a powerful tool for searching physiological biomarkers by its high throughput (3.7,days per one sample analysis) and high performance of finding low abundant proteins from serum or plasma samples. [source] | ||||||||||