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Target Peptide (target + peptide)
Selected AbstractsPreparation, characterization, and binding profile of molecularly imprinted hydrogels for the peptide hepcidinJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 8 2010Vincenzo Abbate Abstract Molecularly imprinted hydrogels for the capture of the peptide hormone hepcidin were prepared by water-in-oil (w/o) suspension polymerization under mild conditions. Spherical and relatively uniformly sized gel beads were routinely obtained after optimization of the synthetic methodology. The polymers were analyzed by Fourier transform infrared spectroscopy, optical microscopy, and scanning electron microscopy. Although the imprinted materials exhibited higher affinity towards the epitope template (hepcidin N -terminus) than their corresponding blank polymers, the full-length target peptide was found strongly bound to all the hydrogels tested. However, by using whole fluorescent hepcidin as the print species, the imprinting effect was more pronounced. Moreover, bovine serum albumin did not bind to the poly N -isopropylacrylamide (PNIPAm)-based polymers. Thus, polymeric "sponges" for biomacromolecules with size-exclusion effect were developed, useful for peptide concentration, immobilization and/or purification from serum samples. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 1721,1731, 2010 [source] Biochemical properties of V91G calmodulin: A calmodulin point mutation that deregulates muscle contraction in DrosophilaPROTEIN SCIENCE, Issue 12 2004Bo Wang Abstract A mutation (Cam7) to the single endogenous calmodulin gene of Drosophila generates a mutant protein with valine 91 changed to glycine (V91G D-CaM). This mutation produces a unique pupal lethal phenotype distinct from that of a null mutation. Genetic studies indicate that the phenotype reflects deregulation of calcium fluxes within the larval muscles, leading to hypercontraction followed by muscle failure. We investigated the biochemical properties of V91G D-CaM. The effects of the mutation on free CaM are minor: Calcium binding, and overall secondary and tertiary structure are indistinguishable from those of wild type. A slight destabilization of the C-terminal domain is detectable in the calcium-free (apo-) form, and the calcium-bound (holo-) form has a somewhat lower surface hydrophobicity. These findings reinforce the indications from the in vivo work that interaction with a specific CaM target(s) underlies the mutant defects. In particular, defective regulation of ryanodine receptor (RyR) channels was indicated by genetic interaction analysis. Studies described here establish that the putative CaM binding region of the Drosophila RyR (D-RyR) binds wild-type D-CaM comparably to the equivalent CaM-RyR interactions seen for the mammalian skeletal muscle RyR channel isoform (RYR1). The V91G mutation weakens the interaction of both apo- and holo-D-CaM with this binding region, and decreases the enhancement of the calcium-binding affinity of CaM that is detectable in the presence of the RyR target peptide. The predicted functional consequences of these changes are consonant with the in vivo phenotype, and indicate that D-RyR is one, if not the major, target affected by the V91G mutation in CaM. [source] Development of an immuno tandem mass spectrometry (iMALDI) assay for EGFR diagnosisPROTEOMICS - CLINICAL APPLICATIONS, Issue 12 2007Jian Jiang Abstract The epidermal growth factor receptor (EGFR) is highly expressed in a variety of tumors, and is therefore an important biomarker for cancer diagnosis and a target for cancer therapy. We have developed a novel peptide-based immuno tandem mass spectrometry (iMALDI) diagnostic assay for highly sensitive, highly specific, and quantitative analysis of EGFR, which we have applied to the detection of the EGFR peptide in three cell lines and in a tumor biopsy sample. This assay is capable of detecting the EGFR target peptide bound to the antibody beads at attomole levels. The ability to directly obtain amino acid sequence data by MS/MS on any affinity-captured peptides provides specificity to this diagnostic technique. This avoids the problem of "false positives" which can result from the nonspecific binding that can occur with any affinity-based technique. The addition of stable-labeled versions of the target peptide (synthesized from stable-isotope coded amino acids) as internal standards allows absolute