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Sample records for peptide labeled fluorescein-5-isothiocyanate

  1. Monoclonal antibody-targeted fluorescein-5-isothiocyanate-labeled biomimetic nanoapatites: a promising fluorescent probe for imaging applications.

    PubMed

    Oltolina, Francesca; Gregoletto, Luca; Colangelo, Donato; Gómez-Morales, Jaime; Delgado-López, José Manuel; Prat, Maria

    2015-02-10

    Multifunctional biomimetic nanoparticles (NPs) are acquiring increasing interest as carriers in medicine and basic research since they can efficiently combine labels for subsequent tracking, moieties for specific cell targeting, and bioactive molecules, e.g., drugs. In particular, because of their easy synthesis, low cost, good biocompatibility, high resorbability, easy surface functionalization, and pH-dependent solubility, nanocrystalline apatites are promising candidates as nanocarriers. This work describes the synthesis and characterization of bioinspired apatite nanoparticles to be used as fluorescent nanocarriers targeted against the Met/hepatocyte growth factor receptor, which is considered a tumor associated cell surface marker of many cancers. To this aim the nanoparticles have been labeled with Fluorescein-5-isothiocyanate (FITC) by simple isothermal adsorption, in the absence of organic, possibly toxic, molecules, and then functionalized with a monoclonal antibody (mAb) directed against such a receptor. Direct labeling of the nanoparticles allowed tracking the moieties with spatiotemporal resolution and thus following their interaction with cells, expressing or not the targeted receptor, as well as their fate in vitro. Cytofluorometry and confocal microscopy experiments showed that the functionalized nanocarriers, which emitted a strong fluorescent signal, were rapidly and specifically internalized in cells expressing the receptor. Indeed, we found that, once inside the cells expressing the receptor, mAb-functionalized FITC nanoparticles partially dissociated in their two components, with some mAbs being recycled to the cell surface and the FITC-labeled nanoparticles remaining in the cytosol. This work thus shows that FITC-labeled nanoapatites are very promising probes for targeted cell imaging applications.

  2. Fluorescein-5-isothiocyanate-conjugated protein-directed synthesis of gold nanoclusters for fluorescent ratiometric sensing of an enzyme-substrate system.

    PubMed

    Ke, Chen-Yi; Wu, Yun-Tse; Tseng, Wei-Lung

    2015-07-15

    This study describes the synthesis of a dual emission probe for the fluorescent ratiometric sensing of hydrogen peroxide (H2O2), enzyme activity, and environmental pH change. Green-emitting fluorescein-5-isothiocyanate (FITC) was conjugated to the amino groups of bovine serum albumin (BSA). This FITC-conjugated BSA acted as a template for the synthesis of red-emitting gold nanoclusters (AuNCs) under alkaline conditions. Under single wavelength excitation, FITC/BSA-stabilized AuNCs (FITC/BSA-AuNCs) emitted fluorescence at 525 and 670nm, which are sensitive to changes in solution pH and H2O2 concentration, respectively. The effective fluorescence quenching of AuNCs by H2O2 enabled FITC/BSA-AuNCs to ratiometrically detect the H2O2 product-related enzyme system and its inhibition, including glucose oxidase-catalyzed oxidation of glucose, acetylcholinesterase/choline oxidase-mediated hydrolysis and oxidation of acetylcholine, and paraoxon-induced inhibition of acetylcholinesterase activity. When pH-insensitive AuNCs were used as an internal standard, FITC/BSA-AuNCs offered a sensitive and reversible ratiometric sensing of a 0.1-pH unit change in the pH range 5.0-8.5. The pH-induced change in FITC fluorescence enabled FITC/BSA-AuNCs to detect an ammonia product-related enzyme system. This was exemplified with the determination of urea in plasma by urease-mediated hydrolysis of urea.

  3. Paramagnetic particles carried by cell-penetrating peptide tracking of bone marrow mesenchymal stem cells, a research in vitro

    SciTech Connect

    Liu Min; Guo Youmin . E-mail: mikie0763@126.com; Wu Qifei; Yang Junle; Wang Peng; Wang Sicen; Guo Xiaojuan; Qiang Yongqian; Duan Xiaoyi

    2006-08-18

    The ability to track the distribution and differentiation of stem cells by high-resolution imaging techniques would have significant clinical and research implications. In this study, a model cell-penetrating peptide was used to carry gadolinium particles for magnetic resonance imaging of the mesenchymal stem cells. The mesenchymal stem cells were isolated from rat bone marrow by Percoll and identified by osteogenic differentiation in vitro. The cell-penetrating peptides labeled with fluorescein-5-isothiocyanate and gadolinium were synthesized by a solid-phase peptide synthesis method and the relaxivity of cell-penetrating peptide-gadolinium paramagnetic conjugate on 400 MHz nuclear magnetic resonance was 5.7311 {+-} 0.0122 mmol{sup -1} s{sup -1}, higher than that of diethylenetriamine pentaacetic acid gadolinium (p < 0.05). Fluorescein imaging confirmed that this new peptide could internalize into the cytoplasm and nucleus. Gadolinium was efficiently internalized into mesenchymal stem cells by the peptide in a time- or concentration-dependent fashion, resulting in intercellular T1 relaxation enhancement, which was obviously detected by 1.5 T magnetic resonance imaging. Cytotoxicity assay and flow cytometric analysis showed the intercellular contrast medium incorporation did not affect cell viability and membrane potential gradient. The research in vitro suggests that the newly constructed peptides could be a vector for tracking mesenchymal stem cells.

  4. Purification of the labeled cyanogen bromide peptides of the. cap alpha. polypeptide from sodium and potassium ion-activated adenosinetriphosphatase modified with N-(/sup 3/H)ethylmaleimide

    SciTech Connect

    Le, D.T.

    1985-01-01

    Sodium and potassium ion-activated adenosinetriphosphatase, isolated from canine kidney, was reacted with N-(/sup 3/H)ethylmaleimide under three different conditions, defined by particular concentrations of ligands for the enzyme, such that after the same amount of time the remaining activity of then enzyme varied from 90% to 30%. The conformation of the enzyme also differed among the three conditions. In all cases, the ..cap alpha..-polypeptide was purified and subjected to cyanogen bromide digestion. Two distinct, radioactive peptides were separated by gel filtration of the cyanogen bromide digest on a column of Sephadex LH-60 equilibrated with 95% ethanol: 88% formic acid:4:1. One of the radioactive peptides was shown to contain the sulfhydryl residue whose reaction with N-ethylmaleimide inactivates the enzyme. The other radioactive peptide contained a sulfhydryl residue that seems to react with N-ethylmaleimide only when the binding site for ATP is not occupied. Alkylation of this residue, however, does not result in inactivation of enzyme. Both peptides were purified further by high-pressure liquid chromatography, and their amino-terminal sequences were determined by the manual dansyl-Edman or solid-phase techniques. The peptide containing the sulfhydryl protected by ATP has, as its amino terminus, the lysine that reacts exclusively with fluorescein-5'-isothiocyanate.

  5. New method of iodine labelling of peptide hormones

    SciTech Connect

    Escher, E.

    1984-01-01

    Usually peptide hormones and related compounds are radioactively labelled with iodine on tyrosine residues of the peptide. However many peptide hormones do not contain tyrosine or the iodinated tyrosine interferes with the biological properties. In order to circumvent these and other problems, a general method is proposed which allows the introduction of iodine into the para-position of phenylalanine with a modified Sandmeyer procedure. This last-step modification together with HPLC purification permits the obtention of carrier-free and metabolically stable labelled products with maximal specific activity possible. The model has been carried out on several peptide-models like angiotensin II, endorphine and head activator peptide.

  6. Fluorescence turn-on detection of iodide, iodate and total iodine using fluorescein-5-isothiocyanate-modified gold nanoparticles.

    PubMed

    Chen, Yi-Ming; Cheng, Tian-Lu; Tseng, Wei-Lung

    2009-10-01

    Selective turn-on fluorescence detection of I(-) was accomplished using fluorescein isothiocyanate-decorated gold nanoparticles (FITC-AuNPs). FITC molecules, which fluoresce strongly in an alkaline solution, were severely quenched when they were attached to the surface of AuNPs through their isothiocyanate group. Upon the addition of I(-), FITC molecules were detached because of I(-) adsorption on the surface of AuNPs. As a result, released FITC molecules were restored to their original fluorescence intensity. Because I(-) has a higher binding affinity to the surface of Au than do Br(-), Cl(-), or F(-), the FITC-AuNPs obviously have a higher selectivity toward I(-) than toward these other anions. Meanwhile, after IO(3)(-) was reduced to I(-) with ascorbic acid, the detection of IO(3)(-) was successfully achieved using the FITC-AuNPs. Under an optimum pH and AuNP concentration, the lowest detectable concentrations of I(-) and IO(3)(-) using this probe were 10.0 and 50.0 nM, respectively. The FITC-AuNPs provide a number of advantages, including easy preparation, selectivity, sensitivity, and low cost. This unique probe was applied to an analysis of the total iodine in edible salt and seawater.

  7. Dynamics and aggregation of the peptide ion channel alamethicin. Measurements using spin-labeled peptides.

    PubMed Central

    Archer, S J; Ellena, J F; Cafiso, D S

    1991-01-01

    Two spin-labeled derivatives of the ion conductive peptide alamethicin were synthesized and used to examine its binding and state of aggregation. One derivative was spin labeled at the C-terminus and the other, a leucine analogue, was spin labeled at the N-terminus. In methanol, both the C and N terminal labeled peptides were monomeric. In aqueous solution, the C-terminal derivative was monomeric at low concentrations, but aggregated at higher concentrations with a critical concentration of 23 microM. In the membrane, the C-terminal label was localized to the membrane-aqueous interface using 13C-NMR, and could assume more than one orientation. The membrane binding of the C-terminal derivative was examined using EPR, and it exhibited a cooperativity seen previously for native alamethicin. However, this cooperativity was not the result of an aggregation of the peptide in the membrane. When the spectra of either the C or N-terminal labeled peptide were examined over a wide range of membrane lipid to peptide ratios, no evidence for aggregation could be found and the peptides remained monomeric under all conditions examined. Because electrical measurements on this peptide provide strong evidence for an ion-conductive aggregate, the ion-conductive form of alamethicin likely represents a minor fraction of the total membrane bound peptide. PMID:1717016

  8. Peptide-membrane Interactions by Spin-labeling EPR

    PubMed Central

    Smirnova, Tatyana I.; Smirnov, Alex I.

    2016-01-01

    Site-directed spin labeling (SDSL) in combination with Electron Paramagnetic Resonance (EPR) spectroscopy is a well-established method that has recently grown in popularity as an experimental technique, with multiple applications in protein and peptide science. The growth is driven by development of labeling strategies, as well as by considerable technical advances in the field, that are paralleled by an increased availability of EPR instrumentation. While the method requires an introduction of a paramagnetic probe at a well-defined position in a peptide sequence, it has been shown to be minimally destructive to the peptide structure and energetics of the peptide-membrane interactions. In this chapter, we describe basic approaches for using SDSL EPR spectroscopy to study interactions between small peptides and biological membranes or membrane mimetic systems. We focus on experimental approaches to quantify peptide-membrane binding, topology of bound peptides, and characterize peptide aggregation. Sample preparation protocols including spin-labeling methods and preparation of membrane mimetic systems are also described. PMID:26477253

  9. Peptide-Membrane Interactions by Spin-Labeling EPR.

    PubMed

    Smirnova, Tatyana I; Smirnov, Alex I

    2015-01-01

    Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy is a well-established method that has recently grown in popularity as an experimental technique, with multiple applications in protein and peptide science. The growth is driven by development of labeling strategies, as well as by considerable technical advances in the field, that are paralleled by an increased availability of EPR instrumentation. While the method requires an introduction of a paramagnetic probe at a well-defined position in a peptide sequence, it has been shown to be minimally destructive to the peptide structure and energetics of the peptide-membrane interactions. In this chapter, we describe basic approaches for using SDSL EPR spectroscopy to study interactions between small peptides and biological membranes or membrane mimetic systems. We focus on experimental approaches to quantify peptide-membrane binding, topology of bound peptides, and characterize peptide aggregation. Sample preparation protocols including spin-labeling methods and preparation of membrane mimetic systems are also described.

  10. Hydrazide Reactive Peptide Tags for Site-Specific Protein Labeling

    PubMed Central

    Eldridge, Glenn M.; Weiss, Gregory A.

    2011-01-01

    New site-specific protein labeling (SSPL) reactions for targeting specific, short peptides could be useful for the real time detection of proteins inside of living cells. One SSPL approach matches bioorthogonal reagents with complementary peptides. Here, hydrazide reactive peptides were selected from phage-displayed libraries using reaction-based selections. Selection conditions included washes of varying pH and treatment with NaCNBH3 in order to specifically select reactive carbonyl containing peptides. Selected peptides were fused to T4 lysozyme or synthesized on filter paper for colorimetric assays of the peptide-hydrazide interaction. A peptide-lysozyme protein fusion demonstrated specific, covalent labeling by the Hydrazide Reactive (HyRe) peptides in crude bacterial cell lysates, sufficient for the specific detection of an over-expressed protein fusion. Chemical synthesis of a short HyRe tag variant and subsequent reaction with two structurally distinct hydrazide probes produced covalent adducts observable by MALDI-TOF MS and MS/MS. Rather than isolating reactive carbonyl-containing peptides, we observed reaction with the N–terminal His of HyRe tag 114, amino acid sequence HKSNHSSKNRE, which attacks the hydrazide carbonyl at neutral pH. However, at the pH used during selection wash steps (<6.0), an alternative imine-containing product is formed that can be reduced with sodium cyanoborohydride. MSMS further reveals that this low pH product forms an adduct on Ser6. Further optimization of the novel bimolecular reaction described here could provide a useful tool for in vivo protein labeling and bioconjugate synthesis. The reported selection and screening methods could be widely applicable to the identification of peptides capable of other site-specific protein labeling reactions with bioorthogonal reagents. PMID:21905743

  11. Cancer therapy with alpha-emitters labeled peptides.

    PubMed

    Dadachova, Ekaterina

    2010-05-01

    Actively targeted alpha-particles offer specific tumor cell killing action with less collateral damage to surrounding normal tissues than beta-emitters. During the last decade, radiolabeled peptides that bind to different receptors on the tumors have been investigated as potential therapeutic agents both in the preclinical and clinical settings. Advantages of radiolabeled peptides over antibodies include relatively straightforward chemical synthesis, versatility, easier radiolabeling, rapid clearance from the circulation, faster penetration and more uniform distribution into tissues, and less immunogenicity. Rapid internalization of the radiolabeled peptides with equally rapid re-expression of the cell surface target is a highly desirable property that enhances the total delivery of these radionuclides into malignant sites. Peptides, such as octreotide, alpha-melanocyte-stimulating hormone analogues, arginine-glycine-aspartic acid-containing peptides, bombesin derivatives, and others may all be feasible for use with alpha-emitters. The on-going preclinical work has primarily concentrated on octreotide and octreotate analogues labeled with Bismuth-213 and Astatine-211. In addition, alpha-melanocyte-stimulating hormone analogue has been labeled with Lead-212/Bismuth-212 in vivo generator and demonstrated the encouraging therapeutic efficacy in treatment of experimental melanoma. Obstacles that continue to obstruct widespread acceptance of alpha-emitter-labeled peptides are primarily the supply of these radionuclides and concerns about potential kidney toxicity. New sources and methods for production of these medically valuable radionuclides and better understanding of mechanisms related to the peptide renal uptake and clearance should speed up the introduction of alpha-emitter-labeled peptides into the clinic. PMID:20350629

  12. The recovery of the polymerizability of Lys-61-labelled actin by the addition of phalloidin. Fluorescence polarization and resonance-energy-transfer measurements.

    PubMed

    Miki, M

    1987-04-01

    Modification of Lys-61 in actin with fluorescein-5-isothiocyanate (FITC) blocks actin polymerization [Burtnick, L. D. (1984) Biochim. Biophys. Acta 791, 57-62]. FITC-labelled actin recovered its ability to polymerize on addition of phalloidin. The polymers had the same characteristic helical thread-like structure as normal F-actin and the addition of myosin subfragment-1 to the polymers formed the characteristic arrowhead structure in electron microscopy. The polymers activated the ATPase activity of myosin subfragment-1 as efficiently as normal F-actin. These results indicate that Lys-61 is not directly involved in an actin-actin binding region nor in myosin binding site. From static fluorescence polarization measurements, the rotational relaxation time of FITC-labelled actin filaments was calculated to be 20 ns as the value reduced in water at 20 degrees C, while any rotational relaxation time of 1,5-IAEDANS bound to Cys-374 on F-actin in the presence of a twofold molar excess of phalloidin could not be detected by static polarization measurements under the same conditions. This indicates that the Lys-61 side chain is extremely mobile even in the filamentous structure. Fluorescence resonance energy transfer between the donor 1,5-IAEDANS bound to SH1 of myosin subfragment-1 and the acceptor fluorescein-5-isothiocyanate bound to Lys-61 of actin in the rigor complex was measured. The transfer efficiency was 0.39 +/- 0.05 which corresponds to the distance of 5.2 +/- 0.1 nm, assuming that the energy donor and acceptor rotate rapidly relative to the fluorescence lifetime and that the transfer occurs between a single donor and an acceptor.

  13. A method for the 32P labeling of peptides or peptide nucleic acid oligomers

    NASA Technical Reports Server (NTRS)

    Kozlov, I. A.; Nielsen, P. E.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

    1998-01-01

    A novel approach to the radioactive labeling of peptides and PNA oligomers is described. It is based on the conjugation of a deoxynucleoside 3'-phosphate with the terminal amine of the substrate, followed by phosphorylation of the 5'-hydroxyl group of the nucleotide using T4 polynucleotide kinase and [gamma-32P]ATP.

  14. Lutetium-177 Labeled Peptides: The European Institute of Oncology Experience.

    PubMed

    Carollo, Angela; Papi, Stefano; Chinol, Marco

    2016-01-01

    Peptide receptor radionuclide therapy (PRRT) using radiolabeled somatostatin analogues has shown encouraging results in various somatostatin receptor positive tumors. Partial remission rates up to 30% have been documented as well as significant improvements in quality of life and survival. This treatment takes advantage of the high specific binding of the radiolabeled peptide to somatostatin receptors overexpressed by the tumors thus being more effective on the tumor cells with less systemic side-effects. The development of macrocyclic chelators conjugated to peptides made possible the stable binding with various radionuclides. In particular 177Lu features favourable physical characteristics with a half-life of 6.7 days, emission of β- with energy of 0.5 MeV for treatment and γ-emissions suitable for imaging. The present contribution describes the learning process achieved at the European Institute of Oncology (IEO) since the first application of 90Y labeled peptides to the therapy of neuroendocrine tumors back in 1997. Continuous improvements led to the preparation of a safe 177Lu labeled peptide for human use. Our learning curve began with the identification of the optimal characteristics of the isotope paying attention to its chemical purity and specific activity along with the optimization of the parameters involved in the radiolabeling procedure. Also the radiation protection issues have been improved along the years and recently more and more attention has been devoted to the pharmaceutical aspects involved in the preparation. The overall issue of the quality has now been completed by drafting an extensive documentation with the goal to deliver a safe and reliable product to our patients.

  15. Accurate quantitation of MHC-bound peptides by application of isotopically labeled peptide MHC complexes.

    PubMed

    Hassan, Chopie; Kester, Michel G D; Oudgenoeg, Gideon; de Ru, Arnoud H; Janssen, George M C; Drijfhout, Jan W; Spaapen, Robbert M; Jiménez, Connie R; Heemskerk, Mirjam H M; Falkenburg, J H Frederik; van Veelen, Peter A

    2014-09-23

    Knowledge of the accurate copy number of HLA class I presented ligands is important in fundamental and clinical immunology. Currently, the best copy number determinations are based on mass spectrometry, employing single reaction monitoring (SRM) in combination with a known amount of isotopically labeled peptide. The major drawback of this approach is that the losses during sample pretreatment, i.e. immunopurification and filtration steps, are not well defined and must, therefore, be estimated. In addition, such losses can vary for individual peptides. Therefore, we developed a new approach in which isotopically labeled peptide-MHC monomers (hpMHC) are prepared and added directly after cell lysis, i.e. before the usual sample processing. Using this approach, all losses during sample processing can be accounted for and allows accurate determination of specific MHC class I-presented ligands. Our study pinpoints the immunopurification step as the origin of the rather extreme losses during sample pretreatment and offers a solution to account for these losses. Obviously, this has important implications for accurate HLA-ligand quantitation. The strategy presented here can be used to obtain a reliable view of epitope copy number and thus allows improvement of vaccine design and strategies for immunotherapy.

  16. Macrocyclization and labeling of helix-loop-helix peptide with intramolecular bis-thioether linkage.

    PubMed

    Nishihara, Toshio; Kitada, Hidekazu; Fujiwara, Daisuke; Fujii, Ikuo

    2016-11-01

    Conformationally constrained peptides have been developed as an inhibitor for protein-protein interactions (PPIs), and we have de novo designed cyclized helix-loop-helix (cHLH) peptide with a disulfide bond consisting of 40 amino acids to generate molecular-targeting peptides. However, synthesis of long peptides has sometimes resulted in low yield according to the respective amino acid sequences. Here we developed a method for efficient synthesis and labeling for cHLH peptides. First, we synthesized two peptide fragments and connected them by the copper-mediated alkyne and azide cycloaddition (CuAAC) reaction. Cyclization was performed by bis-thioether linkage using 1,3-dibromomethyl-5-propargyloxybenzene, and subsequently, the cHLH peptide was labeled with an azide-labeled probe. Finally, we designed and synthesized a peptide inhibitor for the p53-HDM2 interaction using a structure-guided design and successfully labeled it with a fluorescent probe or a functional peptide, respectively, by click chemistry. This macrocyclization and labeling method for cHLH peptide would facilitate the discovery of de novo bioactive ligands and therapeutic leads. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 415-421, 2016. PMID:26917088

  17. Development of a general methodology for labelling peptide-morpholino oligonucleotide conjugates using alkyne-azide click chemistry.

    PubMed

    Shabanpoor, Fazel; Gait, Michael J

    2013-11-11

    We describe a general methodology for fluorescent labelling of peptide conjugates of phosphorodiamidate morpholino oligonucleotides (PMOs) by alkyne functionalization of peptides, subsequent conjugation to PMOs and labelling with a fluorescent compound (Cy5-azide). Two peptide-PMO (PPMO) examples are shown. No detrimental effect of such labelled PMOs was seen in a biological assay.

  18. Lutetium-177 Labeled Bombesin Peptides for Radionuclide Therapy.

    PubMed

    Reynolds, Tamila Stott; Bandari, Rajendra P; Jiang, Zongrun; Smith, Charles J

    2016-01-01

    in 177Lu-labeled bombesin peptides for targeted radiotherapy that includes agonist, antagonist, and multivalent cell-targeting agents. In vitro, in vivo translational, and in vivo human clinical investigations are described.

  19. Lutetium-labelled peptides for therapy of neuroendocrine tumours.

    PubMed

    Kam, B L R; Teunissen, J J M; Krenning, E P; de Herder, W W; Khan, S; van Vliet, E I; Kwekkeboom, D J

    2012-02-01

    Treatment with radiolabelled somatostatin analogues is a promising new tool in the management of patients with inoperable or metastasized neuroendocrine tumours. Symptomatic improvement may occur with (177)Lu-labelled somatostatin analogues that have been used for peptide receptor radionuclide therapy (PRRT). The results obtained with (177)Lu-[DOTA(0),Tyr(3)]octreotate (DOTATATE) are very encouraging in terms of tumour regression. Dosimetry studies with (177)Lu-DOTATATE as well as the limited side effects with additional cycles of (177)Lu-DOTATATE suggest that more cycles of (177)Lu-DOTATATE can be safely given. Also, if kidney-protective agents are used, the side effects of this therapy are few and mild and less than those from the use of (90)Y-[DOTA(0),Tyr(3)]octreotide (DOTATOC). Besides objective tumour responses, the median progression-free survival is more than 40 months. The patients' self-assessed quality of life increases significantly after treatment with (177)Lu-DOTATATE. Lastly, compared to historical controls, there is a benefit in overall survival of several years from the time of diagnosis in patients treated with (177)Lu-DOTATATE. These findings compare favourably with the limited number of alternative therapeutic approaches. If more widespread use of PRRT can be guaranteed, such therapy may well become the therapy of first choice in patients with metastasized or inoperable neuroendocrine tumours.

  20. Biosynthesized (/sup 35/S)methionine-labeled pro-opiomelanocortin peptides as novel recovery markers in radioimmunoassay of peptide hormones

    SciTech Connect

    Rosendale, B.E.; Jarrett, D.B.

    1985-12-01

    Hormones are extracted from plasma with varying efficiency. Thus, markers or internal standards are often needed, to monitor and correct for extraction losses. To do so is difficult in the case of peptide hormones because radioactive recovery markers either have a low specific activity or, if labeled with iodine, may not be fully representative because of alterations in their size and charge. More importantly, markers labeled with /sup 125/I can interact in, and thus compromise, the subsequent radioimmunoassay. AtT-20 mouse pituitary tumor cells, which can be stimulated to synthesize and secrete pro-opiomelanocortin peptides, can biosynthetically label beta-lipotropin (beta-LPH) with (/sup 35/S)methionine. The labeled peptide, which is co-eluted with unlabeled beta-LPH in high-performance liquid chromatography, is fully immunoprecipitable and has a specific activity of 34 Ci/g. We use this labeled peptide to monitor the recovery of beta-LPH in silicic acid extraction from plasma. This peptide is an ideal marker of analytical recovery because it does not interfere in subsequent radioimmunoassays.

  1. Improved proteome coverage by using iTRAQ labelling and peptide OFFGEL fractionation

    PubMed Central

    Ernoult, Emilie; Gamelin, Erick; Guette, Catherine

    2008-01-01

    Background The development of mass spectrometric techniques and fractionation methods now allows the investigation of very complex protein mixtures ranging from subcellular structures to tissues. Nevertheless, this work is particularly difficult due to the wide dynamic range of protein concentration in eukaryotic tissues. In this paper, we present a shotgun method whereby the peptides are fractionated using OFFGEL electrophoresis after iTRAQ labelling. Results We demonstrated that iTRAQ peptide labelling enhances MALDI ionisation and that the OFFGEL fractionation of the labelled peptides introduces a supplementary criterion (pI) useful for validation and identification of proteins. We showed that iTRAQ samples allowed lower-concentrated proteins identification in comparison with free-labelled samples. Conclusion The combined use of iTRAQ labelling and OFFGEL fractionation allows a considerable increase in proteome coverage of very complex samples prepared from total cell extracts and supports the low-concentrated protein identification. PMID:18851748

  2. Rhenium labeled peptides and antibodies for cancer therapy. CRADA final report

    SciTech Connect

    Knapp, Jr., F. F.; Rhodes, B. A.

    1996-08-12

    This CRADA involved development of optimal methods for attachment of rhenium radioisotopes to antibodies and peptides which can be used for cancer treatment. Rhenium-186 and the tungsten-188/rhenium-188 generators were provided from ORNL to RhoMed for peptide labeling studies. The rhenium-186 and carrier-free rhenium-188 were then used to optimize the labeling of various small peptides....A system has been developed at ORNL which provides the rhenium-188 radioisotope, which has excellent therapeutic properties for cancer treatment.

