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At least 649 records · Page 36Linked to original sources
PubMed2005

Analysis of phosphatase and tensin homolog tumor suppressor interacting proteins by in vitro and in silico proteomics.

The phosphatase and tensin homolog (PTEN) tumor suppressor is a multifunctional protein deregulated in many types of cancer. To date, a comprehensive documentation of PTEN interacting proteins has not been performed. The goal of our study was to characterize the PTEN interactome using affinity pull-down and tandem mass spectrometry (MS/MS). Wild-type PTEN cDNA was inserted into pTRC-His2 vector to create a 6-His tagged protein, which was expressed in Escherichia coli. Lysate from a human lymphoma cell line was used in pull-down assays, utilizing affinity for nickel-agarose beads. Bound proteins were eluted with imidazole, digested and analyzed on an LCQ DecaXP ion trap mass spectrometer. The nickel affinity pull-down efficiency was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Acquired data were searched against the NCBI nr.fasta nonredundant protein database using the SEQUEST algorithm and screened using INTERACT and ProteinProphet. All experiments were performed in duplicate with 6-His-lacZ serving as control. A total of 79 proteins were identified in the wild-type 6-His-PTEN pull-down by MS/MS. We further validated a subset of the proteins present in the PTEN interactome by performing immunoprecipitation using an anti-PTEN antibody and establishing the presence of the proteins in the immunocomplex by Western blot analysis. A search of published PTEN interactions was also performed using Online Mendelian Inheritance in Man, Human Protein Reference Database, the IntAct Project database, and PubMed. This in silico analysis confirmed 42 out of 79 (53%) of the proteins identified by MS/MS. The remaining 37 proteins represent probable PTEN interactions not previously documented in public databases or reported in the literature. These results highlight the value of combining both in vitro biochemical approaches with in silico analyses for a comprehensive study of protein-protein interactions.

Algorithms↗
PubMed2002

Isolation of protein subpopulations undergoing protein-protein interactions.

A new method is described for isolating and identifying proteins participating in protein-protein interactions in a complex mixture. The method uses a cyanogen bromide-activated Sepharose matrix to isolate proteins that are non-covalently bound to other proteins. Because the proteins are accessible to chemical manipulation, mass spectrometric identification of the proteins can yield information on specific classes of interacting proteins, such as calcium-dependent or substrate-dependent protein interactions. This permits selection of a subpopulation of proteins from a complex mixture on the basis of specified interaction criteria. The new method has the advantage of screening the entire proteome simultaneously, unlike the two-hybrid system or phage display, which can only detect proteins binding to a single bait protein at a time. The method was tested by selecting rat brain extract for proteins exhibiting calcium-dependent protein interactions. Of 12 proteins identified by mass spectrometry, eight were either known calcium-binding proteins or proteins with known calcium-dependent protein interactions, indicating that the method is capable of enriching a subpopulation of proteins from a complex mixture on the basis of a specific class of protein interactions. Because only naturally occurring interactions of proteins in their native state are observed, this method will have wide applicability to studies of protein interactions in tissue samples and autopsy specimens, for screening for perturbations of protein-protein interactions by signaling molecules, pharmacological agents or toxins, and screening for differences between cancerous and untransformed cells.

Animals↗
PubMed2005

Intact-protein-based high-resolution three-dimensional quantitative analysis system for proteome profiling of biological fluids.

The substantial complexity and vast dynamic range of protein abundance in biological fluids, notably serum and plasma, present a formidable challenge for comprehensive protein analysis. Integration of multiple technologies is required to achieve high-resolution and high-sensitivity proteomics analysis of biological fluids. We have implemented an orthogonal three-dimensional intact-protein analysis system (IPAS), coupled with protein tagging and immunodepletion of abundant proteins, to quantitatively profile the human plasma proteome. Following immunodepletion, plasma proteins in each of paired samples are concentrated and labeled with a different Cy dye, before mixing. Proteins are subsequently separated in three dimensions according to their charge, hydrophobicity, and molecular mass. Differences in the abundance of resolved proteins are determined based on Cy dye ratios. We have applied this strategy to profile the plasma proteome for changes that occur with acute graft-versus-host disease (GVHD), following allogeneic bone marrow transplantation (BMT). Using capillary HPLC ESI Q-TOF MS, we identified 75 proteins in the micromolar to femtomolar range that exhibited quantitative differences between the pre- and post-GVHD samples. These proteins included serum amyloid A, apolipoproteins A-I/A-IV, and complement C3 that are well-known acute-phase reactants likely reflecting the post-BMT inflammatory state. In addition, we identified some potentially interesting immunologically relevant molecules including vitamin D-binding protein, fetuin, vitronectin, proline-rich protein 3 and 4, integrin-alpha, and leukocyte antigen CD97. IPAS provides a combination of comprehensive profiling and quantitative analysis, with a substantial dynamic range, for disease-related applications.