quantitation of the target protein. [source] Highly efficient and selective enrichment of peptide subsets combining fluorous chemistry with reversed-phase chromatographyRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 24 2009Wantao Ying The selective capture of target peptides poses a great challenge to modern chemists and biologists, especially when enriching them from proteome samples possessing extremes in concentration dynamic range and sequence diversity. While approaches based on traditional techniques such as biotin-avidin pairing offer versatile tools to design strategies for selective enrichment, problems are still encountered due to sample loss or poor selectivity of enrichment. Here we show that the recently introduced fluorous chemistry approach has attractive properties as an alternative method for selective enrichment. Through appending a perfluorine group to the target peptide, it is possible to dramatically increase the peptide's hydrophobicity and thus enable facile separation of labeled from non-labeled peptides. Use of reversed-phase chromatography allowed for improved peptide recovery in comparison with results obtained using the formerly reported fluorous bonded phase methods. Furthermore, this approach also allowed for on-line separation and identification of both labeled and unlabeled peptides in a single experiment. The net result is an increase in the confidence of protein identification by tandem mass spectrometry (MS2) as all peptides and subsequent information are retained. Successful off-line and on-line enrichment of cysteine-containing peptides was obtained, and high quality MS2 spectra were obtained by tandem mass spectrometry due to the stability of the tag, allowing for facile identification via standard database searching. We believe that this strategy holds great promise for selective enrichment and identification of low abundance target proteins or peptides. Copyright © 2009 John Wiley & Sons, Ltd. [source] Induction of leukemia-specific antibodies by immunotherapy with leukemia-cell-derived heat shock protein 70CANCER SCIENCE, Issue 7 2008Junko Jimbo Cancer immunotherapy using heat shock protein (HSP) derived from autologous tumor requires cluster of differentiation (CD)4+ as well as CD8+ T-cells for the prolongation of patient survival, suggesting that a humoral immune response through CD4+ T-cells is important in addition to cellular immunity. However, the role of humoral responses in HSP-based autologous tumor immunotherapy remains unclear. In the present study, we investigated whether leukemia-specific antibodies and antibody-mediated cytotoxicity against autologous leukemia cells have a crucial role in a mouse A20 leukemia model by immunizing A20-derived HSP70. Immunization with A20-derived HSP70 induced the production of anti-A20-antibodies and the antibodies recognized HSP70-binding peptides derived from A20. One of those was a major histocompatibility complex (MHC) class-I binding peptide, which has been clarified as the target peptide of CD8+ cytotoxic T-cells (CTL) against A20. The anti-A20-antibodies produced by immunization with A20-derived HSP70 induced complement-dependent cytotoxicity (CDC) against A20 in vitro. In addition, immunization with A20-derived HSP70 increased intracellular interleukin-4 (IL4)-production of CD4+ T-cells, confirming the activation of type-2 helper T-cells. Taken together, immunization with leukemia-cell-derived HSP70 induces antibodies against leukemia-cell-specific peptides and might play a crucial role in the eradication of leukemia cells by CDC in mice. These findings will enable future establishment of a novel therapeutic strategy using antileukemia antibodies in HSP-based autologous tumor immunotherapy. (Cancer Sci 2008; 99: 1427,1434) [source] "Click Peptides",Chemical Biology-Oriented Synthesis of Alzheimer's Disease-Related Amyloid , Peptide (A,) Analogues Based on the "O- Acyl Isopeptide Method"CHEMBIOCHEM, Issue 10 2006Youhei Sohma Abstract A clear understanding of the pathological mechanism of amyloid , peptide (A,) 1,42, a currently unexplained process, would be of great significance for the discovery of novel drug targets for Alzheimer's disease (AD) therapy. To date, though, the elucidation of these A,1,42 dynamic events has been a difficult issue because of uncontrolled polymerization, which also poses a significant obstacle in establishing experimental systems with which to clarify the pathological