  3. Label-free peptide profiling of Orbitrap™ full mass spectra

    PubMed Central

    2011-01-01

    Background We developed a new version of the open source software package Peptrix that can yet compare large numbers of Orbitrap™ LC-MS data. The peptide profiling results for Peptrix on MS1 spectra were compared with those obtained from a small selection of open source and commercial software packages: msInspect, Sieve™ and Progenesis™. The properties compared in these packages were speed, total number of detected masses, redundancy of masses, reproducibility in numbers and CV of intensity, overlap of masses, and differences in peptide peak intensities. Reproducibility measurements were taken for the different MS1 software applications by measuring in triplicate a complex peptide mixture of immunoglobulin on the Orbitrap™ mass spectrometer. Values of peptide masses detected from the high intensity peaks of the MS1 spectra by peptide profiling were verified with values of the MS2 fragmented and sequenced masses that resulted in protein identifications with a significant score. Findings Peptrix finds about the same number of peptide features as the other packages, but peptide masses are in some cases approximately 5 to 10 times less redundant present in the peptide profile matrix. The Peptrix profile matrix displays the largest overlap when comparing the number of masses in a pair between two software applications. The overlap of peptide masses between software packages of low intensity peaks in the spectra is remarkably low with about 50% of the detected masses in the individual packages. Peptrix does not differ from the other packages in detecting 96% of the masses that relate to highly abundant sequenced proteins. MS1 peak intensities vary between the applications in a non linear way as they are not processed using the same method. Conclusions Peptrix is capable of peptide profiling using Orbitrap™ files and finding differential expressed peptides in body fluid and tissue samples. The number of peptide masses detected in Orbitrap™ files can be

  4. Short peptide tag for covalent protein labeling based on coiled coils.

    PubMed

    Wang, Jianpeng; Yu, Yongsheng; Xia, Jiang

    2014-01-15

    To label proteins covalently, one faces a trade-off between labeling a protein specifically and using a small tag. Often one must compromise one parameter for the other or use additional components, such as an enzyme, to satisfy both requirements. Here, we report a new reaction that covalently labels proteins by using engineered coiled-coil peptides. Harnessing the concept of "proximity-induced reactivity", the 21-amino-acid three-heptad peptides CCE/CCK were modified with a nucleophilic cysteine and an α-chloroacetyl group at selected positions. When pairs of coiled coils associated, an irreversible covalent bond spontaneously formed between the peptides. The specificity of the cross-linking reaction was characterized, the probes were improved by making them bivalent, and the system was used to label a protein in vitro and receptors on the surface of mammalian cells. PMID:24341800

  5. Construction and characterization of kilobasepair densely labeled peptide-DNA.

    PubMed

    Kovacic, Suzana; Samii, Laleh; Lamour, Guillaume; Li, Hongbin; Linke, Heiner; Bromley, Elizabeth H C; Woolfson, Derek N; Curmi, Paul M G; Forde, Nancy R

    2014-11-10

    Directed assembly of biocompatible materials benefits from modular building blocks in which structural organization is independent of introduced functional modifications. For soft materials, such modifications have been limited. Here, long DNA is successfully functionalized with dense decoration by peptides. Following introduction of alkyne-modified nucleotides into kilobasepair DNA, measurements of persistence length show that DNA mechanics are unaltered by the dense incorporation of alkynes (∼1 alkyne/2 bp) and after click-chemistry attachment of a tunable density of peptides. Proteolytic cleavage of densely tethered peptides (∼1 peptide/3 bp) demonstrates addressability of the functional groups, showing that this accessible approach to creating hybrid structures can maintain orthogonality between backbone mechanics and overlaid function. The synthesis and characterization of these hybrid constructs establishes the groundwork for their implementation in future applications, such as building blocks in modular approaches to a range of problems in synthetic biology.

  6. Construction and characterization of kilobasepair densely labeled peptide-DNA.

    PubMed

    Kovacic, Suzana; Samii, Laleh; Lamour, Guillaume; Li, Hongbin; Linke, Heiner; Bromley, Elizabeth H C; Woolfson, Derek N; Curmi, Paul M G; Forde, Nancy R

    2014-11-10

    Directed assembly of biocompatible materials benefits from modular building blocks in which structural organization is independent of introduced functional modifications. For soft materials, such modifications have been limited. Here, long DNA is successfully functionalized with dense decoration by peptides. Following introduction of alkyne-modified nucleotides into kilobasepair DNA, measurements of persistence length show that DNA mechanics are unaltered by the dense incorporation of alkynes (∼1 alkyne/2 bp) and after click-chemistry attachment of a tunable density of peptides. Proteolytic cleavage of densely tethered peptides (∼1 peptide/3 bp) demonstrates addressability of the functional groups, showing that this accessible approach to creating hybrid structures can maintain orthogonality between backbone mechanics and overlaid function. The synthesis and characterization of these hybrid constructs establishes the groundwork for their implementation in future applications, such as building blocks in modular approaches to a range of problems in synthetic biology. PMID:25233124

  7. Development of a Multifunctional Benzophenone Linker for Peptide Stapling and Photoaffinity Labelling

    PubMed Central

    Wu, Yuteng; Olsen, Lasse B.; Lau, Yu Heng; Jensen, Claus Hatt; Rossmann, Maxim; Baker, Ysobel R.; Sore, Hannah F.; Collins, Súil

    2016-01-01

    Abstract Photoaffinity labelling is a useful method for studying how proteins interact with ligands and biomolecules, and can help identify and characterise new targets for the development of new therapeutics. We present the design and synthesis of a novel multifunctional benzophenone linker that serves as both a photo‐crosslinking motif and a peptide stapling reagent. Using double‐click stapling, we attached the benzophenone to the peptide via the staple linker, rather than by modifying the peptide sequence with a photo‐crosslinking amino acid. When applied to a p53‐derived peptide, the resulting photoreactive stapled peptide was able to preferentially crosslink with MDM2 in the presence of competing protein. This multifunctional linker also features an extra alkyne handle for downstream applications such as pull‐down assays, and can be used to investigate the target selectivity of stapled peptides. PMID:26919579

  8. Development of a Multifunctional Benzophenone Linker for Peptide Stapling and Photoaffinity Labelling.

    PubMed

    Wu, Yuteng; Olsen, Lasse B; Lau, Yu Heng; Jensen, Claus Hatt; Rossmann, Maxim; Baker, Ysobel R; Sore, Hannah F; Collins, Súil; Spring, David R

    2016-04-15

    Photoaffinity labelling is a useful method for studying how proteins interact with ligands and biomolecules, and can help identify and characterise new targets for the development of new therapeutics. We present the design and synthesis of a novel multifunctional benzophenone linker that serves as both a photo-crosslinking motif and a peptide stapling reagent. Using double-click stapling, we attached the benzophenone to the peptide via the staple linker, rather than by modifying the peptide sequence with a photo-crosslinking amino acid. When applied to a p53-derived peptide, the resulting photoreactive stapled peptide was able to preferentially crosslink with MDM2 in the presence of competing protein. This multifunctional linker also features an extra alkyne handle for downstream applications such as pull-down assays, and can be used to investigate the target selectivity of stapled peptides. PMID:26919579

  9. Tritium labeling of amino acids and peptides with liquid and solid tritium

    SciTech Connect

    Peng, C.T.; Hua, R.L.; Souers, P.C.; Coronado, P.R.

    1988-01-01

    Amino acids and peptides were labeled with liquid and solid tritium at 21 K and 9 K. At these low temperatures radiation degradation is minimal, and tritium incorporation increases with tritium concentration and exposure time. Ring saturation in L-phenyl-alanine does not occur. Peptide linkage in oligopeptides is stable toward tritium. Deiodination in 3-iodotyrosine and 3,5-diiodotyrosine occurs readily and proceeds in steps by losing one iodine atom at a time. Nickel and noble metal supported catalysts when used as supports for dispersion of the substrate promote tritium labeling at 21 K. Our study shows that both liquid and solid tritium are potentially useful agents for labeling peptides and proteins. 11 refs., 1 fig., 3 tabs.

  10. Tritium labeling of amino acids and peptides with liquid and solid tritium

    SciTech Connect

    Souers, P.C.; Coronado, P.R.; Peng, C.T.; Hua, R.L.

    1988-01-01

    Amino acids and peptides were labeled with liquid and solid tritium at 21/degree/K and 9/degree/K. At these low temperatures radiation degradation is minimal, and tritium incorporation increases with tritium concentration and exposure time. Ring saturation in L-phenylalanine does not occur. Peptide linkage in oligopeptides is stable toward tritium. Deiodination in 3-iodotyrosine and 3,5-diiodotyrosine occurs readily and proceeds in steps by losing one iodine atom at a time. Nickel and noble metal supported catalysts when used as supports for dispersion of the substrate promote tritium labeling at 21 K. Our study shows that both liquid and solid tritiums are potentially useful agents for labeling peptides and proteins.

  11. Tritium labeling of amino acids and peptides with liquid and solid tritium

    SciTech Connect

    Peng, C.T.; Hua, R.L.; Souers, P.C.; Coronado, P.R.

    1988-09-01

    Amino acids and peptides were labeled with liquid and solid tritium at 21 K and 9 K. At these low temperatures radiation degradation is minimal, and tritium incorporation increases with tritium concentration and exposure time. Ring saturation in L-phenyl-alanine does not occur. Peptide linkage in oligopeptides is stable toward tritium. Deiodination in 3-iodotyrosine and 3,5-diiodotyrosine occurs readily and proceeds in steps by losing one iodine atom at a time. Nickel and noble metal supported catalysts when sued as supports for dispersion of the substrate promote tritium labeling at 21 K. The authors study shows that both liquid and solid tritium are potentially useful agents for labeling peptides and proteins.

  12. Tetrazine-Containing Amino Acid for Peptide Modification and Live Cell Labeling.

    PubMed

    Ni, Zhongqiu; Zhou, Lanxia; Li, Xu; Zhang, Jing; Dong, Shouliang

    2015-01-01

    A novel amino acid derivative 3-(4-(1, 2, 4, 5-tetrazine-3-yl) phenyl)-2-aminopropanoic acid was synthesized in this study. The compound possessed better water-solubility and was synthesized more easily compared with the well-known and commercially available 3-(p-benzylamino)-1, 2, 4, 5-tetrazine. Tetrazine-containing amino acid showed excellent stability in biological media and might be used for cancer cell labeling. Moreover, the compound remained relatively stable in 50% TFA/DCM with little decomposition after prolonged exposure at room temperature. The compound could be utilized as phenylalanine or tyrosine analogue in peptide modification, and the tetrazine-containing peptide demonstrated more significant biological activity than that of the parent peptide. The combination of tetrazine group and amino acid offered broad development prospects of the bioorthogonal labeling and peptide synthesis.

  13. Tetrazine-Containing Amino Acid for Peptide Modification and Live Cell Labeling

    PubMed Central

    Ni, Zhongqiu; Zhou, Lanxia; Li, Xu; Zhang, Jing; Dong, Shouliang

    2015-01-01

    A novel amino acid derivative 3-(4-(1, 2, 4, 5-tetrazine-3-yl) phenyl)-2-aminopropanoic acid was synthesized in this study. The compound possessed better water-solubility and was synthesized more easily compared with the well-known and commercially available 3-(p-benzylamino)-1, 2, 4, 5-tetrazine. Tetrazine-containing amino acid showed excellent stability in biological media and might be used for cancer cell labeling. Moreover, the compound remained relatively stable in 50% TFA/DCM with little decomposition after prolonged exposure at room temperature. The compound could be utilized as phenylalanine or tyrosine analogue in peptide modification, and the tetrazine-containing peptide demonstrated more significant biological activity than that of the parent peptide. The combination of tetrazine group and amino acid offered broad development prospects of the bioorthogonal labeling and peptide synthesis. PMID:26536589

  14. Improved 2-nitrobenzenesulfenyl method: optimization of the protocol and improved enrichment for labeled peptides.

    PubMed

    Matsuo, Ei-Ichi; Toda, Chikako; Watanabe, Makoto; Iida, Tetsuo; Masuda, Taro; Minohata, Toshikazu; Ando, Eiji; Tsunasawa, Susumu; Nishimura, Osamu

    2006-01-01

    We have developed the NBS (2-nitrobenzenesulfenyl) method, a quantitative proteome analysis method utilizing stable isotope labeling followed by mass spectrometry. The potential of this method was reported previously, and the procedure has now been further optimized. Here, we describe a procedure utilizing urea or guanidine hydrochloride as a protein denaturant, in conjunction with an improved chromatographic enrichment method for the NBS-labeled peptides using a phenyl resin column. By using this new protocol, both sample loss throughout the protocol and the elution of unwanted unlabeled peptides can be minimized, improving the efficiency of the analysis significantly.

  15. Molecular level studies on binding modes of labeling molecules with polyalanine peptides

    NASA Astrophysics Data System (ADS)

    Mao, Xiaobo; Wang, Chenxuan; Ma, Xiaojing; Zhang, Min; Liu, Lei; Zhang, Lan; Niu, Lin; Zeng, Qindao; Yang, Yanlian; Wang, Chen

    2011-04-01

    In this work, the binding modes of typical labeling molecules (thioflavin T (ThT), Congo red (CR) and copper(ii) phthalocyanine tetrasulfonic acid tetrasodium salt (PcCu(SO3Na)4)) on pentaalanine, which is a model peptide segment of amyloidpeptides, have been resolved at the molecular level by using scanning tunneling microscopy (STM). In the STM images, ThT molecules are predominantly adsorbed parallel to the peptide strands and two binding modes could be identified. It was found that ThT molecules are preferentially binding on top of the peptide strand, and the mode of intercalated between neighboring peptides also exists. The parallel binding mode of CR molecules can be observed with pentaalaninepeptides. Besides the binding modes of labeling molecules, the CR and PcCu(SO3Na)4 display different adsorption affinity with the pentaalaninepeptides. The results could be beneficial for obtaining molecular level insight of the interactions between labeling molecules and peptides.In this work, the binding modes of typical labeling molecules (thioflavin T (ThT), Congo red (CR) and copper(ii) phthalocyanine tetrasulfonic acid tetrasodium salt (PcCu(SO3Na)4)) on pentaalanine, which is a model peptide segment of amyloidpeptides, have been resolved at the molecular level by using scanning tunneling microscopy (STM). In the STM images, ThT molecules are predominantly adsorbed parallel to the peptide strands and two binding modes could be identified. It was found that ThT molecules are preferentially binding on top of the peptide strand, and the mode of intercalated between neighboring peptides also exists. The parallel binding mode of CR molecules can be observed with pentaalaninepeptides. Besides the binding modes of labeling molecules, the CR and PcCu(SO3Na)4 display different adsorption affinity with the pentaalaninepeptides. The results could be beneficial for obtaining molecular level insight of the interactions between labeling molecules and peptides. Electronic

  16. Robust MS quantification method for phospho-peptides using 18O/16O labeling

    PubMed Central

    Andersen, Claus A; Gotta, Stefano; Magnoni, Letizia; Raggiaschi, Roberto; Kremer, Andreas; Terstappen, Georg C

    2009-01-01

    Background Quantitative measurements of specific protein phosphorylation sites, as presented here, can be used to investigate signal transduction pathways, which is an important aspect of cell dynamics. The presented method quantitatively compares peptide abundances from experiments using 18O/16O labeling starting from elaborated MS spectra. It was originally developed to study signaling cascades activated by amyloid-β treatment of neurons used as a cellular model system with relevance to Alzheimer's disease, but is generally applicable. Results The presented method assesses, in complete cell lysates, the degree of phosphorylation of specific peptide residues from MS spectra using 18O/16O labeling. The abundance of each observed phospho-peptide from two cell states was estimated from three overlapping isotope contours. The influence of peptide-specific labeling efficiency was removed by performing a label swapped experiment and assuming that the labeling efficiency was unchanged upon label swapping. Different degrees of phosphorylation were reported using the fold change measure which was extended with a confidence interval found to reflect the quality of the underlying spectra. Furthermore a new way of method assessment using simulated data is presented. Using simulated data generated in a manner mimicking real data it was possible to show the method's robustness both with increasing noise levels and with decreasing labeling efficiency. Conclusion The fold change error assessable on simulated data was on average 0.16 (median 0.10) with an error-to-signal ratio and labeling efficiency distributions similar to the ones found in the experimentally observed spectra. Applied to experimentally observed spectra a very good match was found to the model (<10% error for 85% of spectra) with a high degree of robustness, as assessed by data removal. This new method can thus be used for quantitative signal cascade analysis of total cell extracts in a high throughput mode

  17. Protein C-Terminal Labeling and Biotinylation Using Synthetic Peptide and Split-Intein

    PubMed Central

    Volkmann, Gerrit; Liu, Xiang-Qin

    2009-01-01

    Background Site-specific protein labeling or modification can facilitate the characterization of proteins with respect to their structure, folding, and interaction with other proteins. However, current methods of site-specific protein labeling are few and with limitations, therefore new methods are needed to satisfy the increasing need and sophistications of protein labeling. Methodology A method of protein C-terminal labeling was developed using a non-canonical split-intein, through an intein-catalyzed trans-splicing reaction between a protein and a small synthetic peptide carrying the desired labeling groups. As demonstrations of this method, three different proteins were efficiently labeled at their C-termini with two different labels (fluorescein and biotin) either in solution or on a solid surface, and a transferrin receptor protein was labeled on the membrane surface of live mammalian cells. Protein biotinylation and immobilization on a streptavidin-coated surface were also achieved in a cell lysate without prior purification of the target protein. Conclusions We have produced a method of site-specific labeling or modification at the C-termini of recombinant proteins. This method compares favorably with previous protein labeling methods and has several unique advantages. It is expected to have many potential applications in protein engineering and research, which include fluorescent labeling for monitoring protein folding, location, and trafficking in cells, and biotinylation for protein immobilization on streptavidin-coated surfaces including protein microchips. The types of chemical labeling may be limited only by the ability of chemical synthesis to produce the small C-intein peptide containing the desired chemical groups. PMID:20027230

  18. A Double-Clicking Bis-Azide Fluorogenic Dye for Bioorthogonal Self-Labeling Peptide Tags.

    PubMed

    Demeter, Orsolya; Fodor, Eszter A; Kállay, Mihály; Mező, Gábor; Németh, Krisztina; Szabó, Pál T; Kele, Péter

    2016-04-25

    Herein, we give the very first example for the development of a fluorogenic molecular probe that combines the two-point binding specificity of biarsenical-based dyes with the robustness of bioorthogonal click-chemistry. This proof-of-principle study reports on the synthesis and fluorogenic characterization of a new, double-quenched, bis-azide fluorogenic probe suitable for bioorthogonal two-point tagging of small peptide tags by double strain-promoted azide-alkyne cycloaddition. The presented probe exhibits remarkable increase in fluorescence intensity when reacted with bis-cyclooctynylated peptide sequences, which could also serve as possible self-labeling small peptide tag motifs. PMID:27010966

  19. Application of REDOR subtraction for filtered MAS observation of labeled backbone carbons of membrane-bound fusion peptides

    NASA Astrophysics Data System (ADS)

    Yang, Jun; Parkanzky, Paul D.; Bodner, Michele L.; Duskin, Craig A.; Weliky, David P.

    2002-12-01

    Clean MAS observation of 13C-labeled carbons in membrane-bound HIV-1 and influenza fusion peptides was made by using a rotational-echo double-resonance spectroscopy (REDOR) filter of directly bonded 13C- 15N pairs. The clean filtering achieved with the REDOR approach is superior to filtering done with sample difference spectroscopy. In one labeling approach, the peptide had labels at a single 13C carbonyl and its directly bonded 15N. The resulting chemical shift distribution of the filtered signal is used to assess the distribution of local secondary structures at the labeled carbonyl. For the influenza peptide, the Leu-2 carbonyl chemical shift distribution is shown to vary markedly with lipid and detergent composition, as well as peptide:lipid ratio, suggesting that the local peptide structure also has a strong dependence on these factors. Because most carboxylic- and amino-labeled amino acids are commercially available, this REDOR approach should have broad applicability to chemically synthesized peptides as well as bacterially synthesized proteins. In a second labeling approach, the HIV-1 fusion peptide had U- 13C, 15N labeling over three sequential residues. When a 1.6 ms REDOR dephasing time is used, only backbone 13C signals are observed. The resulting spectra are used to determine spectral linewidths and to assess feasibility of assignment of uniformly labeled peptide.

  20. Established theory of radiation-induced decay is not generalizable to Bolton-Hunter labeled peptides.

    PubMed

    Doran, Amanda C; Wan, Yieh-Ping; Kopin, Alan S; Beinborn, Martin

    2003-05-01

    Peptide hormones radiolabeled with 125I are widely used for the pharmacological characterization of cognate receptors. As a prerequisite for calculating ligand affinities from competition binding assays, and for estimating receptor densities from such studies, it is necessary to know the concentration of bioactive radioligand that is used in respective experiments. It has been demonstrated previously that radioiodinated peptides undergo decay catastrophe, i.e. disintegration of the radioactive label leads to the concomitant destruction of the carrier peptide. Here, we demonstrate that decay catastrophe does not apply to two peptide hormones that are iodinated by Bolton-Hunter conjugation: cholecystokinin octapeptide and glucagon-like peptide 2. The function of aged samples of these radioligands at corresponding recombinantly expressed receptors was assessed by measuring ligand-induced inositol phosphate production or generation of cyclic AMP, respectively. Both of the tested compounds, although predicted by decay catastrophe to contain little or subthreshold remaining bioactivity, stimulated an unexpectedly high level of receptor-mediated second messenger signaling. Quantitative comparison of observed functions with those of corresponding unlabeled peptides suggested that the bioactivity of each radioligand had been largely conserved despite the radioactive decay of the iodine label. Consistent with an apparent absence of decay catastrophe, we noted that the specific radioactivity, when determined immediately following peptide iodination, was close to the theoretical maximum but exponentially decreased over time. These findings raise the possibility that attachment of a Bolton-Hunter conjugate may shield labeled peptides from radiation-induced damage, a scenario that should be considered when performing radioligand binding experiments.

  1. Acetone-Linked Peptides: A Convergent Approach for Peptide Macrocyclization and Labeling.

    PubMed

    Assem, Naila; Ferreira, David J; Wolan, Dennis W; Dawson, Philip E

    2015-07-20

    Macrocyclization is a broadly applied approach for overcoming the intrinsically disordered nature of linear peptides. Herein, it is shown that dichloroacetone (DCA) enhances helical secondary structures when introduced between peptide nucleophiles, such as thiols, to yield an acetone-linked bridge (ACE). Aside from stabilizing helical structures, the ketone moiety embedded in the linker can be modified with diverse molecular tags by oxime ligation. Insights into the structure of the tether were obtained through co-crystallization of a constrained S-peptide in complex with RNAse S. The scope of the acetone-linked peptides was further explored through the generation of N-terminus to side chain macrocycles and a new approach for generating fused macrocycles (bicycles). Together, these studies suggest that acetone linking is generally applicable to peptide macrocycles with a specific utility in the synthesis of stabilized helices that incorporate functional tags.

  2. Targeted therapy of colorectal neoplasia with rapamycin in peptide-labeled pegylated octadecyl lithocholate micelles

    PubMed Central

    Khondee, Supang; Rabinsky, Emily F.; Owens, Scott R.; Joshi, Bishnu P.; Qiu, Zhen; Duan, Xiyu; Zhao, Lili; Wang, Thomas D.

    2015-01-01

    Many powerful drugs have limited clinical utility because of poor water solubility and high systemic toxicity. Here, we formulated a targeted nanomedicine, rapamycin encapsulated in pegylated octadecyl lithocholate micelles labeled with a new ligand for colorectal neoplasia, LTTHYKL peptide. CPC;Apc mice that spontaneously develop colonic adenomas were treated with free rapamycin, plain rapamycin micelles, and peptide-labeled rapamycin micelles via intraperitoneal injection for 35 days. Endoscopy was performed to monitor adenoma regression in vivo. We observed complete adenoma regression at the end of therapy. The mean regression rate for peptide-labeled rapamycin micelles was significantly greater than that for plain rapamycin micelles, P<0.01. On immunohistochemistry, we observed a significant reduction in phospho-S6 but not β-catenin expression and reduced tumor cell proliferation, suggesting greater inhibition of downstream mTOR signaling. We observed significantly reduced renal toxicity for peptide-labeled rapamycin micelles compared to that of free drug, and no other toxicities were found on chemistries. Together, this unique targeted micelle represents a potential therapeutic for colorectal neoplasia with comparable therapeutic efficacy to rapamycin free drug and significantly less systemic toxicity. PMID:25483425

  3. (18)F- and (68)Ga-Labeled Neurotensin Peptides for PET Imaging of Neurotensin Receptor 1.

    PubMed

    Maschauer, Simone; Einsiedel, Jürgen; Hübner, Harald; Gmeiner, Peter; Prante, Olaf

    2016-07-14

    The neurotensin (NT) receptor-1 (NTS1) is overexpressed in a variety of carcinomas and is therefore an interesting target for imaging with positron emission tomography (PET). The aim of this study was the development of new NT derivatives based on the metabolically stable peptide sequence NLys-Lys-Pro-Tyr-Tle-Leu suitable for PET imaging. The NT peptides were synthesized by solid-phase supported peptide synthesis and elongated with respective chelators (NODA-GA, DOTA) for (68)Ga-labeling or propargylglycine for (18)F-labeling via copper-catalyzed azide-alkyne cycloaddition. Receptor affinities of the peptides for NTS1 were in the range of 19-110 nM. Biodistribution studies using HT29 tumor-bearing mice showed highest tumor uptake for [(68)Ga]6 and [(68)Ga]8 and specific binding in small-animal PET studies. The tumor uptake of (68)Ga-labeled peptides in vivo significantly correlated with the in vitro Ki values for NTS1. [(68)Ga]8 displayed an excellent tumor-to-background ratio and could therefore be considered as an appropriate molecular probe for NTS1 imaging by PET. PMID:27336295

  4. Interaction of short peptides with FITC-labeled wheat histones and their complexes with deoxyribooligonucleotides.

    PubMed

    Fedoreyeva, L I; Smirnova, T A; Kolomijtseva, G Ya; Khavinson, V Kh; Vanyushin, B F

    2013-02-01

    Judging from fluorescence modulation (quenching), short peptides (Ala-Glu-Asp-Gly, Glu-Asp-Arg, Ala-Glu-Asp-Leu, Lys-Glu-Asp-Gly, Ala-Glu-Asp-Arg, and Lys-Glu-Asp-Trp) bind with FITC-labeled wheat histones H1, H2B, H3, and H4. This results from the interaction of the peptides with the N-terminal histone regions that contain respective and seemingly homologous peptide-binding motifs. Because homologous amino acid sequences in wheat core histones were not found, the peptides seem to bind with some core histone regions having specific conformational structure. Peptide binding with histones and histone-deoxyribooligonucleotide complexes depends on the nature of the histone and the primary structures of the peptides and oligonucleotides; thus, it is site specific. Histones H1 bind preferentially with single-stranded oligonucleotides by homologous sites in the C-terminal region of the protein. Unlike histone H1, the core histones bind predominantly with double-stranded methylated oligonucleotides and methylated DNA. Stern-Volmer constants of interaction of histone H1 and core histones with double-stranded hemimethylated oligonucleotides are higher compared with that of binding with unmethylated ones. DNA or deoxyribooligonucleotides in a complex with histones can enhance or inhibit peptide binding. It is suggested that site-specific interactions of short biologically active peptides with histone tails can serve in chromatin as control epigenetic mechanisms of regulation of gene activity and cellular differentiation.

  5. High-affinity no-carrier-added 99mTc-labeled chemotactic peptides for studies of inflammation in vivo.

    PubMed

    Baidoo, K E; Scheffel, U; Stathis, M; Finley, P; Lever, S Z; Zhan, Y; Wagner, H N

    1998-01-01

    Nalpha-for-Nle-Leu-Phe-Nle-Tyr-Lys, a chemotactic peptide that binds with high affinity to the chemoattractant receptor on granulocytes and monocytes, was labeled with 99mTc using the diaminedithiol (DADT) chelating system to coordinate the Tc. 99mTc labeling of the DADT-coupled peptide was accomplished in 84% overall yield (room temperature for 10 min) using [99mTc]glucoheptonate as the donor of prereduced Tc. HPLC analysis showed two major 99mTc-labeled peptide peaks, 99mTc-DADT-Pep-I and 99mTc-DADT-Pep-II, were obtained in a ratio of 1:0.85. Using an iodoacetamide-derivatized gel to remove unlabeled peptide from the 99mTc labeling mixtures, essentially no-carrier-added (nca) high-specific activity 99mTc-labeled chemotactic peptides were obtained. The 99Tc analogues of the peptides were synthesized (72% yield) in a similar fashion and correlated with 99mTc complexes I and II by HPLC. In vitro competitive receptor binding assays of the isolated 99Tc analogues were performed against the tritiated chemotactic peptide [3H]N-for-Met-Leu-Phe ([3H]fMLF) using isolated granulocytes. The 99Tc-derivatized peptides showed similar binding affinities to the chemoattractant receptor as the unlabeled Nalpha-for-Nle-Leu-Phe-Nle-Tyr-Lys. The nca 99mTc-labeled peptides gave high contrast images of experimental inflammation in rabbits without causing neutropenia. Thus, it is feasible to attach the Tc-DADT chelate to low-molecular weight receptor binding chemotactic peptides and retain substantial binding to the receptor. Chemotactic peptides labeled with 99mTc via the DADT ligand system have the potential for imaging focal sites of inflammation without toxic effects, an important consideration in the successful utilization of chemotactic peptide agonists.