Adult↗
PubMed2004

Affinity-based screening techniques for enhancing lead discovery.

Contemporary, rational small-molecule lead discovery methods, comprising target identification, assay development, high-throughput screening (HTS), hit characterization and medicinal chemistry optimization, dominate early-stage drug discovery strategies in many pharmaceutical companies. There is a growing disparity between the increasing cost of funding these methods and the decreasing number of new drugs reaching the market. New strategies must be adopted to reverse this trend. The use of genomics- and proteomics-based target discovery efforts can aid the process by dramatically increasing the number of novel, more highly validated targets entering the discovery process, but HTS must meet this increased demand with faster, cheaper technologies. Although activity-based screening strategies are typically efficient, allowing one scientist to interrogate tens of thousands of compounds per day, affinity-based screening strategies can allow much greater efficiency in the overall process. Affinity-based methods can play a role in both facilitating the screening of a greater number of targets and in efficiently characterizing the primary hits discovered.

Drug Design↗
PubMed2002

Proteomics gives insight into the regulatory function of chloroplast thioredoxins.

Thioredoxins are small multifunctional redox active proteins widely if not universally distributed among living organisms. In chloroplasts, two types of thioredoxins (f and m) coexist and play central roles in regulating enzyme activity. Reduction of thioredoxins in chloroplasts is catalyzed by an iron-sulfur disulfide enzyme, ferredoxin-thioredoxin reductase, that receives photosynthetic electrons from ferredoxin, thereby providing a link between light and enzyme activity. Chloroplast thioredoxins function in the regulation of the Calvin cycle and associated processes. However, the relatively small number of known thioredoxin-linked proteins (about 16) raised the possibility that others remain to be identified. To pursue this opportunity, we have mutated thioredoxins f and m, such that the buried cysteine of the active disulfide has been replaced by serine or alanine, and bound them to affinity columns to trap target proteins of chloroplast stroma. The covalently linked proteins were eluted with DTT, separated on gels, and identified by mass spectrometry. This approach led to the identification of 15 potential targets that function in 10 chloroplast processes not known to be thioredoxin linked. Included are proteins that seem to function in plastid-to-nucleus signaling and in a previously unrecognized type of oxidative regulation. Approximately two-thirds of these targets contained conserved cysteines. We also identified 11 previously unknown and 9 confirmed target proteins that are members of pathways known to be regulated by thioredoxin. In contrast to results with individual enzyme assays, specificity for thioredoxin f or m was not observed on affinity chromatography.

Chloroplasts↗
PubMed2005

Proteomics analysis of growth hormone isoforms in the human pituitary.

In order to elucidate the roles of human growth hormone (hGH) in the normal (control) pituitary and in adenomas, the hGH isoforms in the human pituitary were analyzed with two-dimensional gel electrophoresis, immobilized metal affinity column (Ga(+3)) chromatography, mass spectrometry (MS), and bioinformatics. Twenty-four hGH-containing proteins, with significantly different expression proportions of their isoforms were found. The proportions of isoforms were as follows: isoform 1 (87.5%) > isoform 2 (8.1%) > isoform 3 (3.3%) > isoform 4 (1.1%). Deamidation of asparagine to aspartate was identified with matrix-assisted laser desorption/ionization-time of flight MS. Tandem mass spectrometry data demonstrated that hGH is a phosphoprotein (spot 6); phosphorylation was found at Ser-77 in the tryptic peptide (68)YSFLQNPQTSLCFSESIPTPSNR(90), at Ser-176 in the tryptic peptide (172)FDTNSHNDDALLK(184), and at Ser-132 in the peptide (126)SLVYGASDSNVYDLLK(141). The phosphorylation sites at Ser-77 and Ser-176 were consistent with computer-program predictions (NetPhos). These results provide novel clues for further studies of the functions, and mechanisms of action, of hGH in the human pituitary and in growth hormone-related diseases.

Alternative Splicing↗
PubMed2006

Proteomic identification of glucocorticoid receptor interacting proteins.