function of A,1,42. We have recently developed chemical biology-oriented pH- or phototriggered "click peptide" isoform precursors of A,1,42, based on the "O -acyl isopeptide method", in which a native amide bond at a hydroxyamino acid residue, such as Ser, is isomerized to an ester bond, the target peptide subsequently being generated by an O,N intramolecular acyl migration reaction. These click peptide precursors did not exhibit any self-assembling character under physiological conditions, thanks to the presence of the one single ester bond, and were able to undergo migration to give the target A,1,42 in a quick and easy, one-way (so-called "click")conversion reaction. The use of click peptides could be a useful strategy to investigate the biological functions of A,1,42 in AD through inducible activation of A,1,42 self-assembly. [source] Methodology for Multi-Site Ligand,Protein Docking Identification Developed for the Optimization of Spirostenol Inhibition of , -Amyloid-Induced NeurotoxicityCHEMISTRY & BIODIVERSITY, Issue 11 2005Spirostenol steroids have been found to inhibit , -amyloid-induced neurotoxicity. We have evaluated in parallel experimental and molecular-modeling studies the relative effectiveness of 17 (22R)-hydroxycholesterol derivatives in binding to the target peptide. Our results support the previous evidence that , -amyloid offers multiple docking sites for these steroids. Molecular modeling allowed for the correlation of spirostenol candidate structural differences with a choice of proposed active sites. A multi-site identification technique based on a Site-Identifier Matrix (SIM) was developed that clearly showed the uniqueness of our lead (maximum neurotoxicity inhibition) candidate SP233, with a nearly equal docking affinity for two sites. [source] Redox-regulated affinity of the third PDZ domain in the phosphotyrosine phosphatase PTP-BL for cysteine-containing target peptidesFEBS JOURNAL, Issue 13 2005Lieke C. J. Van Den Berk PDZ domains are protein,protein interaction modules that are crucial for the assembly of structural and signalling complexes. They specifically bind to short C-terminal peptides and occasionally to internal sequences that structurally resemble such peptide termini. The binding of PDZ domains is dominated by the residues at the P0 and P,2 position within these C-terminal targets, but other residues are also important in determining specificity. In this study, we analysed the binding specificity of the third PDZ domain of protein tyrosine phosphatase BAS-like (PTP-BL) using a C-terminal combinatorial peptide phage library. Binding of PDZ3 to C-termini is preferentially governed by two cysteine residues at the P,1 and P,4 position and a valine residue at the P0 position. Interestingly, we found that this binding is lost upon addition of the reducing agent dithiothrietol, indicating that the interaction is disulfide-bridge-dependent. Site-directed mutagenesis of the single cysteine residue in PDZ3 revealed that this bridge formation does not occur intermolecularly, between peptide and PDZ3 domain, but rather is intramolecular. These data point to a preference of PTP-BL PDZ3 for cyclic C-terminal targets, which may suggest a redox state-sensing role at the cell cortex. [source] Mass spectrometric identification of the trypsin cleavage pathway in lysyl-proline containing oligotuftsin peptidesJOURNAL OF PEPTIDE SCIENCE, Issue 4 2007Marilena Manea Abstract Trypsin cleaves specifically peptide bonds at the C -terminal side of lysine and arginine residues, except for -Arg-Pro- and -Lys-Pro- bonds which are normally resistant to proteolysis. Here we report evidence for a -Lys-Pro- tryptic cleavage in modified oligotuftsin derivatives, Ac-[TKPKG]4 -NH2) (1), using high-resolution mass spectrometry and HPLC as primary methods for analysis of proteolytic reactions. The proteolytic susceptibility of -Lys-Pro- bonds was strongly dependent on flanking residues, and the flexibility of the peptide backbone might be a prerequisite for this unusual cleavage. While -Lys-Gly- bonds in 1 were rapidly cleaved, the modification of these Lys residues by the attachment of a ß-amyloid(4,10) epitope to yield -Lys(X)-Gly derivatives prevented cleavage of this bond, and provided trypsin cleavage of -Lys-Pro- bonds, the pathway of this degradation being independent on the type of Lys- N, -side chains (acetyl group, amino acid, peptide). Substitution of the Lys residues by Ala at the P,2 positions decreased the tryptic cleavage, while replacement of the bulky side chain of Thr at the P2 positions strongly increased the cleavage of -Lys-Pro- bonds. Circular dichroism (CD) data of the modified oligotuftsin derivatives are in accord with enhanced flexibility of the peptide backbone, as a prerequisite for increased susceptibility to cleavage of -Lys-Pro- bonds. These results obtained of oligotuftsin derivatives might have implications for the proteolytic degradation of target peptides that require specific conformational preconditions. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd. [source] The solution structure of the Mg2+ form of soybean calmodulin isoform 4 reveals unique features of plant calmodulins in resting cellsPROTEIN SCIENCE, Issue 3 2010Hao Huang Abstract Soybean calmodulin isoform 4 (sCaM4) is a plant calcium-binding protein, regulating cellular responses to the second messenger Ca2+. We have found that the metal ion free (apo-) form of sCaM4 possesses a half unfolded structure, with the N-terminal domain unfolded and the C-terminal domain folded. This result was unexpected as the apo-forms of both soybean calmodulin isoform 1 (sCaM1) and mammalian CaM (mCaM) are fully folded. Because of the fact that free Mg2+ ions are always present at high concentrations in cells (0.5,2 mM), we suggest that Mg2+ should be bound to sCaM4 in nonactivated cells. CD studies revealed that in the presence of Mg2+ the initially unfolded N-terminal domain of sCaM4 folds into an ,-helix-rich structure, similar to the Ca2+ form. We have used the NMR backbone residual dipolar coupling restraints 1DNH, 1DC,H,, and 1DC,C, to determine the solution structure of the N-terminal domain of Mg2+ -sCaM4 (Mg2+ -sCaM4-NT). Compared with the known structure of Ca2+ -sCaM4, the structure of the Mg2+ -sCaM4-NT does not fully open the hydrophobic pocket, which was further confirmed by the use of the fluorescent probe ANS. Tryptophan fluorescence experiments were used to study the interactions between Mg2+ -sCaM4 and CaM-binding peptides derived from smooth muscle myosin light chain kinase and plant glutamate decarboxylase. These results suggest that Mg2+ -sCaM4 does not bind to Ca2+ -CaM target peptides and therefore is functionally similar to apo-mCaM. The Mg2+ - and apo-structures of the sCaM4-NT provide unique insights into the structure and function of some plant calmodulins in resting cells. [source] On-plate-selective enrichment of glycopeptides using boronic acid-modified gold nanoparticles for direct MALDI-QIT-TOF MS analysisPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 22 2009Jia Tang Abstract In this study, an on-plate-selective enrichment method is developed for fast and efficient glycopeptide investigation. Gold nanoparticles were first spotted and sintered on a stainless-steel plate, then modified with 4-mercaptophenylboronic acid to provide porous substrate with large specific surface and dual functions. These spots were used to selectively capture glycopeptides from peptide mixtures and the captured target peptides could be analyzed by MALDI-MS simply by deposition of 2,5-dihydroxybenzoic acid matrix. Horseradish peroxidase was employed as a standard glycoprotein to investigate the enrichment efficiency. In this way, the enrichment, washing and detection steps can all be fulfilled on a single MALDI target plate. The relatively small sample amount needed, low detection limit and rapid selective enrichment have made this on-plate strategy promising for online enrichment of glycopeptides, which could be applied in high-throughput proteome research. [source] Determination of growth hormone secretagogue pralmorelin (GHRP-2) and its metabolite in human urine by liquid chromatography/electrospray ionization tandem mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 14 2010Masato Okano GHRP-2 (pralmorelin, D-Ala-D-(,-naphthyl)-Ala-Ala-Trp-D-Phe-Lys-NH2), which belongs to a class of growth hormone secretagogue (GHS), is intravenously used to diagnose growth hormone (GH) deficiency. Because it may be misused in expectation of a growth-promoting effect by athletes, the illicit use of GHS by athletes has been prohibited by the World Anti-Doping Agency (WADA). Therefore, the mass spectrometric identification of urinary GHRP-2 and its metabolite D-Ala-D-(,-naphthyl)-Ala-Ala-OH (AA-3) was studied using liquid chromatography/electrospray ionization tandem mass spectrometry for doping control purposes. The