  6. Interconversion of Peptide Mass Spectral Libraries Derivatized with iTRAQ or TMT Labels.

    PubMed

    Zhang, Zheng; Yang, Xiaoyu; Mirokhin, Yuri A; Tchekhovskoi, Dmitrii V; Ji, Weihua; Markey, Sanford P; Roth, Jeri; Neta, Pedatsur; Hizal, Deniz Baycin; Bowen, Michael A; Stein, Stephen E

    2016-09-01

    Derivitization of peptides with isobaric tags such as iTRAQ and TMT is widely employed in proteomics due to their compatibility with multiplex quantitative measurements. We recently made publicly available a large peptide library derived from iTRAQ 4-plex labeled spectra. This resource has not been used for identifying peptides labeled with related tags with different masses, because values for virtually all masses of precursor and most product ions would differ for ions containing the different tags as well as containing different tag-specific peaks. We describe a method for interconverting spectra from iTRAQ 4-plex to TMT (6- and 10-plex) and to iTRAQ 8-plex. We interconvert spectra by appropriately mass shifting sequence ions and discarding derivative-specific peaks. After this "cleaning" of search spectra, we demonstrate that the converted libraries perform well in terms of peptide spectral matches. This is demonstrated by comparing results using sequence database searches as well as by comparing search effectiveness using original and converted libraries. At 1% FDR TMT labeled query spectra match 97% as many spectra against a converted iTRAQ library as compared to an original TMT library. Overall this interconversion strategy provides a practical way to extend results from one derivatization method to others that share related chemistry and do not significantly alter fragmentation profiles. PMID:27386737

  7. Interconversion of Peptide Mass Spectral Libraries Derivatized with iTRAQ or TMT Labels.

    PubMed

    Zhang, Zheng; Yang, Xiaoyu; Mirokhin, Yuri A; Tchekhovskoi, Dmitrii V; Ji, Weihua; Markey, Sanford P; Roth, Jeri; Neta, Pedatsur; Hizal, Deniz Baycin; Bowen, Michael A; Stein, Stephen E

    2016-09-01

    Derivitization of peptides with isobaric tags such as iTRAQ and TMT is widely employed in proteomics due to their compatibility with multiplex quantitative measurements. We recently made publicly available a large peptide library derived from iTRAQ 4-plex labeled spectra. This resource has not been used for identifying peptides labeled with related tags with different masses, because values for virtually all masses of precursor and most product ions would differ for ions containing the different tags as well as containing different tag-specific peaks. We describe a method for interconverting spectra from iTRAQ 4-plex to TMT (6- and 10-plex) and to iTRAQ 8-plex. We interconvert spectra by appropriately mass shifting sequence ions and discarding derivative-specific peaks. After this "cleaning" of search spectra, we demonstrate that the converted libraries perform well in terms of peptide spectral matches. This is demonstrated by comparing results using sequence database searches as well as by comparing search effectiveness using original and converted libraries. At 1% FDR TMT labeled query spectra match 97% as many spectra against a converted iTRAQ library as compared to an original TMT library. Overall this interconversion strategy provides a practical way to extend results from one derivatization method to others that share related chemistry and do not significantly alter fragmentation profiles.

  8. Peptide tag/probe pairs based on the coordination chemistry for protein labeling.

    PubMed

    Uchinomiya, Shohei; Ojida, Akio; Hamachi, Itaru

    2014-02-17

    Protein-labeling methods serve as essential tools for analyzing functions of proteins of interest under complicated biological conditions such as in live cells. These labeling methods are useful not only to fluorescently visualize proteins of interest in biological systems but also to conduct protein and cell analyses by harnessing the unique functions of molecular probes. Among the various labeling methods available, an appropriate binding pair consisting of a short peptide and a de novo designed small molecular probe has attracted attention because of its wide utility and versatility. Interestingly, most peptide tag/probe pairs exploit metal-ligand coordination interactions as the main binding force responsible for their association. Herein, we provide an overview of the recent progress of these coordination-chemistry-based protein-labeling methods and their applications for fluorescence imaging and functional analysis of cellular proteins, while highlighting our originally developed labeling methods. These successful examples clearly exemplify the utility and versatility of metal coordination chemistry in protein functional analysis.

  9. Synthesis and optical properties of pyrrolidinyl peptide nucleic acid carrying a clicked Nile red label

    PubMed Central

    Yotapan, Nattawut; Charoenpakdee, Chayan; Wathanathavorn, Pawinee; Ditmangklo, Boonsong

    2014-01-01

    Summary DNA or its analogues with an environment-sensitive fluorescent label are potentially useful as a probe for studying the structure and dynamics of nucleic acids. In this work, pyrrolidinyl peptide nucleic acid (acpcPNA) was labeled at its backbone with Nile red, a solvatochromic benzophenoxazine dye, by means of click chemistry. The optical properties of the Nile red-labeled acpcPNA were investigated by UV–vis and fluorescence spectroscopy in the absence and in the presence of DNA. In contrast to the usual quenching observed in Nile red-labeled DNA, the hybridization with DNA resulted in blue shifting and an enhanced fluorescence regardless of the neighboring bases. More pronounced blue shifts and fluorescence enhancements were observed when the DNA target carried a base insertion in close proximity to the Nile red label. The results indicate that the Nile red label is located in a more hydrophobic environment in acpcPNA–DNA duplexes than in the single-stranded acpcPNA. The different fluorescence properties of the acpcPNA hybrids of complementary DNA and DNA carrying a base insertion are suggestive of different interactions between the Nile red label and the duplexes. PMID:25246975

  10. Lu-177 labelled peptide treatment for radioiodine refractory differentiated thyroid carcinoma.

    PubMed

    Elboğa, Umut; Özkaya, Mesut; Sayiner, Zeynel A; Çelen, Yusuf Zeki

    2016-01-01

    Differentiated thyroid carcinoma (DTC) has good prognosis but 5% of the patients already have distant metastasis at the diagnosis. Tumour cells can lose their iodine uptake ability and enter a state of dedifferentiation. Treatment for differentiated thyroid carcinoma that is not suitable for the local surgery and unresponsive to radioactive iodine uptake is not always easy for physicians. We present a case of a 64-year-old man who had total thyroidectomy surgery and central lymph node dissection with diagnosis of multinodular goitre disease. Histopathological evaluation was papillary thyroid cancer with tall cell variant. Treatment using 150 mCi radioiodine was administered to the patient three times but could not effect a cure. We performed Ga-68 labelled DOTATE (synthetic somatostatin analogue peptide). This provided a good outcome. As evident from our case, Lu-177 radionuclide labelled synthetic somatostatin analogue peptides have therapeutic effect on radioiodine refractory DTC, as an alternative treatment modality. PMID:26957032

  11. The Effect of Peptide Identification Search Algorithms on MS2-Based Label-Free Protein Quantification

    PubMed Central

    Degroeve, Sven; Staes, An; De Bock, Pieter-Jan

    2012-01-01

    Abstract Several approaches exist for the quantification of proteins in complex samples processed by liquid chromatography-mass spectrometry followed by fragmentation analysis (MS2). One of these approaches is label-free MS2-based quantification, which takes advantage of the information computed from MS2 spectrum observations to estimate the abundance of a protein in a sample. As a first step in this approach, fragmentation spectra are typically matched to the peptides that generated them by a search algorithm. Because different search algorithms identify overlapping but non-identical sets of peptides, here we investigate whether these differences in peptide identification have an impact on the quantification of the proteins in the sample. We therefore evaluated the effect of using different search algorithms by examining the reproducibility of protein quantification in technical repeat measurements of the same sample. From our results, it is clear that a search engine effect does exist for MS2-based label-free protein quantification methods. As a general conclusion, it is recommended to address the overall possibility of search engine-induced bias in the protein quantification results of label-free MS2-based methods by performing the analysis with two or more distinct search engines. PMID:22804230

  12. Plasma protein binding of (99m)Tc-labeled hydrazino nicotinamide derivatized polypeptides and peptides.

    PubMed

    Ono, M; Arano, Y; Mukai, T; Uehara, T; Fujioka, Y; Ogawa, K; Namba, S; Nakayama, M; Saga, T; Konishi, J; Horiuchi, K; Yokoyama, A; Saji, H

    2001-02-01

    6-Hydrazinopyridine-3-carboxylic acid (HYNIC) constitutes one of the most attractive reagents to prepare (99m)Tc-labeled polypeptides and peptides of various molecular weights in combination with two tricine molecules as coligands. Indeed, (99m)Tc-HYNIC-conjugated IgG showed biodistribution of radioactivity similar to that of (111)In-DTPA-conjugated IgG. However, recent studies indicated significant plasma protein binding when the (99m)Tc labeling procedure was expanded to low molecular weight peptides. In this study, pharmacokinetics of (99m)Tc-HYNIC-conjugated IgG, Fab and RC160 using tricine were compared with their radioiodinated counterparts to evaluate this (99m)Tc-labeling method. In mice, [(99m)Tc](HYNIC-IgG)(tricine)(2) and [(99m)Tc](HYNIC-Fab)(tricine)(2) showed persistent localization of radioactivity in tissues when compared with their (125)I-labeled counterparts. [(99m)Tc](HYNIC-IgG)(tricine)(2) eliminated from the blood at a rate similar to that of (125)I-labeled IgG, while [(99m)Tc](HYNIC-Fab)(tricine)(2) showed significantly slower clearance of the radioactivity than (125)I-labeled Fab. On size-exclusion HPLC analyses, little changes were observed in radiochromatograms after incubation of [(99m)Tc](HYNIC-IgG)(tricine)(2) in murine plasma. However, [(99m)Tc](HYNIC-Fab)(tricine)(2) and [(99m)Tc](HYNIC-RC160)(tricine)(2) demonstrated significant increases in the radioactivity in higher molecular weight fractions in plasma. Formation of higher molecular weight species was reduced when [(99m)Tc](HYNIC-RC160)(tricine)(2) was stabilized with nicotinic acid (NIC) to generate [(99m)Tc](HYNIC-RC160)(tricine)(NIC). [(99m)Tc](HYNIC-RC160)(tricine)(NIC) also demonstrated significantly faster clearance of the radioactivity from the blood than [(99m)Tc](HYNIC-RC160)(tricine)(2). These findings suggested that one of the tricine coligands in (99m)Tc-HYNIC-labeled (poly)peptides would be replaced with plasma proteins to generate higher molecular weight species that

  13. Catalytic center of lecithin:cholesterol acyltransferase: isolation and sequence of diisopropyl fluorophosphate-labeled peptides

    SciTech Connect

    Park, Y.B.; Yueksel, U.G.; Gracy, R.W.; Lacko, A.G.

    1987-02-27

    Lecithin:cholesterol acyltransferase (LCAT) was purified from hog plasma and subsequently reacted with (/sup 3/H)-Diisopropyl fluorophosphate (DFP). The labeled enzyme was digested with pepsin and the peptides separated by high performance liquid chromatography (HPLC). Two radioactive peptides were isolated, subjected to automated amino acid sequencing and yielded the following data: A) Ile-Ser-Leu-Gly-Ala-Pro-Trp-Gly-Gly-Ser, and B) Tyr-Ile-Phe-Asp-x-Gly-Phe-Pro-Tyr-x-Asp-Pro-Val. Both of these sequences represent very highly conserved regions of the enzyme when compared to the sequence of human LCAT. Peptide (A) is considered to represent the catalytic center of LCAT based on comparisons with data reported in the literature.

  14. Interaction of fluorescently labeled triethyleneglycol and peptide derivatives with β-cyclodextrin.

    PubMed

    Alouini, Mohamed-Anis; Moustoifa, El-Farouck; Rubio-Albenque, Sandra; Berthelot, Thomas; Fery-Forgues, Suzanne; Déléris, Gérard

    2014-02-24

    A triethyleneglycol (TEG) chain, a linear peptide, and a cyclic peptide labeled with 7-methoxycoumarin-3-carboxylic acid (MC) and 7-diethylaminocoumarin-3-carboxylic acid (DAC) were used to thoroughly study Förster resonance energy transfer (FRET) in inclusion complexes. (1) H NMR evidence was given for the formation of a 1:1 inclusion complex between β-cyclodextrin (β-CD) and the fluorophore moieties of model compounds. The binding constant was 20 times higher for DAC than for MC derivatives. Molecular modeling provided additional information. The UV/Vis absorption and fluorescence properties were studied and the energy transfer process was quantified. Fluorescence quenching was particularly strong for the peptide derivatives. The presence of β-CDs reduced the FRET efficiency slightly. Dye-labeled peptide derivatives can thus be used to form inclusion complexes with β-CDs and retain most of their FRET properties. This paves the way for their subsequent use in analytical devices that are designed to measure the activity of matrix metalloproteinases.

  15. Improved 18F Labeling of Peptides with a Fluoride-Aluminum-Chelate Complex

    PubMed Central

    McBride, William J.; D’Souza, Christopher A.; Sharkey, Robert M.; Karacay, Habibe; Rossi, Edmund A.; Chang, Chien-Hsing; Goldenberg, David M.

    2010-01-01

    We reported previously the feasibility to radiolabel peptides with fluorine-18 (18F) using a rapid, one-pot, method that first mixes 18F− with Al3+, and then binds the (Al18F)2+ complex to a NOTA ligand on the peptide. In this report, we examined several new NOTA ligands and determined how temperature, reaction time, and reagent concentration affected the radiolabeling yield. Four structural variations of the NOTA ligand had isolated radiolabeling yields ranging from 5.8% to 87% under similar reaction conditions. All of the Al18F NOTA complexes were stable in vitro in human serum and those that were tested in vivo also were stable. The radiolabeling reactions were performed at 100°C and the peptides could be labeled in as little as five minutes. The IMP467 peptide could be labeled up to 115 GBq/μmol (3100 Ci/mmol), with a total reaction and purification time of 30 min without chromatographic purification. PMID:20540570

  16. Label-free detection of pathogenic bacteria via immobilized antimicrobial peptides.

    PubMed

    Dong, Zong-Mu; Zhao, Guang-Chao

    2015-05-01

    A novel label-free strategy for the detection of bacteria was developed by using a specific antimicrobial peptide (AMP)-functionalized quartz crystal microbalance (QCM) electrode. This electrode interface was successfully applied to detect pathogenic Escherichia coli O157:H7 based on the specific affinity between the small synthetic antimicrobial peptide and the bacterial cell of pathogenic E. coli O157:H7. The concentrations of pathogenic E. coli O157:H7 were sensitively measured by the frequency response of the QCM with a detection limit of 0.4 cfu μL(-1). The detection can be fulfilled within 10 min because it does not require germiculture process. On the other hand, if the specific antimicrobial peptides were immobilized on a gold electrode, this label-free strategy can also be performed by electrochemical impedance spectroscopy (EIS). Compared with QCM technique, the EIS measurement gives a lower sensitivity and needs a longer assay time. The combination of antimicrobial peptides with the real-time responses of QCM, as well as electronic read-out monitoring of EIS, may open a new way for the direct detection of bacteria.

  17. Differential 14N/15N-Labeling of Peptides Using N-Terminal Charge Derivatization with a High-Proton Affinity for Straightforward de novo Peptide Sequencing

    PubMed Central

    Nihashi, Yoichiro; Miyashita, Masahiro; Awane, Hiroyuki; Miyagawa, Hisashi

    2013-01-01

    While de novo peptide sequencing is essential in many situations, it remains a difficult task. This is because peptide fragmentation results in complicated and often incomplete product ion spectra. In a previous study, we demonstrated that N-terminal charge derivatization with 4-amidinobenzoic acid (Aba) resulted in improved peptide fragmentation under low-energy CID conditions. However, even with this derivatization, some ambiguity exists, due to difficulties in discriminating between N- and C-terminal fragments. In this study, to specifically identify b-ions from complex product ion spectra, the differential 14N/15N-labeling of peptides was performed using Aba derivatization. 15N-Labeled Aba was synthesized in the form of a succinimide ester. Peptides were derivatized individually with 14N-Aba or 15N-Aba and analyzed by ESI-MS/MS using a linear ion trap-Orbitrap hybrid FTMS system. The N-terminal fragments (i.e., b-ions) were then identified based on m/z differences arising from isotope labeling. By comparing the spectra between 14N- and 15N-Aba derivatized peptides, b-ions could be successfully identified based on the m/z shifts, which provided reliable sequencing results for all of the peptides examined in this study. The method developed in this study allows the easy and reliable de novo sequencing of peptides, which is useful in peptidomics and proteomics studies. PMID:24860714

  18. Synchrotron X-ray fluorescence studies of a bromine-labelled cyclic RGD peptide interacting with individual tumor cells.

    PubMed

    Sheridan, Erin J; Austin, Christopher J D; Aitken, Jade B; Vogt, Stefan; Jolliffe, Katrina A; Harris, Hugh H; Rendina, Louis M

    2013-03-01

    The first example of synchrotron X-ray fluorescence imaging of cultured mammalian cells in cyclic peptide research is reported. The study reports the first quantitative analysis of the incorporation of a bromine-labelled cyclic RGD peptide and its effects on the biodistribution of endogenous elements (for example, K and Cl) within individual tumor cells.

  19. Preparation of ⁶⁸Ga-labelled DOTA-peptides using a manual labelling approach for small-animal PET imaging.

    PubMed

    Romero, Eduardo; Martínez, Alfonso; Oteo, Marta; García, Angel; Morcillo, Miguel Angel

    2016-01-01

    (68)Ga-DOTA-peptides are a promising PET radiotracers used in the detection of different tumours types due to their ability for binding specifically receptors overexpressed in these. Furthermore, (68)Ga can be produced by a (68)Ge/(68)Ga generator on site which is a very good alternative to cyclotron-based PET isotopes. Here, we describe a manual labelling approach for the synthesis of (68)Ga-labelled DOTA-peptides based on concentration and purification of the commercial (68)Ga/(68)Ga generator eluate using an anion exchange-cartridge. (68)Ga-DOTA-TATE was used to image a pheochromocytoma xenograft mouse model by a microPET/CT scanner. The method described provides satisfactory results, allowing the subsequent (68)Ga use to label DOTA-peptides. The simplicity of the method along with its implementation reduced cost, makes it useful in preclinical PET studies. PMID:26492321

  20. Preparation of ⁶⁸Ga-labelled DOTA-peptides using a manual labelling approach for small-animal PET imaging.

    PubMed

    Romero, Eduardo; Martínez, Alfonso; Oteo, Marta; García, Angel; Morcillo, Miguel Angel

    2016-01-01

    (68)Ga-DOTA-peptides are a promising PET radiotracers used in the detection of different tumours types due to their ability for binding specifically receptors overexpressed in these. Furthermore, (68)Ga can be produced by a (68)Ge/(68)Ga generator on site which is a very good alternative to cyclotron-based PET isotopes. Here, we describe a manual labelling approach for the synthesis of (68)Ga-labelled DOTA-peptides based on concentration and purification of the commercial (68)Ga/(68)Ga generator eluate using an anion exchange-cartridge. (68)Ga-DOTA-TATE was used to image a pheochromocytoma xenograft mouse model by a microPET/CT scanner. The method described provides satisfactory results, allowing the subsequent (68)Ga use to label DOTA-peptides. The simplicity of the method along with its implementation reduced cost, makes it useful in preclinical PET studies.

  1. GTP-binding peptide of beta-tubulin. Localization by direct photoaffinity labeling and comparison with nucleotide-binding proteins

    SciTech Connect

    Linse, K.; Mandelkow, E.M.

    1988-10-15

    The binding site of the guanine moiety of GTP on beta-tubulin was located within the peptide consisting of residues 63-77, AILVDLEPGTMDSVR. The result was obtained using direct photoaffinity labeling, peptide sequencing, and limited proteolysis. Peptides were identified by end-labeling with a monoclonal antibody against beta-tubulin whose epitope was located between 3 and 4 kDa from the C terminus. The sequence of the GTP-binding site is consistent with predictions from other GTP-binding proteins such as elongation factor Tu or ras p21.

  2. Copper-62 labeled ReCCMSH peptide analogs for melanoma PET imaging.

    PubMed

    Zhang, Xiuli; Yue, Zhiwei; Lu, Bao-Yuan; Vazquez-Flores, Gerson J; Yuen, Johnny; Figueroa, Said Daibes; Gallazzi, Fabio; Cutler, Cathy; Quinn, Thomas P; Lacy, Jeffrey L

    2012-10-01

    High-specific activity radiolabeled melanocortin peptide preparations are necessary for optimal melanoma imaging due to the relatively low number of melanocortin-1 receptors (MC1-Rs) per tumor cell. In this study, a one-step synthesis of 62Cu-labeled MC1-R targeting peptide Re(Arg11)CCMSH was developed, which yielded high specific activity radiolabeled peptide preparations that required no post-labeling purification. DOTA and NOTA conjugated Re(Arg11)CCMSH peptides were synthesized and examined for 62Cu radiolabeling and cell binding properties. Biodistribution and PET imaging studies were performed to assess the in vivo tumor targeting and imaging characteristics of the optimal radiolabeled peptide. Melanoma cell binding affinities for NOTA-, NOTA-GGG-, and NOTA-GSG- conjugated Re(Arg11)CCMSH were determined to be 1.3×10-9 M, 1.9×10-9 M and 6.0×10-9 M. The 62Cu radiolabeling efficiencies of DOTA- and NOTA- conjugated Re(Arg11)CCMSH analogs were 30% and > 98% after 2 min at 24° C, while 0.5 μg of NOTA-GGG-peptide could be labeled to > 95% with a maximum specific activity of 138 Ci/μmol. Tumor uptake of 62Cu- NOTA-GGG-Re(Arg11)CCMSH in B16/F1 melanoma bearing mice was 4.65±0.48% ID/g and 9.43±2.69% ID/g at 20 and 40 min post injection and was visualized by PET imaging. High specific activity 62Cu-NOTA-GGG-Re(Arg11)CCMSH was prepared in a one-step procedure at 24°C in 6 min. 62Cu-NOTA-GGG-Re(Arg11)CCMSH exhibited MC1-R selective binding and rapid tumor uptake in B16/F1 melanoma bearing mice that was confirmed by PET imaging studies. High specific activity 62Cu from a 62Zn/62Cu generator coupled with simple one step radiolabeling procedures makes 62Cu an attractive radionuclide for PET imaging of low-density receptor targets.

  3. Single molecule resolution of the antimicrobial action of quantum dot-labeled sushi peptide on live bacteria

    PubMed Central

    Leptihn, Sebastian; Har, Jia Yi; Chen, Jianzhu; Ho, Bow; Wohland, Thorsten; Ding, Jeak Ling

    2009-01-01

    Background Antimicrobial peptides are found in all kingdoms of life. During the evolution of multicellular organisms, antimicrobial peptides were established as key elements of innate immunity. Most antimicrobial peptides are thought to work by disrupting the integrity of cell membranes, causing pathogen death. As antimicrobial peptides target the membrane structure, pathogens can only acquire resistance by a fundamental change in membrane composition. Hence, the evolution of pathogen resistance has been a slow process. Therefore antimicrobial peptides are valuable alternatives to classical antibiotics against which multiple drug-resistant bacteria have emerged. For potential therapeutic applications as antibiotics a thorough knowledge of their mechanism of action is essential. Despite the increasingly comprehensive understanding of the biochemical properties of these peptides, the actual mechanism by which antimicrobial peptides lyse microbes is controversial. Results Here we investigate how Sushi 1, an antimicrobial peptide derived from the horseshoe crab (Carcinoscorpius rotundicauda), induces lysis of Gram-negative bacteria. To follow the entire process of antimicrobial action, we performed a variety of experiments including transmission electron microscopy and fluorescence correlation spectroscopy as well as single molecule tracking of quantum dot-labeled antimicrobial peptides on live bacteria. Since in vitro measurements do not necessarily correlate with the in vivo action of a peptide we developed a novel fluorescent live bacteria lysis assay. Using fully functional nanoparticle-labeled Sushi 1, we observed the process of antimicrobial action at the single-molecule level. Conclusion Recently the hypothesis that many antimicrobial peptides act on internal targets to kill the bacterium has been discussed. Here, we demonstrate that the target sites of Sushi 1 are outer and inner membranes and are not cytosolic. Further, our findings suggest four successive

  4. Analysis of Intrinsic Peptide Detectability via Integrated Label-Free and SRM-Based Absolute Quantitative Proteomics.

    PubMed

    Jarnuczak, Andrew F; Lee, Dave C H; Lawless, Craig; Holman, Stephen W; Eyers, Claire E; Hubbard, Simon J

    2016-09-01

    Quantitative mass spectrometry-based proteomics of complex biological samples remains challenging in part due to the variability and charge competition arising during electrospray ionization (ESI) of peptides and the subsequent transfer and detection of ions. These issues preclude direct quantification from signal intensity alone in the absence of a standard. A deeper understanding of the governing principles of peptide ionization and exploitation of the inherent ionization and detection parameters of individual peptides is thus of great value. Here, using the yeast proteome as a model system, we establish the concept of peptide F-factor as a measure of detectability, closely related to ionization efficiency. F-factor is calculated by normalizing peptide precursor ion intensity by absolute abundance of the parent protein. We investigated F-factor characteristics in different shotgun proteomics experiments, including across multiple ESI-based LC-MS platforms. We show that F-factors mirror previously observed physicochemical predictors as peptide detectability but demonstrate a nonlinear relationship between hydrophobicity and peptide detectability. Similarly, we use F-factors to show how peptide ion coelution adversely affects detectability and ionization. We suggest that F-factors have great utility for understanding peptide detectability and gas-phase ion chemistry in complex peptide mixtures, selection of surrogate peptides in targeted MS studies, and for calibration of peptide ion signal in label-free workflows. Data are available via ProteomeXchange with identifier PXD003472. PMID:27454336

  5. Novel fluorescently labeled peptide compounds for detection of oxidized low-density lipoprotein at high specificity.

    PubMed

    Sato, Akira; Yamanaka, Hikaru; Oe, Keitaro; Yamazaki, Yoji; Ebina, Keiichi

    2014-10-01

    The probes for specific detection of oxidized low-density lipoprotein (ox-LDL) in plasma and in atherosclerotic plaques are expected to be useful for the identification, diagnosis, prevention, and treatment for atherosclerosis. In this study, to develop a fluorescent peptide probe for specific detection of ox-LDL, we investigated the interaction of fluorescein isothiocyanate (FITC)-labeled peptides with ox-LDL using polyacrylamide gel electrophoresis. Two heptapeptides (KWYKDGD and KP6) coupled through the ε-amino group of K at the N-terminus to FITC in the presence/absence of 6-amino-n-caproic acid (AC) linker to FITC--(FITC-AC)KP6 and (FITC)KP6--both bound with high specificity to ox-LDL in a dose-dependent manner. In contrast, a tetrapeptide (YKDG) labeled with FITC at the N-terminus and a pentapeptide (YKDGK) coupled through the ε-amino group of K at the C-terminus to FITC did not bind selectively to ox-LDL. Furthermore, (FITC)KP6 and (FITC-AC)KP6 bound with high specificity to the protein in mouse plasma (probably ox-LDL fraction). These findings strongly suggest that (FITC)KP6 and (FITC-AC)KP6 may be effective novel fluorescent probes for specific detection of ox-LDL.

  6. Penetration of short fluorescence-labeled peptides into the nucleus in HeLa cells and in vitro specific interaction of the peptides with deoxyribooligonucleotides and DNA.