The glucocorticoid receptor (GR) acts as a ligand dependent transcription factor but can also cross talk with other signaling pathways via protein-protein interactions. In this paper we describe methods to study novel cytosolic GR interacting proteins, using mAb based immunoaffinity chromatography of GR from rat liver cytosol. Co-purifying proteins were identified by 2-DE in combination with MALDI-TOF-MS. Non-liganded/non-activated and in vitro liganded/activated GR, respectively, co-purifies with specific sets of proteins. Of these 34 were conclusively identified, seven have previously been reported to be part of the GR-complex, revealing 27 new possible interacting candidates for the GR-complex. Of the novel GR interacting proteins the major vault protein, TATA binding interacting protein 49a and glycoprotein PP63 were of special interest. Furthermore, using 2-D DIGE we show that the set of proteins interacting with non-liganded GR is distinctly different in protein amount compared to the proteins found with liganded/activated GR. This suggests the presence of different GR complexes in the cell, which was further substantiated by the finding of several separate GR native protein complexes, "GR-receptosomes", using blue native gel electrophoresis. Our findings suggest the existence of several new mechanisms for GR signaling and regulation.

Animals↗
PubMed2005

Investigating diversity in human plasma proteins.

Plasma proteins represent an important part of the human proteome. Although recent proteomics research efforts focus largely on determining the overall number of proteins circulating in plasma, it is equally important to delineate protein variations among individuals, because they can signal the onset of diseases and be used as biological markers in diagnostics. To date, there has been no systematic proteomics effort to characterize the breadth of structural modifications in individual proteins in the general population. In this work, we have undertaken a population proteomics study to define gene- and protein-level diversity that is encountered in the general population. Twenty-five plasma proteins from a cohort of 96 healthy individuals were investigated through affinity-based mass spectrometric assays. A total of 76 structural forms/variants were observed for the 25 proteins within the samples cohort. Posttranslational modifications were detected in 18 proteins, and point mutations were observed in 4 proteins. The frequency of occurrence of these variations was wide-ranged, with some modifications being observed in only one sample, and others detected in all 96 samples. Even though a relatively small cohort of individuals was investigated, the results from this study illustrate the extent of protein diversity in the human population and can be of immediate aid in clinical proteomics/biomarker studies by laying a basal-level statistical foundation from which protein diversity relating to disease can be evaluated.

Adolescent↗
PubMed2003

Sensitive quantitative predictions of peptide-MHC binding by a 'Query by Committee' artificial neural network approach.

We have generated Artificial Neural Networks (ANN) capable of performing sensitive, quantitative predictions of peptide binding to the MHC class I molecule, HLA-A*0204. We have shown that such quantitative ANN are superior to conventional classification ANN, that have been trained to predict binding vs non-binding peptides. Furthermore, quantitative ANN allowed a straightforward application of a 'Query by Committee' (QBC) principle whereby particularly information-rich peptides could be identified and subsequently tested experimentally. Iterative training based on QBC-selected peptides considerably increased the sensitivity without compromising the efficiency of the prediction. This suggests a general, rational and unbiased approach to the development of high quality predictions of epitopes restricted to this and other HLA molecules. Due to their quantitative nature, such predictions will cover a wide range of MHC-binding affinities of immunological interest, and they can be readily integrated with predictions of other events involved in generating immunogenic epitopes. These predictions have the capacity to perform rapid proteome-wide searches for epitopes. Finally, it is an example of an iterative feedback loop whereby advanced, computational bioinformatics optimize experimental strategy, and vice versa.

HLA-A Antigens↗
PubMed2005

Engineering novel binding proteins from nonimmunoglobulin domains.

Not all adaptive immune systems use the immunoglobulin fold as the basis for specific recognition molecules: sea lampreys, for example, have evolved an adaptive immune system that is based on leucine-rich repeat proteins. Additionally, many other proteins, not necessarily involved in adaptive immunity, mediate specific high-affinity interactions. Such alternatives to immunoglobulins represent attractive starting points for the design of novel binding molecules for research and clinical applications. Indeed, through progress and increased experience in library design and selection technologies, gained not least from working with synthetic antibody libraries, researchers have now exploited many of these novel scaffolds as tailor-made affinity reagents. Significant progress has been made not only in the basic science of generating specific binding molecules, but also in applications of the selected binders in laboratory procedures, proteomics, diagnostics and therapy. Challenges ahead include identifying applications where these novel proteins can not only be an alternative, but can enable approaches so far deemed technically impossible, and delineate those therapeutic applications commensurate with the molecular properties of the respective proteins.

Binding Sites↗
PubMed2004

New thioredoxin targets in the unicellular photosynthetic eukaryote Chlamydomonas reinhardtii.