method consists of solid-phase extraction using stable-isotope-labeled GHRP-2 as an internal standard and subsequent ultra-performance liquid chromatography/tandem mass spectrometry, and the two target peptides were determined at urinary concentrations of 0.5,10,ng/mL. The recoveries ranged from 84 to 101%, and the assay precisions were calculated as 1.6,3.8% (intra-day) and 1.9,4.3% (inter-day). Intravenous administration of GHRP-2 in ten male volunteers was studied to demonstrate the applicability of the method. In all ten cases, unchanged GHRP-2 and its specific metabolite AA-3 were detected in urine. Copyright © 2010 John Wiley & Sons, Ltd. [source] Highly efficient and selective enrichment of peptide subsets combining fluorous chemistry with reversed-phase chromatographyRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 24 2009Wantao Ying The selective capture of target peptides poses a great challenge to modern chemists and biologists, especially when enriching them from proteome samples possessing extremes in concentration dynamic range and sequence diversity. While approaches based on traditional techniques such as biotin-avidin pairing offer versatile tools to design strategies for selective enrichment, problems are still encountered due to sample loss or poor selectivity of enrichment. Here we show that the recently introduced fluorous chemistry approach has attractive properties as an alternative method for selective enrichment. Through appending a perfluorine group to the target peptide, it is possible to dramatically increase the peptide's hydrophobicity and thus enable facile separation of labeled from non-labeled peptides. Use of reversed-phase chromatography allowed for improved peptide recovery in comparison with results obtained using the formerly reported fluorous bonded phase methods. Furthermore, this approach also allowed for on-line separation and identification of both labeled and unlabeled peptides in a single experiment. The net result is an increase in the confidence of protein identification by tandem mass spectrometry (MS2) as all peptides and subsequent information are retained. Successful off-line and on-line enrichment of cysteine-containing peptides was obtained, and high quality MS2 spectra were obtained by tandem mass spectrometry due to the stability of the tag, allowing for facile identification via standard database searching. We believe that this strategy holds great promise for selective enrichment and identification of low abundance target proteins or peptides. Copyright © 2009 John Wiley & Sons, Ltd. [source] Refolding of Npro fusion proteins,BIOTECHNOLOGY & BIOENGINEERING, Issue 4 2009Waltraud Kaar Abstract The autoprotease Npro significantly enhances expression of fused peptides and proteins and drives the formation of inclusion bodies during protein expression. Upon refolding, the autoprotease becomes active and cleaves itself specifically at its own C-terminus releasing the target protein with its authentic N-terminus. Npro wild-type and its mutant EDDIE, respectively, were fused N-terminally to the model proteins green fluorescent protein, staphylococcus Protein A domain D, inhibitory peptide of senescence-evasion-factor, and the short 16 amino acid peptide pep6His. In comparison with the Npro wild-type, the tailored mutant EDDIE displayed an increased rate constant for refolding and cleavage from 1.3,×,10,4,s,1 to 3.5,×,10,4,s,1, and allowed a 15-fold higher protein concentration of 1.1,mg/mL when studying pep6His as a fusion partner. For green fluorescent protein, the rate constant was increased from 2.4,×,10,5,s,1 to 1.1,×,10,4,s,1 when fused to EDDIE. When fused to small target peptides, refolding and cleavage yields were independent of initial protein concentration, even at high concentrations of 3.9,mg/mL, although cleavage rates were strongly influenced by the fusion partner. This behavior differed from conventional 1st order refolding kinetics, where yield strongly depends on initial protein concentration due to an aggregation reaction of higher order. Refolding and cleavage of EDDIE fusion proteins follow a monomolecular reaction for the autoproteolytic cleavage over a wide concentration range. At high protein concentrations, deviations from the model assumptions were observed and thus smaller rate constants were required to approximate the data. Biotechnol. Bioeng. 2009; 104: 774,784 © 2009 Wiley Periodicals, Inc. [source] | |||||||||||||