    PubMed

    Fedoreyeva, L I; Kireev, I I; Khavinson, V Kh; Vanyushin, B F

    2011-11-01

    Marked fluorescence in cytoplasm, nucleus, and nucleolus was observed in HeLa cells after incubation with each of several fluorescein isothiocyanate-labeled peptides (epithalon, Ala-Glu-Asp-Gly; pinealon, Glu-Asp-Arg; testagen, Lys-Glu-Asp-Gly). This means that short biologically active peptides are able to penetrate into an animal cell and its nucleus and, in principle they may interact with various components of cytoplasm and nucleus including DNA and RNA. It was established that various initial (intact) peptides differently affect the fluorescence of the 5,6-carboxyfluorescein-labeled deoxyribooligonucleotides and DNA-ethidium bromide complexes. The Stern-Volmer constants characterizing the degree of fluorescence quenching of various single- and double-stranded fluorescence-labeled deoxyribooligonucleotides with short peptides used were different depending on the peptide primary structures. This indicates the specific interaction between short biologically active peptides and nucleic acid structures. On binding to them, the peptides discriminate between different nucleotide sequences and recognize even their cytosine methylation status. Judging from corresponding constants of the fluorescence quenching, the epithalon, pinealon, and bronchogen (Ala-Glu-Asp-Leu) bind preferentially with deoxyribooligonucleotides containing CNG sequence (CNG sites are targets for cytosine DNA methylation in eukaryotes). Epithalon, testagen, and pinealon seem to preferentially bind with CAG- but bronchogen with CTG-containing sequences. The site-specific interactions of peptides with DNA can control epigenetically the cell genetic functions, and they seem to play an important role in regulation of gene activity even at the earliest stages of life origin and in evolution.

  7. Exploring the Potential of (99m)Tc(CO)3-Labeled Triazolyl Peptides for Tumor Diagnosis.

    PubMed

    Gaonkar, Raghuvir H; Ganguly, Soumya; Baishya, Rinku; Dewanjee, Saikat; Sinha, Samarendu; Gupta, Amit; Ganguly, Shantanu; Debnath, Mita C

    2016-04-01

    In recent years the authors have reported on (99m)Tc(CO)3-labeled peptides that serve as carriers for biomolecules or radiopharmaceuticals to the tumors. In continuation of that work they report the synthesis of a pentapeptide (Met-Phe-Phe-Gly-His; pep-1), a hexapeptide (Met-Phe-Phe-Asp-Gly-His; pep-2), and a tetrapeptide (Asp-Gly-Arg-His; pep-3) and the attachment of 3-amino-1,2,4-triazole to the β carboxylic function of the aspartic acid unit of pep-2 and pep-3. The pharmacophores were radiolabeled in high yields with [(99m)Tc(CO)3(H2O)3](+) metal aqua ion, characterized for their stability in serum and saline, as well as in His solution, and found to be substantially stable. B16F10 cell line binding studies showed favorable uptake and internalization. In vivo behavior of the radiolabeled triazolyl peptides was assessed in mice bearing induced tumor. The (99m)Tc(CO)3-triazolyl pep-3 demonstrated rapid urinary clearance and comparatively better tumor uptake. Imaging studies showed visualization of the tumor using (99m)Tc(CO)3-triazolyl pep-3, but due to high abdominal background, low delineation occurred. Based on the results further experiments will be carried out for targeting tumor with triazolyl peptides. PMID:27093344

  8. Imaging inflammation with 99Tcm-labeled chemotactic peptides: analogues with reduced neutropenia.

    PubMed

    Pollak, A; Goodbody, A E; Ballinger, J R; Duncan, G S; Tran, L L; Dunn-Dufault, R; Meghji, K; Lau, F; Andrey, T W; Boxen, I; Sumner-Smith, M

    1996-02-01

    Two 99Tcm-labelled analogues of the chemotactic peptide ForMLF were evaluated as potential agents for imaging inflammation and infection, in the hope that they would be simple to use and would give diagnostically useful images shortly after injection. The peptides differed in the chelation site for 99Tcm and the presence of a hydrophilic spacer. The sequences of RP050 and RP056 were ForNleLFNleYK(G)G-C(Acm)-GPic and ForNleLFNleYKK(DG)GC(Acm)SPic respectively, where Pic is picolinic acid. In in vitro tests of binding to the ForMLF receptor on polymorphonuclear neutrophils and potency for release of myeloperoxidase, RP056 was similar in potency to ForMLF, whereas RP050 was 10 times more potent. When administered in 5-nmol doses to rats, RP050 produced less extensive neutropenia than ForMLF, whereas RP056 produced very little neutropenia. Following labelling by ligand exchange from tartrate or glucoheptonate at 100 degrees C and purification using a C-18 solid-phase extraction cartridge, 4-MBq doses were administered to rats bearing infectious (Escherichia coli) or sterile (zymosan) inflammation sites in the thigh. The inflammation-to-normal muscle ratios at 30 min after injection were 3.9 +/- 0.4 for RP050 and 4.7 +/- 0.3 for RP056 (mean +/- S.E.M., n = 4), and the ratios were maintained for up to 3 h. These peptides are promising agents for imaging inflammation and infection.

  9. Efficient 18F labeling of cysteine-containing peptides and proteins using tetrazine-trans-cyclooctene ligation.

    PubMed

    Liu, Shuanglong; Hassink, Matthew; Selvaraj, Ramajeyam; Yap, Li-Peng; Park, Ryan; Wang, Hui; Chen, Xiaoyuan; Fox, Joseph M; Li, Zibo; Conti, Peter S

    2013-01-01

    18F positron emission tomography (PET) has a number of attributes that make it clinically attractive, including nearly 100% positron efficiency, very high specific radioactivity, and a short half-life of ≈ 110 minutes. However, the short half-life of 18F and the poor nucleophilicity of fluoride introduce challenges for the incorporation of 18F into complex molecules. Recently, the tetrazine-trans-cyclooctene ligation was introduced as a novel 18F labeling method that proceeds with fast reaction rates without catalysis. Herein we report an efficient method for 18F labeling of free cysteines of peptides and proteins based on sequential ligation with a bifunctional tetrazinyl-maleimide and an 18F-labeled trans-cyclooctene. The newly developed method was tested for site-specific labeling of both c(RGDyC) peptide and vascular endothelial growth factor (VEGF)-SH protein. Starting with 4 mCi of 18F-trans-cyclooctene and only 10 μg of tetrazine-RGD (80-100 μM) or 15 μg of tetrazine-VEGF (6.0 μM), 18F-labeled RGD peptide and VEGF protein could be obtained within 5 minutes in 95% yield and 75% yield, respectively. The obtained tracers were then evaluated in mice. In conclusion, a highly efficient method has been developed for site-specific 18F labeling of cysteine-containing peptides and proteins. The special characteristics of the tetrazine-trans-cyclooctene ligation provide unprecedented opportunities to synthesize 18F-labeled probes with high specific activity for PET applications.

  10. Pseudo isobaric peptide termini labelling for relative proteome quantification by SWATH MS acquisition.

    PubMed

    Zhang, Shen; Chen, Lingfan; Shan, Yichu; Sui, Zhigang; Wu, Qi; Zhang, Lihua; Liang, Zhen; Zhang, Yukui

    2016-08-01

    The pseudo isobaric peptide termini labeling (IPTL) method is a remarkable strategy in quantitative proteomics, and has been efficiently applied in biological studies due to its high quantitative accuracy. However, irreproducible precursor ion selection caused by data dependent acquisition and the chromatographic shift caused by isotope effects limit the wide application of this method. Herein, we expand the use of pseudo IPTL to SWATH MS application and develop a novel quantitative strategy, termed SWATH-pseudo-IPTL, by which the relative quantification could be achieved by comparing the "complete" extracted ion chromatogram (XIC) intensity of MS/MS scan instead of a single intensity measurement in DDA-pseudo-IPTL which only reflected the peptide abundances at that given time. The quantitative analysis of various proportions of mixed HeLa samples revealed the strong accuracy and precision of our SWATH-pseudo-IPTL method, both of which were better than that of the DDA-pseudo-IPTL strategy. SWATH-pseudo-IPTL was also applied to the quantitative profiling of the proteome from human hepatocellular carcinoma cell lines with high and low metastatic potential, and most of the differentially expressed proteins were related to tumorigenesis and tumor metastasis, demonstrating the feasibility of this methodology for biological applications.

  11. A silicon-based peptide biosensor for label-free detection of cancer cells

    NASA Astrophysics Data System (ADS)

    Martucci, Nicola M.; Rea, Ilaria; Ruggiero, Immacolata; Terracciano, Monica; De Stefano, Luca; Migliaccio, Nunzia; Dardano, Principia; Arcari, Paolo; Rendina, Ivo; Lamberti, Annalisa

    2015-05-01

    Sensitive and accurate detection of cancer cells plays a crucial role in diagnosis of cancer and minimal residual disease, so being one of the most hopeful approaches to reduce cancer death rates. In this paper, a strategy for highly selective and sensitive detection of lymphoma cells on planar silicon-based biosensor has been evaluated. In this setting an Idiotype peptide, able to specifically bind the B-cell receptor (BCR) of A20 cells in mice engrafted with A20 lymphoma, has been covalently linked to the sensor active surface and used as molecular probe. The biochip here presented showed a coverage efficiency of 85% with a detection efficiency of 8.5×10-3 cells/μm2. The results obtained suggested an efficient way for specific label-free cell detection by using a silicon-based peptide biosensor. In addition, the present recognition strategy, besides being useful for the development of sensing devices capable of monitoring minimal residual disease, could be used to find and characterize new specific receptor-ligand interactions through the screening of a recombinant phage library.

  12. Gd(III)-Labeled Peptide Nanofibers for Reporting on Biomaterial Localization in Vivo

    PubMed Central

    2015-01-01

    Bioactive supramolecular nanostructures are of great importance in regenerative medicine and the development of novel targeted therapies. In order to use supramolecular chemistry to design such nanostructures, it is extremely important to track their fate in vivo through the use of molecular imaging strategies. Peptide amphiphiles (PAs) are known to generate a wide array of supramolecular nanostructures, and there is extensive literature on their use in areas such as tissue regeneration and therapies for disease. We report here on a series of PA molecules based on the well-established β-sheet amino acid sequence V3A3 conjugated to macrocyclic Gd(III) labels for magnetic resonance imaging (MRI). These conjugates were shown to form cylindrical supramolecular assemblies using cryogenic transmission electron microscopy and small-angle X-ray scattering. Using nuclear magnetic relaxation dispersion analysis, we observed that thermal annealing of the nanostructures led to a decrease in water exchange lifetime (τm) of hundreds of nanoseconds only for molecules that self-assemble into nanofibers of high aspect ratio. We interpret this decrease to indicate more solvent exposure to the paramagnetic moiety on annealing, resulting in faster water exchange within angstroms of the macrocycle. We hypothesize that faster water exchange in the nanofiber-forming PAs arises from the dehydration and increase in packing density on annealing. Two of the self-assembling conjugates were selected for imaging PAs after intramuscular injections of the PA C16V3A3E3-NH2 in the tibialis anterior muscle of a murine model. Needle tracts were clearly discernible with MRI at 4 days postinjection. This work establishes Gd(III) macrocycle-conjugated peptide amphiphiles as effective tracking agents for peptide amphiphile materials in vivo over the timescale of days. PMID:24937195

  13. Peptide affinity labels for thrombin and other trypsin-like proteases

    DOEpatents

    Shaw, Elliott N.; Kettner, Charles A.

    1982-03-09

    A peptide affinity label of the formula (I): ##STR1## wherein X is a radical capable of acting as a leaving group in a nucleophilic substitution reaction; A is an aromatic amino acid residue; B is H, or a C.sub.1 -C.sub.4 alkyl group, or aryl; Y is selected from the group consisting of hydrogen, aroyl, C.sub.1 -C.sub.6 acyl, and Q--(A)--.sub.n, wherein Q=hydrogen, aroyl, or C.sub.1 -C.sub.6 acyl, n=1-10, A is an amino acid residue selected from the aliphatic, hydroxy-containing, carboxylic acid group, and amide-thereof-containing, aromatic, sulfur-containing and imino-containing amino acids; and wherein J is selected from the group consisting of --CH.sub.2 --, --CH.sub.2 --CH.sub.2 --,--CH.sub.2 --CH.sub.2 --CH.sub.2 --, --CH.dbd.CH-- and --CH(OH)--CH.sub.2. The affinity label is useful for irreversibly inactivating thrombin and trypsin-like enzymes and may be used as a potential anticlotting agent.

  14. Peptide affinity labels for thrombin and other trypsin-like proteases

    DOEpatents

    Shaw, E.N.; Kettner, C.A.

    1982-03-09

    A peptide affinity label is disclosed of the formula (I): as given in the patent wherein X is a radical capable of acting as a leaving group in a nucleophilic substitution reaction; A is an aromatic amino acid residue; B is H, or a C[sub 1]--C[sub 4] alkyl group, or aryl; Y is selected from the group consisting of hydrogen, aroyl, C[sub 1]--C[sub 6] acyl, and Q--(A)--[sub n], wherein Q = hydrogen, aroyl, or C[sub 1]--C[sub 6] acyl, n = 1--10, A is an amino acid residue selected from the aliphatic, hydroxy-containing, carboxylic acid group, and amide-thereofcontaining, aromatic, sulfur-containing and imino-containing amino acids; and wherein J is selected from the group consisting of --CH[sub 2]--, --CH[sub 2]--CH[sub 2]--, --CH[sub 2]--CH[sub 2]--CH[sub 2]--, --CH[double bond]CH-- and --CH(OH)--CH[sub 2]. The affinity label is useful for irreversibly inactivating thrombin and trypsin-like enzymes and may be used as a potential anticlotting agent. 2 figs.

  15. CW dipolar broadening EPR spectroscopy and mechanically aligned bilayers used to measure distance and relative orientation between two TOAC spin labels on an antimicrobial peptide

    NASA Astrophysics Data System (ADS)

    Sahu, Indra D.; Hustedt, Eric J.; Ghimire, Harishchandra; Inbaraj, Johnson J.; McCarrick, Robert M.; Lorigan, Gary A.

    2014-12-01

    An EPR membrane alignment technique was applied to measure distance and relative orientations between two spin labels on a protein oriented along the surface of the membrane. Previously we demonstrated an EPR membrane alignment technique for measuring distances and relative orientations between two spin labels using a dual TOAC-labeled integral transmembrane peptide (M2δ segment of Acetylcholine receptor) as a test system. In this study we further utilized this technique and successfully measured the distance and relative orientations between two spin labels on a membrane peripheral peptide (antimicrobial peptide magainin-2). The TOAC-labeled magainin-2 peptides were mechanically aligned using DMPC lipids on a planar quartz support, and CW-EPR spectra were recorded at specific orientations. Global analysis in combination with rigorous spectral simulation was used to simultaneously analyze data from two different sample orientations for both single- and double-labeled peptides. We measured an internitroxide distance of 15.3 Å from a dual TOAC-labeled magainin-2 peptide at positions 8 and 14 that closely matches with the 13.3 Å distance obtained from a model of the labeled magainin peptide. In addition, the angles determining the relative orientations of the two nitroxides have been determined, and the results compare favorably with molecular modeling. This study demonstrates the utility of the technique for proteins oriented along the surface of the membrane in addition to the previous results for proteins situated within the membrane bilayer.

  16. Stable isotope N-phosphorylation labeling for Peptide de novo sequencing and protein quantification based on organic phosphorus chemistry.

    PubMed

    Gao, Xiang; Wu, Hanzhi; Lee, Kim-Chung; Liu, Hongxia; Zhao, Yufen; Cai, Zongwei; Jiang, Yuyang

    2012-12-01

    In this paper, we describe the development of a novel stable isotope N-phosphorylation labeling (SIPL) strategy for peptide de novo sequencing and protein quantification based on organic phosphorus chemistry. The labeling reaction could be performed easily and completed within 40 min in a one-pot reaction without additional cleanup procedures. It was found that N-phosphorylation labeling reagents were activated in situ to form labeling intermediates with high reactivity targeting on N-terminus and ε-amino groups of lysine under mild reaction conditions. The introduction of N-terminal-labeled phosphoryl group not only improved the ionization efficiency of peptides and increased the protein sequence coverage for peptide mass fingerprints but also greatly enhanced the intensities of b ions, suppressed the internal fragments, and reduced the complexity of the tandem mass spectrometry (MS/MS) fragmentation patterns of peptides. By using nano liquid chromatography chip/time-of-flight mass spectrometry (nano LC-chip/TOF MS) for the protein quantification, the obtained results showed excellent correlation of the measured ratios to theoretical ratios with relative errors ranging from 0.5% to 6.7% and relative standard deviation of less than 10.6%, indicating that the developed method was reproducible and precise. The isotope effect was negligible because of the deuterium atoms were placed adjacent to the neutral phosphoryl group with high electrophilicity and moderately small size. Moreover, the SIPL approach used inexpensive reagents and was amenable to samples from various sources, including cell culture, biological fluids, and tissues. The method development based on organic phosphorus chemistry offered a new approach for quantitative proteomics by using novel stable isotope labeling reagents.

  17. Cysteinyl peptides labeled by dibromobutanedione in reaction with rabbit muscle pyruvate kinase.

    PubMed Central

    Vollmer, S. H.; Colman, R. F.

    1992-01-01

    The bifunctional reagent 1,4-dibromobutanedione (DBBD) reacts covalently with pyruvate kinase from rabbit muscle to cause inactivation of the enzyme at a rate that is linearly dependent on the reagent concentration, giving a second order rate constant of 444 min-1 M-1. The individual substrates phosphoenolpyruvate (with KCl), ADP, or ATP in the presence of divalent metal cation provide marked protection against inactivation suggesting that reaction occurs in the region of the active site. The limited incorporation of DBBD into pyruvate kinase was measured by reduction of the carbonyl groups of the enzyme-bound reagent using [3H]NaBH4. When pyruvate kinase was reacted with 120 microM DBBD at pH 7.0 for 50 min in the absence of protectants, 1.8 mol of tritium/mol of subunit was incorporated, whereas in the presence of phosphoenolpyruvate with KCl, only 1.0 mol of tritium was incorporated per mole of subunit. Modified peptides were isolated from tryptic digests of pyruvate kinase. Reaction of enzyme in the presence of substrate (showing no activity loss) yielded a single peptide, Asn-Ile-X1-Lys, where X1 corresponds to Cys164 of the known amino acid sequence of muscle pyruvate kinase. In the absence of protectants, reaction for 10 min (when the enzyme retained substantial activity) yielded Asn-Ile-X1-Lys as the major labeled peptide, whereas reaction for 50 min (when the enzyme was 88% inactivated) yielded predominantly Asn-Ile-X1-Lys cross-linked to X2-Asp-Glu-Asn-Ile-Leu-Trp-Leu-Asp-Tyr-Lys, where X2 corresponds to Cys151. Because activity loss correlates with the appearance of the cross-linked peptides but not with formation of Asn-Ile-X1-Lys, inactivation is likely caused by the reaction leading to the cross-link between Cys151 and Cys164. The distance between the alpha-carbons of these residues in the crystal structure is 15.5 A, whereas only 12.0 A can be spanned by the two side chains linked by a dioxobutyl group, suggesting either that pyruvate kinase

  18. Preprocessing significantly improves the peptide/protein identification sensitivity of high-resolution isobarically labeled tandem mass spectrometry data.

    PubMed

    Sheng, Quanhu; Li, Rongxia; Dai, Jie; Li, Qingrun; Su, Zhiduan; Guo, Yan; Li, Chen; Shyr, Yu; Zeng, Rong

    2015-02-01

    Isobaric labeling techniques coupled with high-resolution mass spectrometry have been widely employed in proteomic workflows requiring relative quantification. For each high-resolution tandem mass spectrum (MS/MS), isobaric labeling techniques can be used not only to quantify the peptide from different samples by reporter ions, but also to identify the peptide it is derived from. Because the ions related to isobaric labeling may act as noise in database searching, the MS/MS spectrum should be preprocessed before peptide or protein identification. In this article, we demonstrate that there are a lot of high-frequency, high-abundance isobaric related ions in the MS/MS spectrum, and removing isobaric related ions combined with deisotoping and deconvolution in MS/MS preprocessing procedures significantly improves the peptide/protein identification sensitivity. The user-friendly software package TurboRaw2MGF (v2.0) has been implemented for converting raw TIC data files to mascot generic format files and can be downloaded for free from https://github.com/shengqh/RCPA.Tools/releases as part of the software suite ProteomicsTools. The data have been deposited to the ProteomeXchange with identifier PXD000994. PMID:25435543

  19. Preprocessing significantly improves the peptide/protein identification sensitivity of high-resolution isobarically labeled tandem mass spectrometry data.

    PubMed

    Sheng, Quanhu; Li, Rongxia; Dai, Jie; Li, Qingrun; Su, Zhiduan; Guo, Yan; Li, Chen; Shyr, Yu; Zeng, Rong

    2015-02-01

    Isobaric labeling techniques coupled with high-resolution mass spectrometry have been widely employed in proteomic workflows requiring relative quantification. For each high-resolution tandem mass spectrum (MS/MS), isobaric labeling techniques can be used not only to quantify the peptide from different samples by reporter ions, but also to identify the peptide it is derived from. Because the ions related to isobaric labeling may act as noise in database searching, the MS/MS spectrum should be preprocessed before peptide or protein identification. In this article, we demonstrate that there are a lot of high-frequency, high-abundance isobaric related ions in the MS/MS spectrum, and removing isobaric related ions combined with deisotoping and deconvolution in MS/MS preprocessing procedures significantly improves the peptide/protein identification sensitivity. The user-friendly software package TurboRaw2MGF (v2.0) has been implemented for converting raw TIC data files to mascot generic format files and can be downloaded for free from https://github.com/shengqh/RCPA.Tools/releases as part of the software suite ProteomicsTools. The data have been deposited to the ProteomeXchange with identifier PXD000994.

  20. Direct demonstration of unique mode of natural peptide binding to the type 2 cholecystokinin receptor using photoaffinity labeling

    PubMed Central

    Dong, Maoqing; Miller, Laurence J.

    2013-01-01

    Direct analysis of mode of peptide docking using intrinsic photoaffinity labeling has provided detailed insights for the molecular basis of cholecystokinin (CCK) interaction with the type 1 CCK receptor. In the current work, this technique has been applied to the closely related type 2 CCK receptor that also binds the natural full agonist peptide, CCK, with high affinity. A series of photolabile CCK analogue probes with sites of covalent attachment extending from position 26 through 32 were characterized, with the highest affinity analogues that possessed full biological activity utilized in photoaffinity labeling. The position 29 probe, incorporating a photolabile benzoyl-phenylalanine in that position, was shown to bind with high affinity and to be a full agonist, with potency not different from that of natural CCK, and to covalently label the type 2 CCK receptor in a saturable, specific and efficient manner. Using proteolytic peptide mapping, mutagenesis, and radiochemical Edman degradation sequencing, this probe was shown to establish a covalent bond with type 2 CCK receptor residue Phe120 in the first extracellular loop. This was in contrast to its covalent attachment to Glu345 in the third extracellular loop of the type 1 CCK receptor, directly documenting differences in mode of docking this peptide to these receptors. PMID:23770253

  1. Two-dimensional nuclear magnetic resonance analysis of a labeled peptide bound to a class II major histocompatibility complex molecule.

    PubMed

    Driscoll, P C; Altman, J D; Boniface, J J; Sakaguchi, K; Reay, P A; Omichinski, J G; Appella, E; Davis, M M

    1993-07-20

    The formation of peptide/major histocompatibility complex (MHC) complexes and their subsequent recognition by T cells is a pivotal event in the initiation of an immune response. While X-ray crystal structures are now available for class I MHC/peptide complexes, little detailed structural information is known about the class II MHC equivalent, and there are no solution structure data for either. A 16 amino acid residue moth cytochrome c peptide (residues 88 to 103) was 13C-labeled for two-dimensional isotope-edited NMR analysis. The peptide was labeled either selectively in the methyl groups of alanine residues or uniformly at every carbon position, and bound to unlabeled soluble mouse I-Ek class II MHC molecules. Although alpha-helical in the native cytochrome c protein and with no uniform structure in solution, the peptide is bound to the I-Ek molecule with the alpha-carbon atoms of the 11 C-terminal residues held in the binding groove. This indicates that the class II MHC peptide binding site is somewhat larger than that of class I MHC molecules (> or = 11 amino acid residues versus 8 to 10 amino acid residues), consistent with recent data on eluted peptides. Despite the large size of the complex (approximately 70 kDa), nuclear Overhauser effects are clearly detectable between peptide side-chains and the MHC molecule. Indications of the buried or exposed nature of particular side-chains within the bound peptide are derived from the NMR data and these are used together with information from previous biological studies to propose a crude model of the interaction of the peptide with the groove of the MHC molecule. We find no evidence for a conformational change in the peptide/MHC complex in the spectra at pH 5.0 versus pH 7.0, despite a 40-fold faster on-rate for the peptide at the lower pH value. PMID:8393933

  2. Magnetic immunoaffinity enrichment for selective capture and MS/MS analysis of N-terminal-TMPP-labeled peptides.

    PubMed

    Bland, Céline; Bellanger, Laurent; Armengaud, Jean

    2014-02-01

    Proteogenomics is the alliance of proteomics and genomics with the aim of better annotating structural genes based on experimental, protein-based data items established by tandem mass spectrometry. While, on average, more than one-tenth of protein N-termini are incorrectly annotated, there is a crucial need for methodological approaches to systematically establish the translational starts of polypeptides, and their maturations, such as N-terminal methionine processing and peptide signal excision. Refinement of genome annotation through correction of wrongly annotation initiation start site and detection of unannotated genes can be achieved after enrichment and detection of protein N-termini by mass spectrometry. Here we describe a straightforward strategy to specifically label protein N-termini with a positively charged TMPP label to selectively capture these entities with in-house-developed anti-TMPP antibodies coupled to magnetic beads and to analyze them by nanoLC-MS/MS. While most N-terminomics-oriented approaches are based on the depletion of internal peptides to retrieve N-terminal peptides, this enrichment approach is fast and the results are highly specific for improved, ionizable, TMPP-labeled peptides. The whole proteome of the model marine bacterium, Roseobacter denitrificans, was analyzed, leading to the identification of more than twice the number of N-terminal peptides compared with the nonenriched fraction. A total of 269 proteins were characterized in terms of their N-termini. In addition, three unannotated genes were identified based on multiple, redundant N-terminal peptides. Our strategy greatly simplifies the systematic and automatic proteogenomic annotation of genomes as well as degradomics-oriented approaches, focusing the mass spectrometric efforts on the most crucial enriched fractions. PMID:24313271

  3. Magnetic immunoaffinity enrichment for selective capture and MS/MS analysis of N-terminal-TMPP-labeled peptides.

    PubMed

    Bland, Céline; Bellanger, Laurent; Armengaud, Jean

    2014-02-01

    Proteogenomics is the alliance of proteomics and genomics with the aim of better annotating structural genes based on experimental, protein-based data items established by tandem mass spectrometry. While, on average, more than one-tenth of protein N-termini are incorrectly annotated, there is a crucial need for methodological approaches to systematically establish the translational starts of polypeptides, and their maturations, such as N-terminal methionine processing and peptide signal excision. Refinement of genome annotation through correction of wrongly annotation initiation start site and detection of unannotated genes can be achieved after enrichment and detection of protein N-termini by mass spectrometry. Here we describe a straightforward strategy to specifically label protein N-termini with a positively charged TMPP label to selectively capture these entities with in-house-developed anti-TMPP antibodies coupled to magnetic beads and to analyze them by nanoLC-MS/MS. While most N-terminomics-oriented approaches are based on the depletion of internal peptides to retrieve N-terminal peptides, this enrichment approach is fast and the results are highly specific for improved, ionizable, TMPP-labeled peptides. The whole proteome of the model marine bacterium, Roseobacter denitrificans, was analyzed, leading to the identification of more than twice the number of N-terminal peptides compared with the nonenriched fraction. A total of 269 proteins were characterized in terms of their N-termini. In addition, three unannotated genes were identified based on multiple, redundant N-terminal peptides. Our strategy greatly simplifies the systematic and automatic proteogenomic annotation of genomes as well as degradomics-oriented approaches, focusing the mass spectrometric efforts on the most crucial enriched fractions.