Proteomics were used to identify the proteins from the eukaryotic unicellular green alga Chlamydomonas reinhardtii that can be reduced by thioredoxin. These proteins were retained specifically on a thioredoxin affinity column made of a monocysteinic thioredoxin mutant able to form mixed disulfides with its targets. Of a total of 55 identified targets, 29 had been found previously in higher plants or Synechocystis, but 26 were new targets. Biochemical tests were performed on three of them, showing a thioredoxin-dependent activation of isocitrate lyase and isopropylmalate dehydrogenase and a thioredoxin-dependent deactivation of catalase that is redox insensitive in Arabidopsis. In addition, we identified a Ran protein, a previously uncharacterized nuclear target in a photosynthetic organism. The metabolic and evolutionary implications of these findings are discussed.

Acetates↗
PubMed2003

Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice.

Proteomic approaches require simple and efficient protein purification methodologies that are amenable to high throughput. Biotinylation is an attractive approach for protein complex purification due to the very high affinity of avidin/streptavidin for biotinylated templates. Here, we describe an approach for the single-step purification of transcription factor complex(es) based on specific in vivo biotinylation. We expressed the bacterial BirA biotin ligase in mammalian cells and demonstrated very efficient biotinylation of a hematopoietic transcription factor bearing a small (23-aa) artificial peptide tag. Biotinylation of the tagged transcription factor altered neither the factor's protein interactions or DNA binding properties in vivo nor its subnuclear distribution. Using this approach, we isolated the biotin-tagged transcription factor and at least one other known interacting protein from crude nuclear extracts by direct binding to streptavidin beads. Finally, this method works efficiently in transgenic mice, thus raising the prospect of using biotinylation tagging in protein complex purification directly from animal tissues. Therefore, BirA-mediated biotinylation of tagged proteins provides the basis for the single-step purification of proteins from mammalian cells.

Animals↗
PubMed2006

Increased solubility of integrin betaA domain using maltose-binding protein as a fusion tag.

In proteomics research, generation of recombinant proteins in their native, soluble form with large quantity is often a challenging task. To tackle the expression difficulties, different expression vectors with distinct affinity fusion tags, i.e. pET-43.1a (N-utilization substance A tag), pMAL-cRI (maltose binding protein tag) (MBP tag), pGEX-4T-2 (glutathione S-transferase tag), and pET-15b (hexahistidine tag) were compared for their effects on the productivity and solubility, which were assessed by SDS-PAGE and immunoblotting, of the integrin betaA domain. The incubation temperatures were tested for its effects on these parameters. Our data suggested that MBP tag enhanced the yield and solubility of the betaA domain protein, which can also be recognized using an anti-CD18 antibody, at room temperature incubation. Thus, the nature of fusion partner chosen for expression in bacteria and its incubation temperature would significantly affect the yield and solubility of the recombinant target protein.

Animals↗
PubMed2005

Evidence for the presence of disease-perturbed networks in prostate cancer cells by genomic and proteomic analyses: a systems approach to disease.

Prostate cancer is initially responsive to androgen ablation therapy and progresses to androgen-unresponsive states that are refractory to treatment. The mechanism of this transition is unknown. A systems approach to disease begins with the quantitative delineation of the informational elements (mRNAs and proteins) in various disease states. We employed two recently developed high-throughput technologies, massively parallel signature sequencing (MPSS) and isotope-coded affinity tag, to gain a comprehensive picture of the changes in mRNA levels and more restricted analysis of protein levels, respectively, during the transition from androgen-dependent LNCaP (model for early-stage prostate cancer) to androgen-independent CL1 cells (model for late-stage prostate cancer). We sequenced >5 million MPSS signatures, obtained >142,000 tandem mass spectra, and built comprehensive MPSS and proteomic databases. The integrated mRNA and protein expression data revealed underlying functional differences between androgen-dependent and androgen-independent prostate cancer cells. The high sensitivity of MPSS enabled us to identify virtually all of the expressed transcripts and to quantify the changes in gene expression between these two cell states, including functionally important low-abundance mRNAs, such as those encoding transcription factors and signal transduction molecules. These data enable us to map the differences onto extant physiologic networks, creating perturbation networks that reflect prostate cancer progression. We found 37 BioCarta and 14 Kyoto Encyclopedia of Genes and Genomes pathways that are up-regulated and 23 BioCarta and 22 Kyoto Encyclopedia of Genes and Genomes pathways that are down-regulated in LNCaP cells versus CL1 cells. Our efforts represent a significant step toward a systems approach to understanding prostate cancer progression.