  4. Immobilization-free electrochemical DNA detection with anthraquinone-labeled pyrrolidinyl peptide nucleic acid probe.

    PubMed

    Kongpeth, Jutatip; Jampasa, Sakda; Chaumpluk, Piyasak; Chailapakul, Orawon; Vilaivan, Tirayut

    2016-01-01

    Electrochemical detection provides a simple, rapid, sensitive and inexpensive method for DNA detection. In traditional electrochemical DNA biosensors, the probe is immobilized onto the electrode. Hybridization with the DNA target causes a change in electrochemical signal, either from the intrinsic signal of the probe/target or through a label or a redox indicator. The major drawback of this approach is the requirement for probe immobilization in a controlled fashion. In this research, we take the advantage of different electrostatic properties between PNA and DNA to develop an immobilization-free approach for highly sequence-specific electrochemical DNA sensing on a screen-printed carbon electrode (SPCE) using a square-wave voltammetric (SWV) technique. Anthraquinone-labeled pyrrolidinyl peptide nucleic acid (AQ-PNA) was employed as a probe together with an SPCE that was modified with a positively-charged polymer (poly quaternized-(dimethylamino-ethyl)methacrylate, PQDMAEMA). The electrostatic attraction between the negatively-charged PNA-DNA duplex and the positively-charged modified SPCE attributes to the higher signal of PNA-DNA duplex than that of the electrostatically neutral PNA probe, resulting in a signal change. The calibration curve of this proposed method exhibited a linear range between 0.35 and 50 nM of DNA target with a limit of detection of 0.13 nM (3SD(blank)/Slope). The sub-nanomolar detection limit together with a small sample volume required (20 μL) allowed detection of <10 fmol (<1 ng) of DNA. With the high specificity of the pyrrolidinyl PNA probe used, excellent discrimination between complementary and various single-mismatched DNA targets was obtained. An application of this new platform for a sensitive and specific detection of isothermally-amplified shrimp's white spot syndrome virus (WSSV) DNA was successfully demonstrated. PMID:26695270

  5. Immobilization-free electrochemical DNA detection with anthraquinone-labeled pyrrolidinyl peptide nucleic acid probe.

    PubMed

    Kongpeth, Jutatip; Jampasa, Sakda; Chaumpluk, Piyasak; Chailapakul, Orawon; Vilaivan, Tirayut

    2016-01-01

    Electrochemical detection provides a simple, rapid, sensitive and inexpensive method for DNA detection. In traditional electrochemical DNA biosensors, the probe is immobilized onto the electrode. Hybridization with the DNA target causes a change in electrochemical signal, either from the intrinsic signal of the probe/target or through a label or a redox indicator. The major drawback of this approach is the requirement for probe immobilization in a controlled fashion. In this research, we take the advantage of different electrostatic properties between PNA and DNA to develop an immobilization-free approach for highly sequence-specific electrochemical DNA sensing on a screen-printed carbon electrode (SPCE) using a square-wave voltammetric (SWV) technique. Anthraquinone-labeled pyrrolidinyl peptide nucleic acid (AQ-PNA) was employed as a probe together with an SPCE that was modified with a positively-charged polymer (poly quaternized-(dimethylamino-ethyl)methacrylate, PQDMAEMA). The electrostatic attraction between the negatively-charged PNA-DNA duplex and the positively-charged modified SPCE attributes to the higher signal of PNA-DNA duplex than that of the electrostatically neutral PNA probe, resulting in a signal change. The calibration curve of this proposed method exhibited a linear range between 0.35 and 50 nM of DNA target with a limit of detection of 0.13 nM (3SD(blank)/Slope). The sub-nanomolar detection limit together with a small sample volume required (20 μL) allowed detection of <10 fmol (<1 ng) of DNA. With the high specificity of the pyrrolidinyl PNA probe used, excellent discrimination between complementary and various single-mismatched DNA targets was obtained. An application of this new platform for a sensitive and specific detection of isothermally-amplified shrimp's white spot syndrome virus (WSSV) DNA was successfully demonstrated.

  6. Accurate Proteome-wide Label-free Quantification by Delayed Normalization and Maximal Peptide Ratio Extraction, Termed MaxLFQ *

    PubMed Central

    Cox, Jürgen; Hein, Marco Y.; Luber, Christian A.; Paron, Igor; Nagaraj, Nagarjuna; Mann, Matthias

    2014-01-01

    Protein quantification without isotopic labels has been a long-standing interest in the proteomics field. However, accurate and robust proteome-wide quantification with label-free approaches remains a challenge. We developed a new intensity determination and normalization procedure called MaxLFQ that is fully compatible with any peptide or protein separation prior to LC-MS analysis. Protein abundance profiles are assembled using the maximum possible information from MS signals, given that the presence of quantifiable peptides varies from sample to sample. For a benchmark dataset with two proteomes mixed at known ratios, we accurately detected the mixing ratio over the entire protein expression range, with greater precision for abundant proteins. The significance of individual label-free quantifications was obtained via a t test approach. For a second benchmark dataset, we accurately quantify fold changes over several orders of magnitude, a task that is challenging with label-based methods. MaxLFQ is a generic label-free quantification technology that is readily applicable to many biological questions; it is compatible with standard statistical analysis workflows, and it has been validated in many and diverse biological projects. Our algorithms can handle very large experiments of 500+ samples in a manageable computing time. It is implemented in the freely available MaxQuant computational proteomics platform and works completely seamlessly at the click of a button. PMID:24942700

  7. PET imaging of CXCR4 using copper-64 labeled peptide antagonist.

    PubMed

    Jacobson, Orit; Weiss, Ido D; Szajek, Lawrence P; Niu, Gang; Ma, Ying; Kiesewetter, Dale O; Farber, Joshua M; Chen, Xiaoyuan

    2011-01-01

    Expression of CXCR4 in cancer has been found to correlate with poor prognosis and resistance to chemotherapy. In this study we developed a derivative of the CXCR4 peptide antagonist, T140-2D, that can be labeled easily with the PET isotope copper-64, and thereby enable in vivo visualization of CXCR4 in tumors. T140 was conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono (N-hydroxysuccinimide ester) (DOTA-NHS) to give T140-2D, which contains a DOTA molecule on each of the two lysine residues. (64)Cu-T140-2D was evaluated in vitro by migration and binding experiments, and in vivo by microPET imaging and biodistribution, in mice bearing CXCR4-positive and CXCR4-negative tumor xenografts. T140-2D was labeled with copper-64 to give (64)Cu-T140-2D in a high radiochemical yield of 86 ± 3% (not decay-corrected) and a specific activity of 0.28 - 0.30 mCi/µg (10.36 - 11.1 MBq/µg). (64)Cu-T140-2D had antagonistic and binding characteristics to CXCR4 that were similar to those of T140. In vivo, (64)Cu-T140-2D tended to bind to red blood cells and had to be used in a low specific activity form. In this new form (64)Cu-T140-2D enabled specific imaging of CXCR4-positive, but not CXCR4-negative tumors. Undesirably, however, (64)Cu-T140-2D also displayed high accumulation in the liver and kidneys. In conclusion, (64)Cu-T140-2D was easily labeled and, in its low activity form, enabled imaging of CXCR4 in tumors. It had high uptake, however, in metabolic organs. Further research with imaging tracers targeting CXCR4 is required.

  8. Label-free potentiometry for detecting DNA hybridization using peptide nucleic acid and DNA probes.

    PubMed

    Goda, Tatsuro; Singi, Ankit Balram; Maeda, Yasuhiro; Matsumoto, Akira; Torimura, Masaki; Aoki, Hiroshi; Miyahara, Yuji

    2013-02-07

    Peptide nucleic acid (PNA) has outstanding affinity over DNA for complementary nucleic acid sequences by forming a PNA-DNA heterodimer upon hybridization via Watson-Crick base-pairing. To verify whether PNA probes on an electrode surface enhance sensitivity for potentiometric DNA detection or not, we conducted a comparative study on the hybridization of PNA and DNA probes on the surface of a 10-channel gold electrodes microarray. Changes in the charge density as a result of hybridization at the solution/electrode interface on the self-assembled monolayer (SAM)-formed microelectrodes were directly transformed into potentiometric signals using a high input impedance electrometer. The charge readout allows label-free, reagent-less, and multi-parallel detection of target oligonucleotides without any optical assistance. The differences in the probe lengths between 15- to 22-mer dramatically influenced on the sensitivity of the PNA and DNA sensors. Molecular type of the capturing probe did not affect the degree of potential shift. Theoretical model for charged rod-like duplex using the Gouy-Chapman equation indicates the dominant effect of electrostatic attractive forces between anionic DNA and underlying electrode at the electrolyte/electrode interface in the potentiometry.

  9. Surface plasmon resonance for the label-free detection of Alzheimer's β-amyloid peptide aggregation.

    PubMed

    Palladino, Pasquale; Aura, Angela M; Spoto, Giuseppe

    2016-01-01

    Amyloid peptide oligomers and fibrils are studied as targets for therapy and diagnosis of Alzheimer's disease. They are usually detected by amyloid incubation, but such method is necessarily associated with Aβ1-42 depletion and dye binding or conjugation, which have a complex influence on fibril growth, provide information about fibril elongation over long time periods only, and might lead to false-positive results in amyloid inhibition assay. Surface plasmon resonance (SPR) is used to study with no labelling and in real time the aggregation of Aβ1-42 amyloid on specific antibodies. SPR data show, for the first time by using SPR, a multi-phase association behavior for Aβ1-42 oligomers accounting for a sigmoidal growth of amyloid as a function of time, with two antibody-dependent aggregation patterns. The new method represents an advantageous alternative to traditional procedures for investigating amyloid self-assembly and inhibition from early-stage oligomer association, on the time scale of seconds to minutes, to long-term polymerization, on the time scale of hours to days. PMID:26558762

  10. [The Qualitative Analysis of the Amide Derivative of HLDF-6 Peptide and Its Metabolites with the Use of Tritium- and Deuterium-Labeled Derivatives].

    PubMed

    Zolotarev, A; Dadayan, A K; Kost, N V; Voevodina, M E; Sokolov, O Y; Kozik, V S; Shram, S I; Azev, V N; Bocharov, E V; Bogachouk, A P; Lipkin, V M; Myasoedov, N F

    2015-01-01

    The goal of the study was to elaborate the pharmacokinetics methods of the amide derivative of peptide HLDF-6 (TGENHR-NH2) and its range of nootropic and neuroprotective activity is wide. The hexapeptide 41TGENHR46 is a fragment of the HDLF differentiation factor. It forms the basis for the development of preventive and therapeutic preparations for treating cerebrovascular and neurodegenerative conditions. Pharmacokinetic and molecular mechanisms of the action of the HLDF-6 peptide were studied using tritium- and deuterium-labeled derivatives of this peptide, produced with the use of the high-temperature solid-state catalytic isotope exchange reaction (HSCIE). This reaction was employed to produce the tritium-labeled peptide [3H]TGENHR-NH2 with a molar radioactivity of 230 Ci/mmol and the deuterium-labeled peptide [2H]TGENHR-NH2 with an average deuterium incorporation equal to 10.5 atoms. It was shown by the NMR spectroscopy that the isotope label distribution over the labeled peptide's molecule was uniform, which allowed qualitative analysis ofboth the peptide itself and its fragments in the organism's tissues to be conducted. The newly developed pharmacokinetics method makes it possible to avoid almost completely losses of the peptides under study due to biodegradation during the analysis of tissues. These labeled peptides were used in mice, rats and rabbits to study the pharmacokinetics of the peptide and to calculate the values of its principal pharmacokinetic parameters. Characteristics of its pharmacokinetic profile in the blood were obtained, the hypothesis of pharmacokinetics linearity tested, its metabolism analyzed and its bioavailability value, 34%, calculated. It has been shown that the studied TGENHR-NH2 peptide shows high resistance to hydrolysis in the blood plasma, with dipeptidyl aminopeptidases making the largest contribution to its hydrolysis. PMID:27125017

  11. [The Qualitative Analysis of the Amide Derivative of HLDF-6 Peptide and Its Metabolites with the Use of Tritium- and Deuterium-Labeled Derivatives].

    PubMed

    Zolotarev, A; Dadayan, A K; Kost, N V; Voevodina, M E; Sokolov, O Y; Kozik, V S; Shram, S I; Azev, V N; Bocharov, E V; Bogachouk, A P; Lipkin, V M; Myasoedov, N F

    2015-01-01

    The goal of the study was to elaborate the pharmacokinetics methods of the amide derivative of peptide HLDF-6 (TGENHR-NH2) and its range of nootropic and neuroprotective activity is wide. The hexapeptide 41TGENHR46 is a fragment of the HDLF differentiation factor. It forms the basis for the development of preventive and therapeutic preparations for treating cerebrovascular and neurodegenerative conditions. Pharmacokinetic and molecular mechanisms of the action of the HLDF-6 peptide were studied using tritium- and deuterium-labeled derivatives of this peptide, produced with the use of the high-temperature solid-state catalytic isotope exchange reaction (HSCIE). This reaction was employed to produce the tritium-labeled peptide [3H]TGENHR-NH2 with a molar radioactivity of 230 Ci/mmol and the deuterium-labeled peptide [2H]TGENHR-NH2 with an average deuterium incorporation equal to 10.5 atoms. It was shown by the NMR spectroscopy that the isotope label distribution over the labeled peptide's molecule was uniform, which allowed qualitative analysis ofboth the peptide itself and its fragments in the organism's tissues to be conducted. The newly developed pharmacokinetics method makes it possible to avoid almost completely losses of the peptides under study due to biodegradation during the analysis of tissues. These labeled peptides were used in mice, rats and rabbits to study the pharmacokinetics of the peptide and to calculate the values of its principal pharmacokinetic parameters. Characteristics of its pharmacokinetic profile in the blood were obtained, the hypothesis of pharmacokinetics linearity tested, its metabolism analyzed and its bioavailability value, 34%, calculated. It has been shown that the studied TGENHR-NH2 peptide shows high resistance to hydrolysis in the blood plasma, with dipeptidyl aminopeptidases making the largest contribution to its hydrolysis.

  12. Stable isotope labeling tandem mass spectrometry (SILT): integration with peptide identification and extension to data-dependent scans.

    PubMed

    Elbert, Donald L; Mawuenyega, Kwasi G; Scott, Evan A; Wildsmith, Kristin R; Bateman, Randall J

    2008-10-01

    Quantitation of relative or absolute amounts of proteins by mass spectrometry can be prone to large errors. The use of MS/MS ion intensities and stable isotope labeling, which we term stable isotope labeling tandem mass spectrometry (SILT), decreases the effects of contamination from unrelated compounds. We present a software package (SILTmass) that automates protein identification and quantification by the SILT method. SILTmass has the ability to analyze the kinetics of protein turnover, in addition to relative and absolute protein quantitation. Instead of extracting chromatograms to find elution peaks, SILTmass uses only scans in which a peptide is identified and that meet an ion intensity threshold. Using only scans with identified peptides, the accuracy and precision of SILT is shown to be superior to precursor ion intensities, particularly at high or low dilutions of the isotope labeled compounds or with low amounts of protein. Using example scans, we demonstrate likely reasons for the improvements in quantitation by SILT. The appropriate use of variable modifications in peptide identification is described for measurement of protein turnover kinetics. The combination of identification with SILT facilitates quantitation without peak detection and helps to ensure the appropriate use of variable modifications for kinetics experiments.

  13. Highly accurate quantification of hydroxyproline-containing peptides in blood using a protease digest of stable isotope-labeled collagen.

    PubMed

    Taga, Yuki; Kusubata, Masashi; Ogawa-Goto, Kiyoko; Hattori, Shunji

    2014-12-17

    Collagen-derived hydroxyproline (Hyp)-containing dipeptides and tripeptides, which are known to possess physiological functions, appear in blood at high concentrations after oral ingestion of gelatin hydrolysate. However, highly accurate and sensitive quantification of the Hyp-containing peptides in blood has been challenging because of the analytical interference from numerous other blood components. We recently developed a stable isotope-labeled collagen named "SI-collagen" that can be used as an internal standard in various types of collagen analyses employing liquid chromatography-mass spectrometry (LC-MS). Here we prepared stable isotope-labeled Hyp-containing peptides from SI-collagen using trypsin/chymotrypsin and plasma proteases by mimicking the protein degradation pathways in the body. With the protease digest of SI-collagen used as an internal standard mixture, we achieved highly accurate simultaneous quantification of Hyp and 13 Hyp-containing peptides in human blood by LC-MS. The area under the plasma concentration-time curve of Hyp-containing peptides ranged from 0.663 ± 0.022 nmol/mL·h for Pro-Hyp-Gly to 163 ± 1 nmol/mL·h for Pro-Hyp after oral ingestion of 25 g of fish gelatin hydrolysate, and the coefficient of variation of three separate measurements was <7% for each peptide except for Glu-Hyp-Gly, which was near the detection limit. Our method is useful for absorption/metabolism studies of the Hyp-containing peptides and development of functionally characterized gelatin hydrolysate.

  14. Method for the Determination of ¹⁵N Incorporation Percentage in Labeled Peptides and Proteins.

    PubMed

    Kilpatrick, Eric L

    2016-01-01

    Use of labeled (15)N proteins and peptides as internal standards in isotope-dilution mass spectrometry for the quantification of proteins has been increasing and is now accepted as a gold standard for this analysis. As a necessary reagent in this process, stable heavy isotope-labeled internal standards must be rigorously characterized in a number of ways including identity, concentration, purity, and structure. Additionally, the degree of the incorporation of the heavy isotope is a critical feature to consider. For proteins that are (15)N labeled, the percentage of incorporation is a valid measurement used to assess the fitness-to-purpose of the material. This measurement should be objective, repeatable, and based on empirical analysis. One means of assigning this value is to compare a mass spectrum of the isotopic profile of a peptide against a series of theoretical profiles containing different enrichment rates. This comparison can be made using the Pearson product-moment correlation coefficient (r) to find the best match between the empirical and theoretical profiles. Theoretical profiles can be generated using probability multinomial analysis but are computationally intensive and require the use of computers for practical use. The method described in this chapter describes the development and use of a computer program to calculate the percentage of (15)N enrichment of a labeled internal standard. Additionally, methods will be described for the empirical determination of an isotopic profile using a variety of mass spectrometry techniques.

  15. Evaluating Kinase ATP Uptake and Tyrosine Phosphorylation using Multiplexed Quantification of Chemically Labeled and Post-Translationally Modified Peptides

    PubMed Central

    Fang, Bin; Hoffman, Melissa A.; Mirza, Abu-Sayeef; Mishall, Katie M.; Li, Jiannong; Peterman, Scott M.; Smalley, Keiran S. M.; Shain, Kenneth H.; Weinberger, Paul M.; Wu, Jie; Rix, Uwe; Haura, Eric B.; Koomen, John M.

    2015-01-01

    Cancer biologists and other healthcare researchers face an increasing challenge in addressing the molecular complexity of disease. Biomarker measurement tools and techniques now contribute to both basic science and translational research. In particular, liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) for multiplexed measurements of protein biomarkers has emerged as a versatile tool for systems biology. Assays can be developed for specific peptides that report on protein expression, mutation, or post-translational modification; discovery proteomics data rapidly translated into multiplexed quantitative approaches. Complementary advances in affinity purification enrich classes of enzymes or peptides representing post-translationally modified or chemically labeled substrates. Here, we illustrate the process for the relative quantification of hundreds of peptides in a single LC-MRM experiment. Desthiobiotinylated peptides produced by activity-based protein profiling (ABPP) using ATP probes and tyrosine-phosphorylated peptides are used as examples. These targeted quantification panels can be applied to further understand the biology of human disease. PMID:25782629

  16. A Tc-99m labeled laminin derived peptide, Tc-99m-YIGSR for thrombus specific imaging

    SciTech Connect

    Wang, G.J.; Oster, Z.H.; Som, P.

    1994-05-01

    Laminin derived adhesive peptides were studied as potential agents for thrombus specific imaging. Using a novel peptide Tc-99m labeling method studies were performed in vitro using human whole blood clots and platelets, and in vivo scintigraphy in animals with experimental thrombi. Aliquots of 0.1 ml human blood were placed in inclined Petri dishes until clot was well formed. Clots were rinsed 3x with phosphate buffer and 10 {mu}Ci Tc-99m YIGSR II was added. After incubation at room temperature for 1 hr, clots were again washed 3x. Residual activity was measured. Platelets were harvested using routine methods and incubated with Tc-99m YIGSR II, washed and assayed. Blocking experiments using cold YIGSR II showed that the Tc-99m labeled peptide preparation YIGSR II binds specifically and selectively to clot and platelets as compared to control experiments using nonspecific human Tc-99m IgG. Tissue distribution studies showed rapid blood clearance, urinary excretion and to a lesser degree GI tract excretion. Tc-99m YIGSR II was lower in all organs except kidneys compared to Tc-99m 50 H.19, Tc-99m IgG and Tc-99m YIGSR I. Tc-99m-YIGSR II consistently visualized thrombi within 30 min p.i. In vivo scintigraphic (thrombus/contralateral side) ratio was 3:1 and ex vivo direct counting (thrombosed to nonthrombosed vessel segment) was 5.4: 1. Compared to monoclonal antibodies peptide preparations are non- or minimally immunogenic, preparation is probably less expensive and there is also less danger of viral DNA contamination. These considerations and our data indicate that the Tc-99m-YIGSR II peptide has significant potential as a thrombus imaging agent.

  17. A facile method for expression and purification of (15)N isotope-labeled human Alzheimer's β-amyloid peptides from E. coli for NMR-based structural analysis.

    PubMed

    Sharma, Sudhir C; Armand, Tara; Ball, K Aurelia; Chen, Anna; Pelton, Jeffrey G; Wemmer, David E; Head-Gordon, Teresa

    2015-12-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disease affecting millions of people worldwide. AD is characterized by the presence of extracellular plaques composed of aggregated/oligomerized β-amyloid peptides with Aβ42 peptide representing a major isoform in the senile plaques. Given the pathological significance of Aβ42 in the progression of AD, there is considerable interest in understanding the structural ensembles for soluble monomer and oligomeric forms of Aβ42. This report describes an efficient method to express and purify high quality (15)N isotope-labeled Aβ42 for structural studies by NMR. The protocol involves utilization of an auto induction system with (15)N isotope labeled medium, for high-level expression of Aβ42 as a fusion with IFABP. After the over-expression of the (15)N isotope-labeled IFABP-Aβ42 fusion protein in the inclusion bodies, pure (15)N isotope-labeled Aβ42 peptide is obtained following a purification method that is streamlined and improved from the method originally developed for the isolation of unlabeled Aβ42 peptide (Garai et al., 2009). We obtain a final yield of ∼ 6 mg/L culture for (15)N isotope-labeled Aβ42 peptide. Mass spectrometry and (1)H-(15)N HSQC spectra of monomeric Aβ42 peptide validate the uniform incorporation of the isotopic label. The method described here is equally applicable for the uniform isotope labeling with (15)N and (13)C in Aβ42 peptide as well as its other variants including any Aβ42 peptide mutants.

  18. Cysteinyl peptides of rabbit muscle pyruvate kinase labeled by the affinity label 8-((4-bromo-2,3-dioxobutyl)thio)adenosine 5 prime -triphosphate

    SciTech Connect

    Vollmer, S.H.; Colman, R.F. )

    1990-03-13

    The affinity label 8-((4-bromo-2,3-dioxobutyl)thio)adenosine 5{prime}-triphosphate (8-BDB-TA-5{prime}-TP) reacts covalently with rabbit muscle pyruvate kinase, incorporating 2 mol of reagent/mol of enzyme subunit upon complete inactivation. Protection against inactivation is provided by phosphoenolpyruvate, K{sup +}, and Mn{sup 2+} and only 1 mol of reagent/mol of subunit is incorporated. The authors have now identified the resultant modified residues. After reaction with 8-BDB-TA-5{prime}-TP at pH 7.0, modified enzyme was incubated with ({sup 3}H)NaBH{sub 4} to reduce the carbonyl groups of enzyme-bound 8-BDB-TA-5{prime}-TP and to introduce a radioactive tracer into the modified residues. Following carboxymethylation and digestion with trypsin, the radioactive peptides were separated on a phenylboronate agarose column followed by reverse-phase high-performance liquid chromatography in 0.1% trifluoroacetic acid with an acetonitrile gradient. Gas-phase sequencing gave the cysteine-modified peptides Asn{sup 162}-Ile-Cys-Lys{sup 165} and Cys{sup 151}-Asp-Glu-Asn-Ile-Leu-Trp-Leu-Asp-Tyr-Lys{sup 161}, with a smaller amount of Asn{sup 43}-Thr-Gly-Ile-Ile-Cys-Thr-Ile-Gly-Pro-Ala-Ser-Arg{sup 55}. Reaction in the presence of the protectants phosphoenolpyruvate, K{sup +}, and Mn{sup 2+} yielded Asn-Ile-Cys-Lys as the only labeled peptide, indicating that inactivation is caused by modification of Cys{sup 151} and Cys{sup 48}.

  19. [18F]azadibenzocyclooctyne ([18F]ADIBO): a biocompatible radioactive labeling synthon for peptides using catalyst free [3+2] cycloaddition.

    PubMed

    Arumugam, Selvanathan; Chin, Joshua; Schirrmacher, Ralf; Popik, Vladimir V; Kostikov, Alexey P

    2011-12-01

    N-Terminally azido-modified peptides were labeled with the novel prosthetic labeling synthon [(18)F]azadibenzocyclooctyne ([(18)F]ADIBO) using copper-free azide-alkyne [3+2]-dipolar cycloaddition in high radiochemical yields (RCYs). (18)F-Labeled [(18)F]ADIBO was prepared by nucleophilic substitution of the corresponding tosylate in 21% overall RCY (EOB) in a fully automated synthesis unit within 55 min. [(18)F]ADIBO was incubated with azide-containing peptides at room temperature in the absence of toxic metal catalysts and the formation of the triazole conjugate was confirmed. Finally, the azide-alkyne [3+2]-dipolar cycloaddition was shown to proceed with 95% radiochemical yield in ethanol within 30 min, allowing for a development of a kit-like peptide labeling approach with [(18)F]ADIBO. PMID:22024032

  20. Identification and Quantitation of Newly Synthesized Proteins in Escherichia coli by Enrichment of Azidohomoalanine-labeled Peptides with Diagonal Chromatography

    PubMed Central

    Kramer, Gertjan; Sprenger, Richard R.; Back, JaapWillem; Dekker, Henk L.; Nessen, Merel A.; van Maarseveen, Jan H.; de Koning, Leo J.; Hellingwerf, Klaas J.; de Jong, Luitzen; de Koster, Chris G.

    2009-01-01

    A method is presented to identify and quantify several hundreds of newly synthesized proteins in Escherichia coli upon pulse labeling cells with the methionine analogue azidohomoalanine (azhal). For the first 30 min after inoculation, a methionine-auxotrophic strain grows equally well on azhal as on methionine. Upon a pulse of 15 min and digestion of total protein, azhal-labeled peptides are isolated by a retention time shift between two reversed phase chromatographic runs. The retention time shift is induced by a reaction selective for the azido group in labeled peptides using tris(2-carboxyethyl)phosphine. Selectively modified peptides are identified by reversed phase liquid chromatography and on-line tandem mass spectrometry. We identified 527 proteins representative of all major Gene Ontology categories. Comparing the relative amounts of 344 proteins synthesized in 15 min upon a switch of growth temperature from 37 to 44 °C showed that nearly 20% increased or decreased more than 2-fold. Among the most up-regulated proteins many were chaperones and proteases in accordance with the cells response to unfolded proteins due to heat stress. Comparison of our data with results from previous microarray experiments revealed the importance of regulation of gene expression at the level of transcription of the most elevated proteins under heat shock conditions and enabled identification of several candidate genes whose expression may predominantly be regulated at the level of translation. This work demonstrates for the first time the use of a bioorthogonal amino acid for proteome-wide detection of changes in the amounts of proteins synthesized during a brief period upon variations in cellular growth conditions. Comparison of such data with relative mRNA levels enables assessment of the separate contributions of transcription and translation to the regulation of gene expression. PMID:19321432

  1. Identification and quantitation of newly synthesized proteins in Escherichia coli by enrichment of azidohomoalanine-labeled peptides with diagonal chromatography.

    PubMed

    Kramer, Gertjan; Sprenger, Richard R; Back, JaapWillem; Dekker, Henk L; Nessen, Merel A; van Maarseveen, Jan H; de Koning, Leo J; Hellingwerf, Klaas J; de Jong, Luitzen; de Koster, Chris G

    2009-07-01

    A method is presented to identify and quantify several hundreds of newly synthesized proteins in Escherichia coli upon pulse labeling cells with the methionine analogue azidohomoalanine (azhal). For the first 30 min after inoculation, a methionine-auxotrophic strain grows equally well on azhal as on methionine. Upon a pulse of 15 min and digestion of total protein, azhal-labeled peptides are isolated by a retention time shift between two reversed phase chromatographic runs. The retention time shift is induced by a reaction selective for the azido group in labeled peptides using tris(2-carboxyethyl)phosphine. Selectively modified peptides are identified by reversed phase liquid chromatography and on-line tandem mass spectrometry. We identified 527 proteins representative of all major Gene Ontology categories. Comparing the relative amounts of 344 proteins synthesized in 15 min upon a switch of growth temperature from 37 to 44 degrees C showed that nearly 20% increased or decreased more than 2-fold. Among the most up-regulated proteins many were chaperones and proteases in accordance with the cells response to unfolded proteins due to heat stress. Comparison of our data with results from previous microarray experiments revealed the importance of regulation of gene expression at the level of transcription of the most elevated proteins under heat shock conditions and enabled identification of several candidate genes whose expression may predominantly be regulated at the level of translation. This work demonstrates for the first time the use of a bioorthogonal amino acid for proteome-wide detection of changes in the amounts of proteins synthesized during a brief period upon variations in cellular growth conditions. Comparison of such data with relative mRNA levels enables assessment of the separate contributions of transcription and translation to the regulation of gene expression.

  2. An assessment tumor targeting ability of (177)Lu labeled cyclic CCK analogue peptide by binding with cholecystokinin receptor.

    PubMed

    Cho, Eun-Ha; Lim, Jae Cheong; Lee, So-Young; Jung, Sung-Hee

    2016-07-01

    The cholecystokinin (CCK) receptor is known as a receptor that is overexpressed in many human tumors. The present study was designed to investigate the targeting ability of cyclic CCK analogue in AR42J pancreatic cells. The CCK analogues, DOTA-K(glucose)-Gly-Trp-Nle-Asp-Phe (DOTA-glucose-CCK) and DOTA-Nle-cyclo(Glu-Trp-Nle-Asp-Phe-Lys-NH2) (DOTA-[Nle]-cCCK), were synthesized and radiolabeled with (177)Lu, and competitive binding was evaluated. The binding appearance of synthesized peptide with AR42J cells was evaluated by confocal microscopy. And bio-distribution was performed in AR42J xenografted mice. Synthesized peptides were prepared by a solid phase synthesis method, and their purity was over 98%. DOTA is the chelating agent for (177)Lu-labeling, in which the peptides were radiolabeled with (177)Lu by a high radiolabeling yield. A competitive displacement of (125)I-CCK8 on the AR42J cells revealed that the 50% inhibitory concentration value (IC50) was 12.3 nM of DOTA-glucose-CCK and 1.7 nM of DOTA-[Nle]-cCCK. Radio-labeled peptides were accumulated in AR42J tumor in vivo, and %ID/g of the tumor was 0.4 and 0.9 at 2 h p.i. It was concluded that (177)Lu-DOTA-[Nle]-cCCK has higher binding affinity than (177)Lu-DOTA-glucose-CCK and can be a potential candidate as a targeting modality for a CCK receptor over-expressing tumors. PMID:27430985

  3. Site-specific Orientation of an α-helical Peptide Ovispirin-1 from Isotope Labeled SFG Spectroscopy

    PubMed Central

    Ding, Bei; Laaser, Jennifer E.; Liu, Yuwei; Wang, Pengrui; Zanni, Martin T.; Chen, Zhan

    2013-01-01

    Sum-frequency generation (SFG) vibrational spectroscopy is often used to probe the backbone structures and orientations of polypeptides at surfaces. Using the ovispirin-1 polypeptide at the solid/liquid interface of polystyrene, we demonstrate for the first time that SFG can probe the polarization response of a single isotope labeled residue. To interpret the spectral intensities, we simulated the spectra using an excitonic Hamiltonian approach. We show that the polarization dependence of either the label or the unlabeled amide I band alone does not provide sufficient structural constraints to obtain both the tilt and the twist of the ovispirin helix at a solid/liquid interface, but that both can be determined from the polarization dependence of the complete spectrum. For ovispirin, the detailed analysis of the polarized SFG experimental data shows that the helix axis is tilted at roughly 138 degrees from the surface normal, and the transition dipole of the isotope labeled C=O group is tilted at 23 degrees from the surface normal, with the hydrophobic region facing the polystyrene surface. We further demonstrated that the Hamiltonian approach is able to address the coupling effect and the structural disorder. For comparison, we also collected the FTIR spectrum of ovispirin under similar conditions, which reveals the enhanced sensitivity of SFG for structural studies of single monolayer peptide surfaces. Our study provides insight into how structural and environmental effects appear in SFG spectra of the amide I band and establishes that SFG of isotope labeled peptides will be a powerful technique for elucidating secondary structures with residue-by-residue resolution. PMID:24228619

  4. Recombinant production, isotope labeling and purification of ENOD40B: a plant peptide hormone.

    PubMed

    Chae, Young Kee; Tonneli, Marco; Markley, John L

    2012-08-01

    The plant peptide hormone ENOD40B was produced in a protein production strain of Escherichia coli harboring an induction controller plasmid (Rosetta(DE3)pLysS) as a His6-tagged ubiquitin fusion protein. The fusion protein product was denatured and refolded as part of the isolation procedure and purified by immobilized metal ion chromatography. The peptide hormone was released from its fusion partner by adding yeast ubiquitin hydrolase (YUH) and subsequently purified by reversed phase chromatography. The purity of the resulting peptide fragment was assayed by MALDITOF mass spectrometry and NMR spectroscopy. The final yields of the target peptide were 7.0 mg per liter of LB medium and 3.4 mg per liter of minimal medium.

  5. Theranostic Radiopharmaceuticals Based on Gold Nanoparticles Labeled with (177)Lu and Conjugated to Peptides.

    PubMed

    Ferro-Flores, Guillermina; Ocampo-García, Blanca E; Santos-Cuevas, Clara L; de María Ramírez, Flor; Azorín-Vega, Erika P; Meléndez-Alafort, Laura

    2015-01-01

    Gold nanoparticles (AuNPs) have been proposed for a variety of medical applications such as localized heat sources for cancer treatment and drug delivery systems. The conjugation of peptides to AuNPs produces stable multimeric systems with target-specific molecular recognition. Lutetium- 177 ((177)Lu) has been successfully used in peptide radionuclide therapy. Recently, (177)Lu-AuNPs conjugated to different peptides have been proposed as a new class of theranostic radiopharmaceuticals. These radioconjugates may function simultaneously as molecular imaging agents, radiotherapy systems and thermal-ablation systems. This article covers advancements in the design, synthesis, physicochemical characterization, molecular recognition assessment and preclinical therapeutic efficacy of gold nanoparticles radiolabeled with (177)Lu and conjugated to RGD (-Arg-Gly-Asp-), Lys(3)-Bombesin and Tat(49-57) peptides.

  6. Theranostic Radiopharmaceuticals Based on Gold Nanoparticles Labeled with (177)Lu and Conjugated to Peptides.

    PubMed

    Ferro-Flores, Guillermina; Ocampo-García, Blanca E; Santos-Cuevas, Clara L; de María Ramírez, Flor; Azorín-Vega, Erika P; Meléndez-Alafort, Laura

    2015-01-01

    Gold nanoparticles (AuNPs) have been proposed for a variety of medical applications such as localized heat sources for cancer treatment and drug delivery systems. The conjugation of peptides to AuNPs produces stable multimeric systems with target-specific molecular recognition. Lutetium- 177 ((177)Lu) has been successfully used in peptide radionuclide therapy. Recently, (177)Lu-AuNPs conjugated to different peptides have been proposed as a new class of theranostic radiopharmaceuticals. These radioconjugates may function simultaneously as molecular imaging agents, radiotherapy systems and thermal-ablation systems. This article covers advancements in the design, synthesis, physicochemical characterization, molecular recognition assessment and preclinical therapeutic efficacy of gold nanoparticles radiolabeled with (177)Lu and conjugated to RGD (-Arg-Gly-Asp-), Lys(3)-Bombesin and Tat(49-57) peptides. PMID:25771363

  7. Sub-cellular localisation of a 15N-labelled peptide vector using NanoSIMS imaging

    NASA Astrophysics Data System (ADS)

    Römer, Winfried; Wu, Ting-Di; Duchambon, Patricia; Amessou, Mohamed; Carrez, Danièle; Johannes, Ludger; Guerquin-Kern, Jean-Luc

    2006-07-01

    Dynamic SIMS imaging is proposed to map sub-cellular distributions of isotopically labelled, exogenous compounds. NanoSIMS imaging allows the characterisation of the intracellular transport pathways of exogenous molecules, including peptide vectors employed in innovative therapies, using stable isotopes as molecular markers to detect the compound of interest. Shiga toxin B-subunit (STxB) was chosen as a representative peptide vector. The recombinant protein ( 15N-STxB) was synthesised in Escherichia coli using 15NH 4Cl as sole nitrogen source resulting in 15N enrichment in the molecule. Using the NanoSIMS 50 ion microprobe (Cameca), different ion species ( 12C 14N -, 12C 15N -, 31P -) originating from the same sputtered micro volume were simultaneously detected. High mass resolving power enabled the discrimination of 12C 15N - from its polyatomic isobars of mass 27. We imaged the membrane binding and internalisation of 15N-STxB in HeLa cells at spatial resolutions of less than 100 nm. Thus, the use of rare stable isotopes like 15N with dynamic SIMS imaging permits sub-cellular detection of isotopically labelled, exogenous molecules and imaging of their transport pathways at high mass and spatial resolution. Application of stable isotopes as markers can replace the large and chemically complex tags used for fluorescence microscopy, without altering the chemical and physical properties of the molecule.

  8. Peptide-templated gold nanocluster beacon as a sensitive, label-free sensor for protein post-translational modification enzymes.

    PubMed

    Wen, Qian; Gu, Yi; Tang, Li-Juan; Yu, Ru-Qin; Jiang, Jian-Hui

    2013-12-17

    Protein post-translational modifications (PTMs), which are chemical modifications and most often regulated by enzymes, play key roles in functional proteomics. Detection of PTM enzymes, thus, is critical in the study of cell functioning and development of diagnostic and therapeutic tools. Herein, we develop a simple peptide-templated method to direct rapid synthesis of highly fluorescent gold nanoclusters (AuNCs) and interrogate the effect of enzymatic modifications on their luminescence. A new finding is that enzymes are able to exert chemical modifications on the peptide-templated AuNCs and quench their fluorescence, which furnishes the development of a real-time and label-free sensing strategy for PTM enzymes. Two PTM enzymes, histone deacetylase 1 and protein kinase A, have been employed to demonstrate the feasibility of this enzyme-responsive fluorescent nanocluster beacon. The results reveal that the AuNCs' fluorescence can be dynamically decreased with increasing concentration of the enzymes, and subpicomolar detection limits are readily achieved for both enzymes. The developed strategy can thus offer a useful, label-free biosensor platform for the detection of protein-modifying enzymes and their inhibitors in biomedical applications.

  9. Site-specific labeling of proteins and peptides with trans-cyclooctene containing handles capable of tetrazine ligation.

    PubMed

    Wollack, James W; Monson, Benjamin J; Dozier, Jonathan K; Dalluge, Joseph J; Poss, Kristina; Hilderbrand, Scott A; Distefano, Mark D

    2014-08-01

    There is a growing library of functionalized non-natural substrates for the enzyme protein farnesyltransferase (PFTase). PFTase covalently attaches these functionalized non-natural substrates to proteins ending in the sequence CAAX, where C is a cysteine that becomes alkylated, A represents an aliphatic amino acid, and X is Ser, Met, Ala, or Gln. Reported substrates include a variety of functionalities that allow modified proteins to undergo subsequent bioconjugation reactions. To date the most common strategy used in this approach has been copper catalyzed azide-alkyne cycloaddition (CuAAC). While being fast and bioorthogonal CuAAC has limited use in live cell experiments due to copper's toxicity.(1) Here, we report the synthesis of trans-cyclooctene geranyl diphosphate. This substrate can be synthesized from geraniol in six steps and be enzymatically transferred to peptides and proteins that end in a CAAX sequence. Proteins and peptides site-specially modified with trans-cyclooctene geranyl diphosphate were subsequently targeted for further modification via tetrazine ligation. As tetrazine ligation is bioorthogonal, fast, and is contingent on ring strain rather than the addition of a copper catalyst, this labeling strategy should prove useful for labeling proteins where the presence of copper may hinder solubility or biological reactivity.

  10. Label-free SPR detection of gluten peptides in urine for non-invasive celiac disease follow-up.

    PubMed

    Soler, Maria; Estevez, M-Carmen; Moreno, Maria de Lourdes; Cebolla, Angel; Lechuga, Laura M

    2016-05-15

    Motivated by the necessity of new and efficient methods for dietary gluten control of celiac patients, we have developed a simple and highly sensitive SPR biosensor for the detection of gluten peptides in urine. The sensing methodology enables rapid and label-free quantification of the gluten immunogenic peptides (GIP) by using G12 mAb. The overall performance of the biosensor has been in-depth optimized and evaluated in terms of sensitivity, selectivity and reproducibility, reaching a limit of detection of 0.33 ng mL(-1). Besides, the robustness and stability of the methodology permit the continuous use of the biosensor for more than 100 cycles with excellent repeatability. Special efforts have been focused on preventing and minimizing possible interferences coming from urine matrix enabling a direct analysis in this fluid without requiring extraction or purification procedures. Our SPR biosensor has proven to detect and identify gluten consumption by evaluating urine samples from healthy and celiac individuals with different dietary gluten conditions. This novel biosensor methodology represents a novel approach to quantify the digested gluten peptides in human urine with outstanding sensitivity in a rapid and non-invasive manner. Our technique should be considered as a promising opportunity to develop Point-of-Care (POC) devices for an efficient, simple and accurate gluten free diet (GFD) monitoring as well as therapy follow-up of celiac disease patients.

  11. Absolute quantification of protein and post-translational modification abundance with stable isotope–labeled synthetic peptides

    PubMed Central

    Kettenbach, Arminja N; Rush, John; Gerber, Scott A

    2013-01-01

    In the analysis of biological systems, it is of interest to identify the components of the system and to monitor their changes in abundance under different conditions. The AQUA (for ‘absolute quantification’) method allows sensitive and specific targeted quantification of protein and post-translational modifications in complex protein mixtures using stable isotope–labeled peptides as internal standards. Each AQUA experiment is composed of two stages: method development and application to a biological scenario. In the method development stage, peptides from the protein of interest are chosen and then synthesized with stable isotopes such as 13C, 2H or 15N. The abundance of these internal standards and their endogenous counterparts can be measured by mass spectrometry with selected reaction monitoring or selected ion monitoring methods. Once an AQUA method is established, it can be rapidly applied to a wide range of biological samples, from tissue culture cells to human plasma and tissue. After AQUA peptide synthesis, the development, optimization and application of AQUA analyses to a specific biological problem can be achieved in ~1 week. Here we demonstrate the usefulness of this method by monitoring both Polo-like kinase 1 (Plk1) protein abundance in multiple lung cancer cell lines and the extent of Plk1 activation loop phosphorylation (pThr-210) during release from S phase. PMID:21293459

  12. Summarization vs Peptide-Based Models in Label-Free Quantitative Proteomics: Performance, Pitfalls, and Data Analysis Guidelines.

    PubMed

    Goeminne, Ludger J E; Argentini, Andrea; Martens, Lennart; Clement, Lieven

    2015-06-01

    Quantitative label-free mass spectrometry is increasingly used to analyze the proteomes of complex biological samples. However, the choice of appropriate data analysis methods remains a major challenge. We therefore provide a rigorous comparison between peptide-based models and peptide-summarization-based pipelines. We show that peptide-based models outperform summarization-based pipelines in terms of sensitivity, specificity, accuracy, and precision. We also demonstrate that the predefined FDR cutoffs for the detection of differentially regulated proteins can become problematic when differentially expressed (DE) proteins are highly abundant in one or more samples. Care should therefore be taken when data are interpreted from samples with spiked-in internal controls and from samples that contain a few very highly abundant proteins. We do, however, show that specific diagnostic plots can be used for assessing differentially expressed proteins and the overall quality of the obtained fold change estimates. Finally, our study also illustrates that imputation under the "missing by low abundance" assumption is beneficial for the detection of differential expression in proteins with low abundance, but it negatively affects moderately to highly abundant proteins. Hence, imputation strategies that are commonly implemented in standard proteomics software should be used with care. PMID:25827922

  13. Technetium 99m-labeled VQ peptide: a new imaging agent for the early detection of tumors or premalignancies.

    PubMed

    Shi, Jiyun; Cui, Liyang; Jia, Bing; Liu, Zhaofei; He, Peng; Dong, Chengyan; Jin, Xiaona; Zhao, Huiyun; Li, Fang; Wang, Fan

    2013-01-01

    There is a critical need to develop diagnostic procedures enabling early detection of tumors while at a curable stage. Technetium 99m (99mTc)-labeled VQ peptide (99mTc-HYNIC-VQ) identified through screening phage display peptide libraries against fresh human colonic adenomas was prepared and evaluated for tumor detection. 99mTc-HYNIC-VQ was prepared by a non-SnCl2 method with more than 99% radiochemical purity. The biodistribution in the HT-29 tumor model showed that although the absolute tumor uptake values were relatively low (0.60 ± 0.09, 0.41 ± 0.09, 0.36 ± 0.18, and 0.19 ± 0.08 %ID/g at 0.5, 1, 2, and 4 hours postinjection, respectively), the tumor uptake was higher than that of any of the other organs except for the kidneys at any time point examined, which led to the high tumor to nontarget ratios. The tumors and inflammation were clearly visualized with high contrast. Although the mechanism of accumulation of radiolabeled VQ peptide in tumors and inflammation needs to be further investigated, 99mTc-HYNIC-VQ is a promising imaging agent for the early detection of tumors or premalignancies, at least for screening patients with a high risk of developing cancers.

  14. Protective spin-labeled fluorenes maintain amyloid beta peptide in small oligomers and limit transitions in secondary structure

    SciTech Connect

    Altman, Robin; Ly, Sonny; Hilt, Silvia; Petrlova, Jitka; Maezawa, Izumi; Kálai, Tamás; Hideg, Kálmán; Jin, Lee-Way; Laurence, Ted A.; Voss, John C.

    2015-12-01

    Alzheimer’s disease is characterized by the presence of extracellular plaques comprised of amyloid beta (Aβ) peptides. Soluble oligomers of the Aβ peptide underlie a cascade of neuronal loss and dysfunction associated with Alzheimer's disease. Single particle analyses of Aβ oligomers in solution by fluorescence correlation spectroscopy (FCS) were used to provide real-time descriptions of how spin-labeled fluorenes (SLFs; bi-functional small molecules that block the toxicity of Aβ) prevent and disrupt oligomeric assemblies of Aβ in solution. The FCS results, combined with electron paramagnetic resonance spectroscopy and circular dichroism spectroscopy, demonstrate SLFs can inhibit the growth of Aβ oligomers and disrupt existing oligomers while retaining Aβ in a largely disordered state. Furthermore, while the ability of SLF to block Aβ toxicity correlates with a reduction in oligomer size, our results suggest the conformation of Aβ within the oligomer determines the toxicity of the species. Attenuation of Aβ toxicity, which has been associated primarily with the soluble oligomeric form, can be achieved through redistribution of the peptides into smaller oligomers and arrest of the fractional increase in beta secondary structure.

  15. Measurement of the isotope enrichment of stable isotope-labeled proteins using high-resolution mass spectra of peptides.

    PubMed

    MacCoss, Michael J; Wu, Christine C; Matthews, Dwight E; Yates, John R

    2005-12-01

    Stable isotope-enriched molecules are used as internal standards and as tracers of in vivo substrate metabolism. The accurate conversion of measured ratios in the mass spectrometer to mole ratios is complicated because a polyatomic molecule containing enriched atoms will result in a combinatorial distribution of isotopomers depending on the enrichment and number of "labeled" atoms. This effect could potentially cause a large error in the mole ratio measurement depending on which isotope peak or peaks were used to determine the ratio. We report a computational method that predicts isotope distributions over a range of enrichments and compares the predicted distributions to experimental peptide isotope distributions obtained by Fourier transform ion cyclotron resonance mass spectrometry. Our approach is accurate with measured enrichments within 1.5% of expected isotope distributions. The method is also precise with 4.9, 2.0, and 0.8% relative standard deviations for peptides containing 59, 79, and 99 atom % excess (15)N, respectively. The approach is automated making isotope enrichment calculations possible for thousands of peptides in a single muLC-FTICR-MS experiment.

  16. An (125)I-labeled octavalent peptide fluorescent nanoprobe for tumor-homing imaging in vivo.

    PubMed

    Luo, Haiming; Shi, Jiyun; Jin, Honglin; Fan, Di; Lu, Lisen; Wang, Fan; Zhang, Zhihong

    2012-06-01

    Targeting radiopeptides are promising agents for radio-theranostics. However, in vivo evaluation of their targeting specificity is often obscured by their short biologic half-lives and low binding affinities. Here, we report an approach to efficiently examine targeting radiopeptides with a new class of octavalent peptide fluorescent nanoprobe (Octa-FNP) platform, which is composed of candidate targeting peptides and a tetrameric far-red fluorescent protein (tfRFP) scaffold. To shed light on this process, (125)I-Octa-FNP, (125)I-tfRFP and (125)I-peptide were synthesized, and their targeting functionalities were compared. Both fluorescence imaging and radioactive quantification results confirmed that (125)I-Octa-FNP had a significantly higher cellular binding capability than (125)I-tfRFP. In vivo biodistribution studies show that at 6 h post-injection, (125)I-Octa-FNP had 2-fold and 30-fold higher tumor uptake than that of (125)I-tfRFP and (125)I-peptide, respectively. Moreover, γ-imaging at 24 h post-injection revealed a remarkable accumulation of (125)I-Octa-FNP in the tumor while maintaining an extremely low background contrast, which was further confirmed by immunofluorescence analysis. These data suggested that, as an engineered and multivalent platform, Octa-FNP could enhance the tumor targeting of a designed peptide and provide excellent contrast radioimaging, making it a valuable tool for the evaluation of the targeting ability of specifically designed radiopeptides for cancer theranostics.

  17. Heavy-Atom Labeled Transmembrane β-Peptides: Synthesis, CD-Spectroscopy, and X-ray Diffraction Studies in Model Lipid Multilayer.

    PubMed

    Rost, Ulrike; Xu, Yihui; Salditt, Tim; Diederichsen, Ulf

    2016-08-18

    Transmembrane β-peptides are promising candidates for the design of well-controlled membrane anchors in lipid membranes. Here, we present the synthesis of transmembrane β-peptides with and without tryptophan anchors, as well as a novel iodine-labeled d-β(3) -amino acid. By using one or more of the heavy-atom labeled amino acids as markers, the orientation of the helical peptide was inferred based on the electron-density profile determined by X-ray reflectivity. The β-peptides were synthesized through manual Fmoc-based solid-phase peptide synthesis (SPPS) and reconstituted in unilamellar vesicles forming a right-handed 314 -helix secondary structure, as shown by circular dichroism spectroscopy. We then integrated the β-peptide into solid-supported membrane stacks and carried out X-ray reflectivity and grazing incidence small-angle X-ray scattering to determine the β-peptide orientation and its effect on the membrane bilayers. These β-peptides adopt a well-ordered transmembrane motif in the solid-supported model membrane, maintaining the basic structure of the original bilayer with some distinct alterations. Notably, the helical tilt angle, which accommodates the positive hydrophobic mismatch, induces a tilt of the acyl chains. The tilted chains, in turn, lead to a membrane thinning effect.

  18. Preparation of fluorescently-labeled amyloid-beta peptide assemblies: the effect of fluorophore conjugation on structure and function

    PubMed Central

    Jungbauer, L. M.; Yu, C; Laxton, K. J.; LaDu, M. J.

    2009-01-01

    Recent research has focused on soluble oligomeric assemblies of the 42 amino acid isoform of the amyloid-beta peptide (Aβ42) as the proximal cause of neuronal injury, synaptic loss, and the eventual dementia associated with Alzheimer’s disease (AD). While neurotoxicity, neuroinflammation, and deficits in behavior and memory have all been attributed to oligomeric Aβ42, the specific roles for this assembly in the cellular neuropathology of AD remain poorly understood. In particular, lack of reliable and well-characterized forms of easily detectable Aβ42 oligomers has hindered study of the cellular trafficking of exogenous Aβ42 by neurons in vitro and in vivo. Therefore, the objective of this study is to fluorescently label soluble oligomeric Aβ42 without altering the structure or function of this assembly. Previous studies have demonstrated the advantages of using tapping mode atomic force microscopy (AFM) to characterize the structural assemblies formed by synthetic Aβ42 under specific solution conditions (e.g., oligomers, protofibrils, and fibrils). Here, we extend these methods to establish a strategy for fluorescent labeling of oligomeric Aβ42 assemblies that are structurally comparable to unlabeled oligomeric Aβ42. To compare function, we demon-strate that the uptake of labeled and unlabeled oligomeric Aβ42 by neurons in vitro is similar. AFM-characterized fluorophore-Aβ42 oligomers are an exciting new reagent for use in a variety of studies designed to elucidate critical cellular and molecular mechanisms underlying the functions of this Aβ42 assembly form in AD. PMID:19343729

  19. DIVERSE System: De Novo Creation of Peptide Tags for Non-enzymatic Covalent Labeling by In Vitro Evolution for Protein Imaging Inside Living Cells.

    PubMed

    Kawakami, Takashi; Ogawa, Koji; Goshima, Naoki; Natsume, Tohru

    2015-12-17

    Polypeptide-tag/small-molecule pairs for specific cellular protein labeling are useful for visualizing cellular proteins and controlling their activity. Here, we report the development of an in vitro evolution-based (poly)peptide tag identification system named the DIVERSE (Directed In Vitro Evolution of Reactive peptide tags via Sequential Enrichment) system. In this system, an extremely diverse (10(14)) library of peptide tags, displayed by covalent attachment to their encoding cDNAs, is continuously prepared from the DNA library in a one-pot approach. Using this system, we demonstrated de novo creation of non-enzymatically covalent-labeling peptide tags for a synthetic small-molecule target from a random peptide library. Protein labeling with these tags was applicable to N- and C-terminal fusions, multiple different proteins and fluorophores, and intracellular labeling. The DIVERSE system can be used not only for the de novo creation of polypeptide tags but also sequence optimization of existing polypeptide tags from extremely diverse libraries.

  20. EPR Studies of Functionally Active, Nitroxide Spin-Labeled Peptide Analogs of the C-terminus of a G-Protein Alpha Subunit

    PubMed Central

    Van Eps, Ned; Anderson, Lori L.; Kisselev, Oleg G.; Baranski, Thomas J.; Hubbell, Wayne L.; Marshall, Garland R.

    2010-01-01

    The C-terminal tail of the transducin alpha subunit, Gtα(340–350), is known to bind and stabilize the active conformation of rhodopsin upon photoactivation (R*). Five spin-labeled analogs of Gtα(340–350) demonstrated native-like activity in their ability to bind and stabilize R*. The spin label 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) was employed at interior sites within the peptide, whereas a Proxyl (3-carboxyl-2,2,5,5-tetramethyl-pyrrolidinyloxy) spin label was employed at the amino terminus of the peptide. Upon binding to R*, the electron paramagnetic resonance spectrum of TOAC343-Gtα(340–350) revealed greater immobilization of the nitroxide when compared to that of the N-terminal modified Proxyl-Gtα(340–350) analog. A double-labeled Proxyl/TOAC348-Gtα(340–350) was examined by DEER spectroscopy to determine the distribution of distances between the two nitroxides in the peptides when in solution and when bound to R*. TOAC and Proxyl spin labels in this GPCR-G-protein α-peptide system provide unique biophysical probes that can be used to explore the structure and conformational changes at the rhodopsin-G-protein interface. PMID:20695526

  1. Magneto-immunocapture with on-bead fluorescent labeling of amyloid-β peptides: towards a microfluidized-bed-based operation.