Cell Line, Tumor↗
PubMed2006

Proteomic identification of palmitoylated proteins.

A proteomic method that purifies and identifies palmitoylated proteins from complex protein extracts is described. Using the fatty acid exchange labeling chemistry (described in the preceding report), palmitoyl modifications are exchanged for biotinylated compounds, allowing the subset of palmitoyl-proteins to be affinity-purified and then identified by mass spectroscopic protein identification technologies. The advantages and pitfalls of this new technology are discussed within the context of the recent application of this method in the yeast Saccharomyces cerevisiae.

Alkylation↗
PubMed2004

Pentamerization of single-domain antibodies from phage libraries: a novel strategy for the rapid generation of high-avidity antibody reagents.

We describe a novel type of molecule in which single-domain antibodies (sdAbs) isolated from a nai;ve llama single domain antibody library are linked to an oligomerization domain to generate high-avidity, antigen-binding reagents. An sdAb is fused to the B-subunit of Escherichia coli verotoxin, or shiga-like toxin, which self-assembles to form a homopentamer and results in simultaneous sdAb pentamerization and introduction of avidity. Molecular modeling indicated that this fusion protein (PDB: 1OJF), termed pentabody, has structural flexibility for binding to surface-presented antigen. In the instance of an sdAb specific for a peptide antigen, pentamerization resulted in a dramatic increase in functional affinity for immobilized antigen. The pentabody was expressed in high yield in E.coli in a non-aggregated state, and exhibited excellent thermostability and protease resistance. This technology provides a relatively rapid means of generating novel antigen-binding molecules that bind strongly to immobilized antigen. It is expected that pentavalent sdAbs will have general applicability in proteomics, immunochemical staining, cancer diagnosis and other applications in which antigens are presented multivalently.

Animals↗
PubMed2004

A tagging-via-substrate technology for detection and proteomics of farnesylated proteins.

A recently developed proteomics strategy, designated tagging-via-substrate (TAS) approach, is described for the detection and proteomic analysis of farnesylated proteins. TAS technology involves metabolic incorporation of a synthetic azido-farnesyl analog and chemoselective derivatization of azido-farnesyl-modified proteins by an elegant version of Staudinger reaction, pioneered by the Bertozzi group, using a biotinylated phosphine capture reagent. The resulting protein conjugates can be specifically detected and/or affinity-purified by streptavidin-linked horseradish peroxidase or agarose beads, respectively. Thus, the technology enables global profiling of farnesylated proteins by enriching farnesylated proteins and reducing the complexity of farnesylation subproteome. Azido-farnesylated proteins maintain the properties of protein farnesylation, including promoting membrane association, Ras-dependent mitogen-activated protein kinase kinase activation, and inhibition of lovastatin-induced apoptosis. A proteomic analysis of farnesylated proteins by TAS technology revealed 18 farnesylated proteins, including those with potentially novel farnesylation motifs, suggesting that future use of this method is likely to yield novel insight into protein farnesylation. TAS technology can be extended to other posttranslational modifications, such as geranylgeranylation and myristoylation, thus providing powerful tools for detection, quantification, and proteomic analysis of posttranslationally modified proteins.

Animals↗
PubMed2001

Chemical characterization of the lectin from Amaranthus leucocarpus syn. hypocondriacus by 2-D proteome analysis.

In this work, we characterized chemically the N-acetyl-D-galactosamine specific lectin from Amaranthus leucocarpus syn hypocondriacus lectin (ALL). It is a dimeric glycoprotein composed by three isoforms with pl at 4.8, 4.9, and 5.2. Circular dichroism analysis indicated that the secondary structure of ALL contains 45% of \bibeta-sheet and 5% of \bialpha-helix. Amino acid sequence of the purified lectin and its isoforms was determined from peptides obtained after trypsin digestion by MALDI-TOF (Matrix assisted laser desorption ionization-time of flight). The tryptic peptides prepared from the purified lectin and the three isoforms showed different degrees (80 to 83%) of identity with the amino acid sequence belonging to a previously described high nutritional value protein from A. hypocondriacus not shown at the time to be a lectin. Furthermore, analysis of tryptic peptides obtained from ALL previously treated with peptide N-glycosidase, revealed a 93% identity with the aforementioned protein. Presence of N-glycosidically linked glycans of the oligomannosidic type and, in minor proportion, of the N-acetyllactosaminic type glycans was determined by affinity chromatography on immobilized Con A.

Amaranthus↗