    PubMed

    Mai, Thanh Duc; Pereiro, Iago; Hiraoui, Mohamed; Viovy, Jean-Louis; Descroix, Stéphanie; Taverna, Myriam; Smadja, Claire

    2015-09-01

    A new sample treatment approach for sensitive determination of three amyloid-β peptides (Aβ 1-42, Aβ 1-40 and Aβ 1-38) with capillary electrophoresis coupled with laser induced fluorescent detection is reported herein. These Aβ peptides are considered an important family of biomarkers in the cerebrospinal fluid (CSF) for early diagnosis of Alzheimer's disease (AD). Due to their extremely low abundance in CSF (down to sub nM ranges), batch-wise preconcentration via magneto-immunocapture with enrichment factors up to 100 was implemented. The Aβ peptides were first captured onto magnetic micro-beads. Then, on-beads fluorescent labeling of the captured Aβ peptides were carried out to avoid the unwanted presence of extra fluorescent dye in the eluent as in the case of in-solution labeling. Finally thermal elution was performed and eluted labeled peptides were analyzed off line with CE-LIF. The Aβ-capturing efficiencies of different commercially available antibodies grafted onto magnetic beads were tested. Aβ peptides in CSF samples collected from AD's patients and healthy persons (used as controls) were measured and evaluated. As a proof of concept, the developed strategy was adapted into a miniaturized fluidized bed configuration that has the potential for coupling with a microchip separation system. PMID:26206107

  2. A new (68)Ga-labeled BBN peptide with a hydrophilic linker for GRPR-targeted tumor imaging.

    PubMed

    Pan, Donghui; Xu, Yu Ping; Yang, Rong Hua; Wang, Lizhen; Chen, Fei; Luo, Shineng; Yang, Min; Yan, Yongjun

    2014-06-01

    Bombesin (BBN) is a peptide exhibiting high affinity for the gastrin-releasing peptide receptor (GRPR), which is overexpressed on several types of cancers. Various GRPR antagonists and agonists have been labeled with radiometals for positron emission tomography (PET) imaging of GRPR-positive tumors. However, unfavorable hepatobiliary excretion such as high intestinal activity may prohibit their clinical utility for imaging abdominal cancer. In this study, the modified BBN peptide with a new hydrophilic linker was labeled with (68)Ga for PET imaging of GRPR-expressing PC-3 prostate cancer xenograft model. GRPR antagonists, MATBBN (Gly-Gly-Gly-Arg-Asp-Asn-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHCH2CH3) and ATBBN (D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHCH2CH3), were conjugated with 1,4,7-triazacyclononanetriacetic acid (NOTA) and labeled with (68)Ga. Partition coefficient and in vitro stability were also determined. GRPR binding affinity of both tracers was investigated by competitive radioligand binding assay. The in vivo receptor targeting potential and pharmacokinetic of (68)Ga-NOTA-MATBBN were also evaluated in PC-3 prostate tumor model and compared with those of (68)Ga-NOTA-ATBBN. NOTA-conjugated BBN analogs were labeled with (68)Ga within 20 min with a decay-corrected yield ranging from 90 to 95 % and a radiochemical purity of more than 98 %. The specific activity of (68)Ga-NOTA-MATBBN and (68)Ga-NOTA-ATBBN was at least 16.5 and 11.9 GBq/μmol, respectively. The radiotracers were stable in phosphate-buffered saline and human serum. (68)Ga-NOTA-MATBBN was more hydrophilic than (68)Ga-NOTA-ATBBN, as indicated by their log P values (-2.73 ± 0.02 vs. -1.20 ± 0.03). The IC50 values of NOTA-ATBBN and NOTA-MATBBN were similar (102.7 ± 1.18 and 124.6 ± 1.21 nM). The accumulation of (68)Ga-labeled GRPR antagonists in the subcutaneous PC-3 tumors could be visualized via small animal PET. The tumors were clearly visible, and the tumor uptakes of (68)Ga-NOTA-MATBBN and (68)Ga

  3. Radioiodine and 211At-labeled guanidinomethyl halobenzoyl octreotate conjugates: potential peptide radiotherapeutics for somatostatin receptor-positive cancers.

    PubMed

    Vaidyanathan, Ganesan; Boskovitz, Abraham; Shankar, Sriram; Zalutsky, Michael R

    2004-12-01

    Derivatives of the somatostatin analogues octreotide and octreotate labeled with radioiosotopes are used in the diagnosis and therapy of somatostatin receptor (SSTR)-positive tumors. A method has been devised to synthesize {N-(4-guanidinomethyl-3-iodobenzoyl)-Phe1-octreotate (GMIBO). Receptor binding assay and scatchard analysis yielded a Kd of 4.83 +/- 0.19 nM for this peptide. Derivatives of this peptide labeled with radioiodine ([*I]GMIBO) and the alpha-particle-emitting radiohalogen 211At N-(3-[211At]astato-4-guanidinomethylbenzoyl)-Phe1-octreotate; [211At]AGMBO} were prepared in a single step from a tin precursor in radiochemical yields of 30-35% and 15-20%, respectively. Paired-label internalization assays performed with the SSTR-positive D341 Med human medulloblastoma cell line demonstrated that [125I]GMIBO and [211At]AGMBO were specifically internalized 20-40% more than Nalpha-(1-deoxy-D-fructosyl)-[131I]I-Tyr3-octreotate ([131I]I-Glu-TOCA), the radioiodinated octreotide derivative previously shown to exhibit maximum internalization in this cell line. Uptake of [131I]GMIBO in D341 Med subcutaneous xenografts in a murine model (8.34 +/- 1.82 versus 8.10 +/- 2.23% ID/g at 1h) and SSTR-expressing normal tissues was comparable to that of [125I]I-Glu-TOCA and was shown to be specific. However, the uptake of [131I]GMIBO also was substantially higher in liver (16.9 +/- 3.15 versus 1.39 +/- 0.45% ID/g at 1 h) and in kidneys (44.33 +/- 6.47 versus 3.44 +/- 0.68% ID/g at 1h) compared to that of [125I]I-Glu-TOCA. These data suggest that these novel peptide conjugates retain their specificity for SSTR both in vitro and in vivo; however, because of their higher accumulation in normal tissues they would be best applied in settings amenable to loco-regional administration such as medulloblastoma neoplastic meningitis. PMID:15572196

  4. Elucidation of the Molecular Basis of Cholecystokinin Peptide Docking to Its Receptor Using Site-Specific Intrinsic Photoaffinity Labeling and Molecular Modeling†

    PubMed Central

    Dong, Maoqing; Lam, Polo C.-H.; Pino, Delia I.; Abagyan, Ruben; Miller, Laurence J.

    2012-01-01

    G protein-coupled receptors represent the largest family of receptors and the major target of current drug development efforts. Understanding of the mechanisms of ligand binding and activation of these receptors remains limited, despite recent advances in structural determination of family members. This work focuses on the use of photoaffinity labeling and molecular modeling to elucidate the structural basis of binding a natural peptide ligand to a family A G protein-coupled receptor, the type 1 cholecystokinin receptor. Two photolabile cholecystokinin analogues were developed and characterized as representing high-affinity, fully biologically active probes with sites of covalent attachment at positions 28 and 31. The sites of receptor labeling were identified by purification, proteolytic peptide mapping, and radiochemical sequencing of labeled wild-type and mutant cholecystokinin receptors. The position 28 probe labeled second extracellular loop residue Leu199, while the position 31 probe labeled first extracellular loop residue Phe107. Along with five additional spatial approximation constraints coming from previous photoaffinity labeling studies and 12 distance restraints from fluorescence resonance energy transfer studies, these were built into two homology models of the cholecystokinin receptor, based on the recent crystal structures of the β2-adrenergic receptor and A2a-adenosine receptor. The resultant agonist ligand-occupied receptor models fully accommodate all existing experimental data and represent the best refined models of a peptide hormone receptor in this important family. PMID:19441839

  5. (18)F, (64)Cu, and (68)Ga labeled RGD-bombesin heterodimeric peptides for PET imaging of breast cancer.

    PubMed

    Liu, Zhaofei; Yan, Yongjun; Liu, Shuanglong; Wang, Fan; Chen, Xiaoyuan

    2009-05-20

    Radiolabeled RGD (Arg-Gly-Asp) and bombesin (BBN) radiotracers that specifically target integrin alpha(v)beta(3) and gastrin releasing peptide receptor (GRPR) are both promising radiopharmaceuticals for tumor imaging. We recently designed and synthesized a RGD-BBN heterodimeric peptide with both RGD and BBN motifs in one single molecule. The (18)F-labeled RGD-BBN heterodimer exhibited dual integrin alpha(v)beta(3) and GRPR targeting in a PC-3 prostate cancer model. In this study we investigated whether radiolabeled RGD-BBN tracers can be used to detect breast cancer by using microPET. Cell binding assay demonstrated that the high GRPR expressing breast cancer cells typically express low to moderate level of integrin alpha(v)beta(3), while high integrin alpha(v)beta(3) expressing breast cancer cells have negligible level of GRPR. We labeled RGD-BBN heterodimer with three positron emitting radionuclides (18)F, (64)Cu, and (68)Ga and investigated the corresponding PET radiotracers in both orthotopic T47D (GRPR(+)/low integrin alpha(v)beta(3)) and MDA-MB-435 (GRPR(-)/integrin alpha(v)beta(3)(+)) breast cancer models. The three radiotracers all possessed in vitro dual integrin alpha(v)beta(3) and GRPR binding affinity. The advantages of the RGD-BBN radiotracers over the corresponding BBN analogues are obvious for imaging MDA-MB-435 (GRPR(-)/integrin alpha(v)beta(3)(+)) tumor. (18)F-FB-PEG(3)-RGD-BBN showed lower tumor uptake than (64)Cu-NOTA-RGD-BBN and (68)Ga-NOTA-RGD-BBN but was able to visualize breast cancer tumors with high contrast. Synthesis of (64)Cu-NOTA-RGD-BBN and (68)Ga-NOTA-RGD-BBN is much faster and easier than (18)F-FB-PEG(3)-RGD-BBN. (64)Cu-NOTA-RGD-BBN showed prolonged tumor uptake but also higher liver retention and kidney uptake than (68)Ga-NOTA-RGD-BBN and (18)F-FB-PEG(3)-RGD-BBN. (68)Ga-NOTA-RGD-BBN possessed high tumor signals but also relatively high background uptake compared with the other two radiotracers. In summary, the prosthetic labeling

  6. Examining ubiquitinated peptide enrichment efficiency through an epitope labeled protein.

    PubMed

    Parker, J; Oh, Y; Moazami, Y; Pierce, J G; Loziuk, P L; Dean, R A; Muddiman, D C

    2016-11-01

    Ubiquitination is a dynamic process that is responsible for regulation of cellular responses to stimuli in a number of biological systems. Previous efforts to study this post-translational modification have focused on protein enrichment; however, recent research utilizes the presence of the di-glycine (Gly-Gly) remnants following trypsin digestion to immuno-enrich ubiquitinated peptides. Monoclonal antibodies developed to the cleaved ubiquitin modification epitope, (tert-butoxycarbonyl) glycylglycine (Boc-Gly-Gly-NHS)(1), are used to identify the Gly-Gly signature. Here, we have successfully generated the Boc-Gly-Gly-NHS modification and showed that when conjugated to a lysine containing protein, such as lysozyme, it can be applied as a standard protein to examine ubiquitinated peptide enrichment within a complex background. PMID:27562526

  7. In vivo fluorescence imaging of hepatocellular carcinoma xenograft using near-infrared labeled epidermal growth factor receptor (EGFR) peptide

    PubMed Central

    Li, Z.; Zhou, Q.; Zhou, J.; Duan, X.; Zhu, J.; Wang, T. D.

    2016-01-01

    Minimally-invasive surgery of hepatocellular carcinoma (HCC) can be limited by poor tumor visualization with white light. We demonstrate systemic administration of a Cy5.5-labeled peptide specific for epidermal growth factor receptor (EGFR) to target HCC in vivo in a mouse xenograft model. We attached a compact imaging module to the proximal end of a medical laparoscope to collect near-infrared fluorescence and reflectance images concurrently at 15 frames/sec. We measured a mean target-to-background ratio of 2.99 ± 0.22 from 13 surgically exposed subcutaneous human HCC tumors in vivo in 5 mice. This integrated imaging methodology is promising to guide laparoscopic resection of HCC.

  8. In vivo fluorescence imaging of hepatocellular carcinoma xenograft using near-infrared labeled epidermal growth factor receptor (EGFR) peptide

    PubMed Central

    Li, Z.; Zhou, Q.; Zhou, J.; Duan, X.; Zhu, J.; Wang, T. D.

    2016-01-01

    Minimally-invasive surgery of hepatocellular carcinoma (HCC) can be limited by poor tumor visualization with white light. We demonstrate systemic administration of a Cy5.5-labeled peptide specific for epidermal growth factor receptor (EGFR) to target HCC in vivo in a mouse xenograft model. We attached a compact imaging module to the proximal end of a medical laparoscope to collect near-infrared fluorescence and reflectance images concurrently at 15 frames/sec. We measured a mean target-to-background ratio of 2.99 ± 0.22 from 13 surgically exposed subcutaneous human HCC tumors in vivo in 5 mice. This integrated imaging methodology is promising to guide laparoscopic resection of HCC. PMID:27699089

  9. Comparison of /sup 125/I-labeled and /sup 14/C-Labeled peptides of the major outer membrane protein of Chlamydia Trachomatis Strain L2/434 separated by high-performance liquid chromatography

    SciTech Connect

    Judd, R.C.; Caldwell, H.D.

    1985-01-01

    The objective of this study was to determine if in-gel chloramine-T radioiodination adequately labels OM proteins to allow for accurate and precise structural comparison of these molecules. Therefore, intrinsically /sup 14/C-amino acid labeled proteins and /sup 125/I-labeled proteins were cleaved with two endopeptidic reagents and the peptide fragments separated by HPLC. A comparison of retention times of the fragments, as determined by differential radiation counting, thus indicated whether /sup 125/Ilabeling identified of all the peptide peaks seen in the /sup 14/Clabeled proteins. Results demonstrated that radioiodination yields complete and accurate information about the primary structure of outer membrane proteins. In addition, it permits the use of extremely small amounts of protein allowing for method optimization and multiple separations to insure reproducibility.

  10. Interresidue carbonyl-carbonyl polarization transfer experiments in uniformly 13C, 15N-labeled peptides and proteins

    NASA Astrophysics Data System (ADS)

    Janik, Rafal; Ritz, Emily; Gravelle, Andrew; Shi, Lichi; Peng, Xiaohu; Ladizhansky, Vladimir

    2010-03-01

    In this work, we demonstrate that Homonuclear Rotary Resonance Recoupling (HORROR) can be used to reintroduce carbonyl-carbonyl interresidue dipolar interactions and to achieve efficient polarization transfer between carbonyl atoms in uniformly 13C, 15N-labeled peptides and proteins. We show that the HORROR condition is anisotropically broadened and overall shifted to higher radio frequency intensities because of the CSA effects. These effects are analyzed theoretically using Average Hamiltonian Theory. At spinning frequencies used in this study, 22 kHz, this broadening is experimentally found to be on the order of a kilohertz at a proton field of 600 MHz. To match HORROR condition over all powder orientations, variable amplitude radio frequency (RF) fields are required, and efficient direct transfers on the order of 20-30% can be straightforwardly established. Two- and three-dimensional chemical shift correlation experiments establishing long-range interresidue connectivities (e.g., (N[i]-CO[i - 2])) are demonstrated on the model peptide N-acetyl-valine-leucine, and on the third immunoglobulin binding domain of protein G. Possible future developments are discussed.

  11. Interresidue carbonyl-carbonyl polarization transfer experiments in uniformly 13C,15N-labeled peptides and proteins.

    PubMed

    Janik, Rafal; Ritz, Emily; Gravelle, Andrew; Shi, Lichi; Peng, Xiaohu; Ladizhansky, Vladimir

    2010-03-01

    In this work, we demonstrate that Homonuclear Rotary Resonance Recoupling (HORROR) can be used to reintroduce carbonyl-carbonyl interresidue dipolar interactions and to achieve efficient polarization transfer between carbonyl atoms in uniformly (13)C,(15)N-labeled peptides and proteins. We show that the HORROR condition is anisotropically broadened and overall shifted to higher radio frequency intensities because of the CSA effects. These effects are analyzed theoretically using Average Hamiltonian Theory. At spinning frequencies used in this study, 22kHz, this broadening is experimentally found to be on the order of a kilohertz at a proton field of 600MHz. To match HORROR condition over all powder orientations, variable amplitude radio frequency (RF) fields are required, and efficient direct transfers on the order of 20-30% can be straightforwardly established. Two- and three-dimensional chemical shift correlation experiments establishing long-range interresidue connectivities (e.g., (N[i]-CO[i-2])) are demonstrated on the model peptide N-acetyl-valine-leucine, and on the third immunoglobulin binding domain of protein G. Possible future developments are discussed. PMID:20060344

  12. Reducing renal uptake of 90Y- and 177Lu-labeled alpha-melanocyte stimulating hormone peptide analogues

    SciTech Connect

    Miao, Yubin; Fisher, Darrell R.; Quinn, Thomas P.

    2006-06-15

    The purpose of this study was to improve the tumor-to-kidney uptake ratios of 90Y- and 177Lu-[1,2,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-Re-Cys,D-Phe,Arg]alpha-melanocyte stimulating hormone (DOTA-RE(Arg)CCMSH), through coupling a negatively charged glutamic acid (Glu) to the peptide sequence. A new peptide of DOTA-Re(Glu,Arg)CCMSH was designed, synthesized and labeled with 90Y and 177Lu. Pharmacokinetics of 90Y- and 177Lu-DOTA-RE(Glu,Arg)CCNSH were determined in B16/F1 murine melanoma-bearing C57 mice. Both exhibited significantly less renal uptake than 90Y- and 177Lu-DOTA-Re(Arg)CCMSH at 30 min and at 2, 3, and 24 h after dose administration. The renal uptake values of 90Y- and 177Lu-DOTA-Re(Glu,Arg)CCMSH were 28.16% and 28.81% of those of 90Y- and 177Lu-DOTA-RE(Arg)CCMSH, respectively, at 4 hr post-injection. We also showed higher tumor-to-kidney uptake ratios 2.28 and 1.69 times that of 90Y- and 177Lu-DOTA-Re(Arg)CCMSH, respectively, at 4 h post-injection. The90Y- and 177Lu-DOTA-Re(Glu,Arg)CCMSH activity accumulation was low in normal organs except for kidneys. Coupling a negatively charged amino acid (Glu) to the CCMSH peptide sequence dramatically reduced the renal uptake values and increased the tumor-to-kidney uptake ratios of 90Y- and 177Lu-DOTA-Re(Glu,Arg)CCMSH, facilitating their potential applications as radiopharmaceuticals for targeted radionuclide therapy of melanoma.

  13. Bis(thiosemicarbazones) as bifunctional chelators for the room temperature 64-copper labeling of peptides.

    PubMed

    Hueting, Rebekka; Christlieb, Martin; Dilworth, Jonathan R; García Garayoa, Elisa; Gouverneur, Véronique; Jones, Michael W; Maes, Veronique; Schibli, Roger; Sun, Xin; Tourwé, Dirk A

    2010-04-21

    A range of new carboxylate functionalised bis(thiosemicarbazone) ligands and their Cu(II) complexes have been prepared, fully characterised and radiolabeled in high yield with both (64)Cu and (99m)Tc. Conjugation to a bombesin derivative was achieved using standard solid phase synthetic methodologies and the (64)Cu-labeled conjugate was shown to have good tumour uptake in mice with xenografted PC-3 tumours.

  14. TubStain: a universal peptide-tool to label microtubules.

    PubMed

    Theiss, Carsten; Neuhaus, Alexander; Schliebs, Wolfgang; Erdmann, Ralf

    2012-09-01

    Imaging of the microtubular network is an important strategy to define cell-cycle specific and pathological states of eukaryotic cells. Here, we describe TubStain, a novel recombinant polypeptide allowing direct, non-antibody dependent labeling of microtubules in fixed cells over a broad range of different species and tissues. TubStain has, due to its small size and susceptibility for biochemical and genetic manipulations, high potential as a microtubule marker in state-of-the-art microscopy.

  15. Synthesis and properties of peptide nucleic acid labeled at the N-terminus with HiLyte Fluor 488 fluorescent dye.

    PubMed

    Hnedzko, Dziyana; McGee, Dennis W; Rozners, Eriks

    2016-09-15

    Fluorescently labeled peptide nucleic acids (PNAs) are important tools in fundamental research and biomedical applications. However, synthesis of labeled PNAs, especially using modern and expensive dyes, is less explored than similar preparations of oligonucleotide dye conjugates. Herein, we present a simple procedure for labeling of the PNA N-terminus with HiLyte Fluor 488 as the last step of solid phase PNA synthesis. A minimum excess of 1.25equiv of activated carboxylic acid achieved labeling yields close to 90% providing a good compromise between the price of dye and the yield of product and significant improvement over previous literature procedures. The HiLyte Fluor 488-labeled PNAs retained the RNA binding ability and in live cell fluorescence microscopy experiments were brighter and significantly more photostable than PNA labeled with carboxyfluorescein. In contrast to fluorescein-labeled PNA, the fluorescence of PNAs labeled with HiLyte Fluor 488 was independent of pH in the biologically relevant range of 5-8. The potential of HiLyte Fluor 488-labeling for studies of PNA cellular uptake and distribution was demonstrated in several cell lines.

  16. Synthesis and properties of peptide nucleic acid labeled at the N-terminus with HiLyte Fluor 488 fluorescent dye.

    PubMed

    Hnedzko, Dziyana; McGee, Dennis W; Rozners, Eriks

    2016-09-15

    Fluorescently labeled peptide nucleic acids (PNAs) are important tools in fundamental research and biomedical applications. However, synthesis of labeled PNAs, especially using modern and expensive dyes, is less explored than similar preparations of oligonucleotide dye conjugates. Herein, we present a simple procedure for labeling of the PNA N-terminus with HiLyte Fluor 488 as the last step of solid phase PNA synthesis. A minimum excess of 1.25equiv of activated carboxylic acid achieved labeling yields close to 90% providing a good compromise between the price of dye and the yield of product and significant improvement over previous literature procedures. The HiLyte Fluor 488-labeled PNAs retained the RNA binding ability and in live cell fluorescence microscopy experiments were brighter and significantly more photostable than PNA labeled with carboxyfluorescein. In contrast to fluorescein-labeled PNA, the fluorescence of PNAs labeled with HiLyte Fluor 488 was independent of pH in the biologically relevant range of 5-8. The potential of HiLyte Fluor 488-labeling for studies of PNA cellular uptake and distribution was demonstrated in several cell lines. PMID:27430566

  17. Effect of Fluorescent Labels on Peptide and Amino Acid Sample Dimensionality in Two Dimensional nLC × μFFE Separations.

    PubMed

    Geiger, Matthew; Bowser, Michael T

    2016-02-16

    Multidimensional separations present a unique opportunity for generating the high peak capacities necessary for the analysis of complex biological mixtures. We have coupled nano liquid chromatography with micro free flow electrophoresis (nLC × μFFE) to produce high peak capacity separations of peptide and amino acid mixtures. Currently, μFFE largely relies on laser-induced fluorescence (LIF) detection. We have demonstrated that the choice of fluorescent label significantly affects the fractional coverage and peak capacity of nLC × μFFE separations of peptides and amino acids. Of the labeling reagents assessed, Chromeo P503 performed the best for nLC × μFFE separations of peptides. A nLC × μFFE analysis of a Chromeo P503-labeled BSA tryptic digest produced a 2D separation that made effective use of the available separation space (48%), generating a corrected peak capacity of 521 in a 5 min separation window (104 peaks/min). nLC × μFFE separations of NBD-F-labeled peptides produced similar fractional coverage and peak capacity, but this reagent was able to react with multiple reaction sites, producing an unnecessarily complex analyte mixture. NBD-F performed the best for nLC × μFFE separations of amino acids. NBD-F-labeled amino acids produced a 2D separation that covered 36% of the available separation space, generating a corrected peak capacity of 95 in a 75 s separation window (76 peaks/min). Chromeo P503 and Alexa Fluor 488-labeled amino acids were not effectively separated in the μFFE dimension, giving 2D separations with poor fractional coverage and peak capacity. PMID:26757484

  18. A photoaffinity analogue of discodermolide specifically labels a peptide in beta-tubulin.

    PubMed

    Xia, Shujun; Kenesky, Craig S; Rucker, Paul V; Smith, Amos B; Orr, George A; Horwitz, Susan Band

    2006-10-01

    Discodermolide is a potentially important antitumor agent that stabilizes microtubules and blocks cells at the G2/M phase of the cell cycle in a manner similar to that of Taxol. Discodermolide also has unique properties that distinguish it from Taxol. In the present study, photoaffinity-labeled discodermolide analogues are used to investigate their binding site in tubulin. Three photoaffinity-labeled discodermolide analogues were synthesized, all of which promoted microtubule polymerization in the absence of GTP. The analogue, C19-[4-(4-(3)H-benzoyl-phenyl)-carbamate]-discodermolide (C19-[3H]BPC-discodermolide), was selected for photolabeling studies because it had the highest extent of photoincorporation, approximately 1%, of the three radiolabeled discodermolide analogues explored. Although compared to discodermolide, C19-BPC-discodermolide revealed no hypernucleation effect in the in vitro microtubule polymerization assay, it was more cytotoxic than discodermolide, and, like discodermolide, demonstrated synergism with Taxol. These results suggest that the hypernucleation effect of discodermolide is not involved in its cytotoxic activity. Similar to discodermolide, C19-BPC-discodermolide can effectively displace [3H]Taxol from microtubules, but Taxol cannot effectively displace C19-[3H]BPC-discodermolide binding. Discodermolide can effectively displace C19-[3H]BPC-discodermolide binding. Formic acid hydrolysis, immunoprecipitation experiments, and subtilisin digestion indicate that C19-BPC-discodermolide labels amino acid residues 305-433 in beta-tubulin. Further digestion with Asp-N and Arg-C enzymes suggested that C19-BPC-discodermolide binds to amino acid residues, 355-359, in beta-tubulin, which is in close proximity to the Taxol binding site. Molecular modeling guided by the above evidence led to a putative binding model for C19-BPC-discodermolide in tubulin.

  19. Probing the Quenching of Quantum Dot Photoluminescence by Peptide-Labeled Ruthenium(II) Complexes.

    PubMed

    Scott, Amy M; Algar, W Russ; Stewart, Michael H; Trammell, Scott A; Blanco-Canosa, Juan B; Dawson, Philip E; Deschamps, Jeffrey R; Goswami, Ramasis; Oh, Eunkeu; Huston, Alan L; Medintz, Igor L

    2014-05-01

    Charge transfer processes with semiconductor quantum dots (QDs) have generated much interest for potential utility in energy conversion. Such configurations are generally nonbiological; however, recent studies have shown that a redox-active ruthenium(II)-phenanthroline complex (Ru(2+)-phen) is particularly efficient at quenching the photoluminescence (PL) of QDs, and this mechanism demonstrates good potential for application as a generalized biosensing detection modality since it is aqueous compatible. Multiple possibilities for charge transfer and/or energy transfer mechanisms exist within this type of assembly, and there is currently a limited understanding of the underlying photophysical processes in such biocomposite systems where nanomaterials are directly interfaced with biomolecules such as proteins. Here, we utilize redox reactions, steady-state absorption, PL spectroscopy, time-resolved PL spectroscopy, and femtosecond transient absorption spectroscopy (FSTA) to investigate PL quenching in biological assemblies of CdSe/ZnS QDs formed with peptide-linked Ru(2+)-phen. The results reveal that QD quenching requires the Ru(2+) oxidation state and is not consistent with Förster resonance energy transfer, strongly supporting a charge transfer mechanism. Further, two colors of CdSe/ZnS core/shell QDs with similar macroscopic optical properties were found to have very different rates of charge transfer quenching, by Ru(2+)-phen with the key difference between them appearing to be the thickness of their ZnS outer shell. The effect of shell thickness was found to be larger than the effect of increasing distance between the QD and Ru(2+)-phen when using peptides of increasing persistence length. FSTA and time-resolved upconversion PL results further show that exciton quenching is a rather slow process consistent with other QD conjugate materials that undergo hole transfer. An improved understanding of the QD-Ru(2+)-phen system can allow for the design of more

  20. Probing the Quenching of Quantum Dot Photoluminescence by Peptide-Labeled Ruthenium(II) Complexes

    PubMed Central

    2015-01-01

    Charge transfer processes with semiconductor quantum dots (QDs) have generated much interest for potential utility in energy conversion. Such configurations are generally nonbiological; however, recent studies have shown that a redox-active ruthenium(II)–phenanthroline complex (Ru2+-phen) is particularly efficient at quenching the photoluminescence (PL) of QDs, and this mechanism demonstrates good potential for application as a generalized biosensing detection modality since it is aqueous compatible. Multiple possibilities for charge transfer and/or energy transfer mechanisms exist within this type of assembly, and there is currently a limited understanding of the underlying photophysical processes in such biocomposite systems where nanomaterials are directly interfaced with biomolecules such as proteins. Here, we utilize redox reactions, steady-state absorption, PL spectroscopy, time-resolved PL spectroscopy, and femtosecond transient absorption spectroscopy (FSTA) to investigate PL quenching in biological assemblies of CdSe/ZnS QDs formed with peptide-linked Ru2+-phen. The results reveal that QD quenching requires the Ru2+ oxidation state and is not consistent with Förster resonance energy transfer, strongly supporting a charge transfer mechanism. Further, two colors of CdSe/ZnS core/shell QDs with similar macroscopic optical properties were found to have very different rates of charge transfer quenching, by Ru2+-phen with the key difference between them appearing to be the thickness of their ZnS outer shell. The effect of shell thickness was found to be larger than the effect of increasing distance between the QD and Ru2+-phen when using peptides of increasing persistence length. FSTA and time-resolved upconversion PL results further show that exciton quenching is a rather slow process consistent with other QD conjugate materials that undergo hole transfer. An improved understanding of the QD–Ru2+-phen system can allow for the design of more sophisticated

  1. A Comparative Analysis of Computational Approaches to Relative Protein Quantification Using Peptide Peak Intensities in Label-free LC-MS Proteomics Experiments

    SciTech Connect

    Matzke, Melissa M.; Brown, Joseph N.; Gritsenko, Marina A.; Metz, Thomas O.; Pounds, Joel G.; Rodland, Karin D.; Shukla, Anil K.; Smith, Richard D.; Waters, Katrina M.; McDermott, Jason E.; Webb-Robertson, Bobbie-Jo M.

    2013-02-01

    Liquid chromatography coupled with mass spectrometry (LC-MS) is widely used to identify and quantify peptides in complex biological samples. In particular, label-free shotgun proteomics is highly effective for the identification of peptides and subsequently obtaining a global protein profile of a sample. As a result, this approach is widely used for discovery studies. Typically, the objective of these discovery studies is to identify proteins that are affected by some condition of interest (e.g. disease, exposure). However, for complex biological samples, label-free LC-MS proteomics experiments measure peptides and do not directly yield protein quantities. Thus, protein quantification must be inferred from one or more measured peptides. In recent years, many computational approaches to relative protein quantification of label-free LC-MS data have been published. In this review, we examine the most commonly employed quantification approaches to relative protein abundance from peak intensity values, evaluate their individual merits, and discuss challenges in the use of the various computational approaches.

  2. Site-specific φ- and ψ-torsion angle determination in a uniformly/extensively 13C- and 15N-labeled peptide

    NASA Astrophysics Data System (ADS)

    Wi, Sungsool; Spano, Justin

    2011-10-01

    A solid-state rotational-echo double resonance (REDOR) NMR method was introduced to identify the ϕ- and ψ-torsion angle from a 1H- 15N or 1H- 13C' spin system of alanine-like residues in a selectively, uniformly, or extensively 15N-/ 13C-labeled peptide. When a C α( i) or a 15N peak is site-specifically obtainable in the NMR spectrum of a uniformly 15N/ 13C-labeled sample system, the ψ- or ϕ-torsion angle specified by the conformational structure of peptide geometry involving 15N( i)- 1H αi - 15N( i + 1) or 13C'( i - 1)- 1H Ni- 13C'( i) spin system can be identified based on 13C α- or 15N-detected 1H α- 15N or 1H N- 13C REDOR experiment. This method will conveniently be utilized to identify major secondary motifs, such as α-helix, β-sheet, and β-turn, from a uniformly 15N-/ 13C-labled peptide sample system. When tested on a 13C-/ 15N-labeled model system of a three amino acid peptide Gly-[U- 13C, 15N]Ala-[U- 13C, 15N]Leu, the ψ-angle of alanine obtained experimentally, ψ = -40 ± 30°, agreed reasonably well with the X-ray determined angle, ψ = -39°.

  3. Site-Specific N-Terminal Labeling of Peptides and Proteins using Butelase 1 and Thiodepsipeptide.

    PubMed

    Nguyen, Giang K T; Cao, Yuan; Wang, Wei; Liu, Chuan Fa; Tam, James P

    2015-12-21

    An efficient ligase with exquisite site-specificity is highly desirable for protein modification. Recently, we discovered the fastest known ligase called butelase 1 from Clitoria ternatea for intramolecular cyclization. For intermolecular ligation, butelase 1 requires an excess amount of a substrate to suppress the reverse reaction, a feature similar to other ligases. Herein, we describe the use of thiodepsipeptide substrates with a thiol as a leaving group and an unacceptable nucleophile to render the butelase-mediated ligation reactions irreversible and in high yields. Butelase 1 also accepted depsipeptides as substrates, but unlike a thiodesipeptide, the desipeptide ligation was partially reversible as butelase 1 can tolerate an alcohol group as a poor nucleophile. The thiodesipeptide method was successfully applied in N-terminal labeling of ubiquitin and green fluorescent protein using substrates with or without a biotin group in high yields. PMID:26563575

  4. Fermentation and Cost-Effective 13C/15N Labeling of the Nonribosomal Peptide Gramicidin S for Nuclear Magnetic Resonance Structure Analysis

    PubMed Central

    Berditsch, Marina; Afonin, Sergii; Steineker, Anna; Orel, Nataliia; Jakovkin, Igor; Weber, Christian

    2015-01-01

    Gramicidin S (GS) is a nonribosomally synthesized decapeptide from Aneurinibacillus migulanus. Its pronounced antibiotic activity is attributed to amphiphilic structure and enables GS interaction with bacterial membranes. Despite its medical use for over 70 years, the peptide-lipid interactions of GS and its molecular mechanism of action are still not fully understood. Therefore, a comprehensive structural analysis of isotope-labeled GS needs to be performed in its biologically relevant membrane-bound state, using advanced solid-state nuclear magnetic resonance (NMR) spectroscopy. Here, we describe an efficient method for producing the uniformly 13C/15N-labeled peptide in a minimal medium supplemented by selected amino acids. As GS is an intracellular product of A. migulanus, we characterized the producer strain DSM 5759 (rough-convex phenotype) and examined its biosynthetic activity in terms of absolute and biomass-dependent peptide accumulation. We found that the addition of either arginine or ornithine increases the yield only at very high supplementing concentrations (1% and 0.4%, respectively) of these expensive 13C/15N-labeled amino acids. The most cost-effective production of 13C/15N-GS, giving up to 90 mg per gram of dry cell weight, was achieved in a minimal medium containing 1% 13C-glycerol and 0.5% 15N-ammonium sulfate, supplemented with only 0.025% of 13C/15N-phenylalanine. The 100% efficiency of labeling is corroborated by mass spectrometry and preliminary solid-state NMR structure analysis of the labeled peptide in the membrane-bound state. PMID:25795666

  5. Fermentation and Cost-Effective 13C/15N Labeling of the Nonribosomal Peptide Gramicidin S for Nuclear Magnetic Resonance Structure Analysis.

    PubMed

    Berditsch, Marina; Afonin, Sergii; Steineker, Anna; Orel, Nataliia; Jakovkin, Igor; Weber, Christian; Ulrich, Anne S

    2015-06-01

    Gramicidin S (GS) is a nonribosomally synthesized decapeptide from Aneurinibacillus migulanus. Its pronounced antibiotic activity is attributed to amphiphilic structure and enables GS interaction with bacterial membranes. Despite its medical use for over 70 years, the peptide-lipid interactions of GS and its molecular mechanism of action are still not fully understood. Therefore, a comprehensive structural analysis of isotope-labeled GS needs to be performed in its biologically relevant membrane-bound state, using advanced solid-state nuclear magnetic resonance (NMR) spectroscopy. Here, we describe an efficient method for producing the uniformly (13)C/(15)N-labeled peptide in a minimal medium supplemented by selected amino acids. As GS is an intracellular product of A. migulanus, we characterized the producer strain DSM 5759 (rough-convex phenotype) and examined its biosynthetic activity in terms of absolute and biomass-dependent peptide accumulation. We found that the addition of either arginine or ornithine increases the yield only at very high supplementing concentrations (1% and 0.4%, respectively) of these expensive (13)C/(15)N-labeled amino acids. The most cost-effective production of (13)C/(15)N-GS, giving up to 90 mg per gram of dry cell weight, was achieved in a minimal medium containing 1% (13)C-glycerol and 0.5% (15)N-ammonium sulfate, supplemented with only 0.025% of (13)C/(15)N-phenylalanine. The 100% efficiency of labeling is corroborated by mass spectrometry and preliminary solid-state NMR structure analysis of the labeled peptide in the membrane-bound state.

  6. Site-Specifically Labeled Immunoconjugates for Molecular Imaging--Part 2: Peptide Tags and Unnatural Amino Acids.

    PubMed

    Adumeau, Pierre; Sharma, Sai Kiran; Brent, Colleen; Zeglis, Brian M

    2016-04-01

    Molecular imaging using radioisotope- or fluorophore-labeled antibodies is increasingly becoming a critical component of modern precision medicine. Yet despite this promise, the vast majority of these immunoconjugates are synthesized via the random coupling of amine-reactive bifunctional probes to lysines within the antibody, a process that can result in heterogeneous and poorly defined constructs with suboptimal pharmacological properties. In an effort to circumvent these issues, the last 5 years have played witness to a great deal of research focused on the creation of effective strategies for the site-specific attachment of payloads to antibodies. These chemoselective modification methods yield immunoconjugates that are more homogenous and better defined than constructs created using traditional synthetic approaches. Moreover, site-specifically labeled immunoconjugates have also been shown to exhibit superior in vivo behavior compared to their randomly modified cousins. The over-arching goal of this two-part review is to provide a broad yet detailed account of the various site-specific bioconjugation approaches that have been used to create immunoconjugates for positron emission tomography (PET), single photon emission computed tomography (SPECT), and fluorescence imaging. In Part 1, we covered site-specific bioconjugation techniques based on the modification of cysteine residues and the chemoenzymatic manipulation of glycans. In Part 2, we will detail two families of bioconjugation approaches that leverage biochemical tools to achieve site-specificity. First, we will discuss modification methods that employ peptide tags either as sites for enzyme-catalyzed ligations or as radiometal coordination architectures. And second, we will examine bioconjugation strategies predicated on the incorporation of unnatural or non-canonical amino acids into antibodies via genetic engineering. Finally, we will compare the advantages and disadvantages of the modification

  7. Peptide linkers lead to modification of liver metabolism and improved tumor targeting of copper-67-labeled antibody fragments.

    PubMed

    Novak-Hofer, I; Zimmermann, K; Schubiger, P A

    2001-12-01

    In order to determine if tumor/nontarget tissue ratios of 67Cu-labeled antibody fragments can be improved, modifying the DO3A copper chelate with tripeptide linkers was investigated. The peptide-linked chelates 1,4,7,10-tetraazacyclodecane-N,N',N",N"'-tetraacetate (DOTA)-triglycyl-L-p-isothiocyanato-phenylalanine (DOTA-R1-NCS), DOTA-glycyl-phenylalanyl-glycyl-L-p-isothiocyanato-phenylalanine (DOTA-R2-NCS), DOTA-glycyl-prolyl-glycyl-L-p-isothiocyanato-phenylalanine (DOTA-R3-NCS) and DOTA-glycyl-L-p-isothiocyanato-phenylalanine (DOTA-R4-NCS) were synthesized and coupled to F(ab')2 fragments of anti-colon carcinoma mAb35. In vitro, the 67Cu-labeled antibody fragments were fully immunoreactive and stable in human serum. In vivo in nude mice bearing human colon carcinoma xenografts the conjugates R1 and R3 showed improved tumor uptake and lower levels of radioactivity in the liver compared with the other conjugates. Biodistributions of the DOTA-R2-F(ab')2 showed at early time points after injection higher levels of radioactivity in the liver, lower levels of activity persisting in the blood and lower accumulation of activity in the tumor. When liver homogenates were analyzed 30 min post injection by SDS-PAGE or FPLC gel chromatography, it was found that radioactivity was released more slowly from the triglycine (R1)-F(ab')2 than from the immunoconjugates with the R2 or the R4 linker. The main radioactive metabolites were protein bands at 66 kD, 31 kD and low molecular weight fragments. The results show that the rate of cleavage of the copper complex from F(ab')2 fragments in vivo can be influenced by the amino acid sequence close to the complex, with significant consequences on biodistributions.

  8. Site-Specifically Labeled Immunoconjugates for Molecular Imaging—Part 2: Peptide Tags and Unnatural Amino Acids

    PubMed Central

    Adumeau, Pierre; Sharma, Sai Kiran; Brent, Colleen; Zeglis, Brian M.

    2016-01-01

    Molecular imaging using radioisotope- or fluorophore-labeled antibodies is increasingly becoming a critical component of modern precision medicine. Yet despite this promise, the vast majority of these immunoconjugates are synthesized via the random coupling of amine-reactive bifunctional probes to lysines within the antibody, a process that can result in heterogeneous and poorly defined constructs with suboptimal pharmacological properties. In an effort to circumvent these issues, the last 5 years have played witness to a great deal of research focused on the creation of effective strategies for the site-specific attachment of payloads to antibodies. These chemoselective modification methods yield immunoconjugates that are more homogenous and better defined than constructs created using traditional synthetic approaches. Moreover, site-specifically labeled immunoconjugates have also been shown to exhibit superior in vivo behavior compared to their randomly modified cousins. The over-arching goal of this two-part review is to provide a broad yet detailed account of the various site-specific bioconjugation approaches that have been used to create immunoconjugates for positron emission tomography (PET), single photon emission computed tomography (SPECT), and fluorescence imaging. In Part 1, we covered site-specific bioconjugation techniques based on the modification of cysteine residues and the chemoenzymatic manipulation of glycans. In Part 2, we will detail two families of bioconjugation approaches that leverage biochemical tools to achieve site-specificity. First, we will discuss modification methods that employ peptide tags either as sites for enzyme-catalyzed ligations or as radiometal coordination architectures. And second, we will examine bioconjugation strategies predicated on the incorporation of unnatural or non-canonical amino acids into antibodies via genetic engineering. Finally, we will compare the advantages and disadvantages of the modification

  9. Modification of the catalytic subunit of bovine heart cAMP-dependent protein kinase with affinity labels related to peptide substrates.

    PubMed

    Bramson, H N; Thomas, N; Matsueda, R; Nelson, N C; Taylor, S S; Kaiser, E T

    1982-09-25

    The modification and concomitant inactivation of the catalytic subunit of bovine heart cAMP-dependent protein kinase with affinity analogs of peptide substrates potentially capable of undergoing disulfide interchange with enzyme-bound sulfhydryl groups have been used to probe the active site associated with peptide binding. The regeneration of catalytic activity on treatment of the modified enzymes with dithiothreitol and the observation that prior reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) blocks the modification of the kinase by these reagents are consistent with the proposal that only thiol residues are reacting. The affinity analog Leu-Arg-Arg-Ala-Cys(3-nitro-2-pyridinesulfenyl)-Leu-Gly, 1, and the closely related peptide AcLeu-Arg-Arg-Ala-Cys(3-nitro-2-pyridinesulfenyl)-Leu-Gly-OEt, 3, react with a single sulfhydryl as shown by the stoichiometry of the release of the 3-nitro-2-pyridinesulfenyl group and the amount of label incorporated in the enzyme when the radioactively labeled peptide analog of 3 (peptide 4) is employed as the modifying agent. The kinetics of the reaction of 1 with 4.3 microM catalytic subunit was monophasic (employing substrate in excess conditions), yielding an apparent value of KI of approximately 40 microM and a k2 value of approximately 0.25 s-1. The low value of the observed KI, together with the observation that protein kinase substrates inhibit the modification reactions, suggest strongly that the cysteine residue undergoing reaction is in the vicinity of the active site. By trypsin-catalyzed degradation and identification of the peptide segment modified by covalent attachment of the peptide portion of the radioactive analog 4, the single cysteine modified was identified as cysteine-198.

  10. Photoaffinity labeling of human serum vitamin D binding protein and chemical cleavage of the labeled protein: Identification of an 11. 5-kDa peptide containing the putative 25-hydroxyvitamin D sub 3 binding site

    SciTech Connect

    Ray, R.; Holick, M.F. ); Bouillon, R.; Baelen, H.V. )

    1991-07-30

    In this paper, the authors describe photoaffinity labeling and related studies of human serum vitamin D binding protein (hDBP) with 25-hydroxyvitamin D{sub 3} 3{beta}-3{prime}-(N-(4-azido-2-nitrophenyl)amino)propyl ether (25-ANE) and its radiolabeled counterpart, i.e., 25-hydroxyvitamin D{sub 3} 3{beta}-3{prime}-(N-(4-azido-2-nitro-(3,5-{sup 3}H)phenyl)amino)propyl ether ({sup 3}H-25-ANE). They have carried out studies to demonstrate that (1) 25-ANE competes with 25-OH-D{sub 3} for the binding site of the latter in hDBP and (2) {sup 3}H-25-ANE is capable of covalently labeling the hDBP molecule when exposed ot UV light. Treatment of a sample of purified hDBP, labeled with {sup 3}H-25-ANE, with BNPS-skatole produced two Coomassie Blue stained peptide fragments, and the majority of the radioactivity was assoicated with the smaller of the two peptide fragments (16.5 kDa). On the other hand, cleavage of the labeled protein with cyanogen bromide produced a peptide (11.5 kDa) containing most of the covalently attached radioactivity. Considering the primary amino acid structure of hDBP, this peptide fragment (11.5 kDa) represents the N-terminus through residue 108 of the intact protein. Thus, the results tentatively identify this segment of the protein containing the binding pocket for 25-OH-D{sub 3}.

  11. Fast voxel-level dosimetry for (177)Lu labelled peptide treatments.

    PubMed

    Hippeläinen, E; Tenhunen, M; Sohlberg, A

    2015-09-01

    In peptide receptor radionuclide therapy (PRRT), voxel-level radiation absorbed dose calculations can be performed using several different methods. Each method has it strengths and weaknesses; however, Monte Carlo (MC) simulation is presently considered the most accurate method at providing absorbed dose distributions. Unfortunately MC simulation is time-consuming and often impractical to carry out in a clinical practice. In this work, a fast semi-Monte Carlo (sMC) absorbed dose calculation method for (177)Lu PRRT dosimetry is presented. The sMC method is based on a local electron absorption assumption and fast photon MC simulations. The sMC method is compared against full MC simulation code built on PENELOPE (vxlPen) using digital phantoms to assess the accuracy of these assumptions.Due to the local electron absorption assumption of sMC, the potential errors in cross-fire dose from electrons and photons emitted by (177)Lu were first evaluated using an ellipsoidal kidney model by comparing vxlPen and sMC. The photon cross-fire dose from background to kidney and kidney to background with varying kidney-to-background activity concentration ratios were calculated. In addition, kidney to kidney photon and electron cross-dose with different kidney to kidney distances were studied. Second, extended cardiac-torso (XCAT) phantoms were created with liver lesions and with realistic activity distributions and tissue densities. The XCAT phantoms were used to simulate SPECT projections and 3D activity distribution images were reconstructed using an OSEM algorithm. Image-based dose rate distributions were calculated using vxlPen and sMC. Total doses and dose rate volume histograms (DrVH) produced by the two methods were compared.The photon cross-fire dose from the kidney increased the background's absorbed dose by 5% or more up to 5.8 cm distance with 20 : 1 kidney to background activity concentration ratio. On the other hand, the photon cross-fire dose from the background to

  12. Fast voxel-level dosimetry for 177Lu labelled peptide treatments

    NASA Astrophysics Data System (ADS)

    Hippeläinen, E.; Tenhunen, M.; Sohlberg, A.

    2015-09-01

    In peptide receptor radionuclide therapy (PRRT), voxel-level radiation absorbed dose calculations can be performed using several different methods. Each method has it strengths and weaknesses; however, Monte Carlo (MC) simulation is presently considered the most accurate method at providing absorbed dose distributions. Unfortunately MC simulation is time-consuming and often impractical to carry out in a clinical practice. In this work, a fast semi-Monte Carlo (sMC) absorbed dose calculation method for 177Lu PRRT dosimetry is presented. The sMC method is based on a local electron absorption assumption and fast photon MC simulations. The sMC method is compared against full MC simulation code built on PENELOPE (vxlPen) using digital phantoms to assess the accuracy of these assumptions. Due to the local electron absorption assumption of sMC, the potential errors in cross-fire dose from electrons and photons emitted by 177Lu were first evaluated using an ellipsoidal kidney model by comparing vxlPen and sMC. The photon cross-fire dose from background to kidney and kidney to background with varying kidney-to-background activity concentration ratios were calculated. In addition, kidney to kidney photon and electron cross-dose with different kidney to kidney distances were studied. Second, extended cardiac-torso (XCAT) phantoms were created with liver lesions and with realistic activity distributions and tissue densities. The XCAT phantoms were used to simulate SPECT projections and 3D activity distribution images were reconstructed using an OSEM algorithm. Image-based dose rate distributions were calculated using vxlPen and sMC. Total doses and dose rate volume histograms (DrVH) produced by the two methods were compared. The photon cross-fire dose from the kidney increased the background’s absorbed dose by 5% or more up to 5.8 cm distance with 20 : 1 kidney to background activity concentration ratio. On the other hand, the photon cross-fire dose from the background to

  13. Fast voxel-level dosimetry for (177)Lu labelled peptide treatments.

    PubMed

    Hippeläinen, E; Tenhunen, M; Sohlberg, A

    2015-09-01

    In peptide receptor radionuclide therapy (PRRT), voxel-level radiation absorbed dose calculations can be performed using several different methods. Each method has it strengths and weaknesses; however, Monte Carlo (MC) simulation is presently considered the most accurate method at providing absorbed dose distributions. Unfortunately MC simulation is time-consuming and often impractical to carry out in a clinical practice. In this work, a fast semi-Monte Carlo (sMC) absorbed dose calculation method for (177)Lu PRRT dosimetry is presented. The sMC method is based on a local electron absorption assumption and fast photon MC simulations. The sMC method is compared against full MC simulation code built on PENELOPE (vxlPen) using digital phantoms to assess the accuracy of these assumptions.Due to the local electron absorption assumption of sMC, the potential errors in cross-fire dose from electrons and photons emitted by (177)Lu were first evaluated using an ellipsoidal kidney model by comparing vxlPen and sMC. The photon cross-fire dose from background to kidney and kidney to background with varying kidney-to-background activity concentration ratios were calculated. In addition, kidney to kidney photon and electron cross-dose with different kidney to kidney distances were studied. Second, extended cardiac-torso (XCAT) phantoms were created with liver lesions and with realistic activity distributions and tissue densities. The XCAT phantoms were used to simulate SPECT projections and 3D activity distribution images were reconstructed using an OSEM algorithm. Image-based dose rate distributions were calculated using vxlPen and sMC. Total doses and dose rate volume histograms (DrVH) produced by the two methods were compared.The photon cross-fire dose from the kidney increased the background's absorbed dose by 5% or more up to 5.8 cm distance with 20 : 1 kidney to background activity concentration ratio. On the other hand, the photon cross-fire dose from the background to

  14. Inhibition of /sup 125/I-labeled ristocetin binding to Micrococcus luteus cells by the peptides related to bacterial cell wall mucopeptide precursors: quantitative structure-activity relationships

    SciTech Connect

    Kim, K.H.; Martin, Y.; Otis, E.; Mao, J.

    1989-01-01

    Quantitative structure-activity relationships (QSAR) of N-Ac amino acids, N-Ac dipeptides, and N-Ac tripeptides in inhibition of /sup 125/I-labeled ristocetin binding to Micrococcus luteus cell wall have been developed to probe the details of the binding between ristocetin and N-acetylated peptides. The correlation equations indicate that (1) the binding is stronger for peptides in which the side chain of the C-terminal amino acid has a large molar refractivity (MR) value, (2) the binding is weaker for peptides with polar than for those with nonpolar C-terminal side chains, (3) the N-terminal amino acid in N-Ac dipeptides contributes 12 times that of the C-terminal amino acid to binding affinity, and (4) the interactions between ristocetin and the N-terminal amino acid of N-acetyl tripeptides appear to be much weaker than those with the first two amino acids.

  15. Development of novel radiogallium-labeled bone imaging agents using oligo-aspartic acid peptides as carriers.

    PubMed

    Ogawa, Kazuma; Ishizaki, Atsushi; Takai, Kenichiro; Kitamura, Yoji; Kiwada, Tatsuto; Shiba, Kazuhiro; Odani, Akira

    2013-01-01

    (68)Ga (T 1/2 = 68 min, a generator-produced nuclide) has great potential as a radionuclide for clinical positron emission tomography (PET). Because poly-glutamic and poly-aspartic acids have high affinity for hydroxyapatite, to develop new bone targeting (68)Ga-labeled bone imaging agents for PET, we used 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) as a chelating site and conjugated aspartic acid peptides of varying lengths. Subsequently, we compared Ga complexes, Ga-DOTA-(Asp)n (n = 2, 5, 8, 11, or 14) with easy-to-handle (67)Ga, with the previously described (67)Ga-DOTA complex conjugated bisphosphonate, (67)Ga-DOTA-Bn-SCN-HBP. After synthesizing DOTA-(Asp)n by a Fmoc-based solid-phase method, complexes were formed with (67)Ga, resulting in (67)Ga-DOTA-(Asp)n with a radiochemical purity of over 95% after HPLC purification. In hydroxyapatite binding assays, the binding rate of (67)Ga-DOTA-(Asp)n increased with the increase in the length of the conjugated aspartate peptide. Moreover, in biodistribution experiments, (67)Ga-DOTA-(Asp)8, (67)Ga-DOTA-(Asp)11, and (67)Ga-DOTA-(Asp)14 showed high accumulation in bone (10.5 ± 1.5, 15.1 ± 2.6, and 12.8 ± 1.7% ID/g, respectively) but were barely observed in other tissues at 60 min after injection. Although bone accumulation of (67)Ga-DOTA-(Asp)n was lower than that of (67)Ga-DOTA-Bn-SCN-HBP, blood clearance of (67)Ga-DOTA-(Asp)n was more rapid. Accordingly, the bone/blood ratios of (67)Ga-DOTA-(Asp)11 and (67)Ga-DOTA-(Asp)14 were comparable with those of (67)Ga-DOTA-Bn-SCN-HBP. In conclusion, these data provide useful insights into the drug design of (68)Ga-PET tracers for the diagnosis of bone disorders, such as bone metastases. PMID:24391942

  16. Stable-isotope-labeled Histone Peptide Library for Histone Post-translational Modification and Variant Quantification by Mass Spectrometry *

    PubMed Central

    Lin, Shu; Wein, Samuel; Gonzales-Cope, Michelle; Otte, Gabriel L.; Yuan, Zuo-Fei; Afjehi-Sadat, Leila; Maile, Tobias; Berger, Shelley L.; Rush, John; Lill, Jennie R.; Arnott, David; Garcia, Benjamin A.

    2014-01-01

    To facilitate accurate histone variant and post-translational modification (PTM) quantification via mass spectrometry, we present a library of 93 synthetic peptides using Protein-Aqua™ technology. The library contains 55 peptides representing different modified forms from histone H3 peptides, 23 peptides representing H4 peptides, 5 peptides representing canonical H2A peptides, 8 peptides representing H2A.Z peptides, and peptides for both macroH2A and H2A.X. The PTMs on these peptides include lysine mono- (me1), di- (me2), and tri-methylation (me3); lysine acetylation; arginine me1; serine/threonine phosphorylation; and N-terminal acetylation. The library was subjected to chemical derivatization with propionic anhydride, a widely employed protocol for histone peptide quantification. Subsequently, the detection efficiencies were quantified using mass spectrometry extracted ion chromatograms. The library yields a wide spectrum of detection efficiencies, with more than 1700-fold difference between the peptides with the lowest and highest efficiencies. In this paper, we describe the impact of different modifications on peptide detection efficiencies and provide a resource to correct for detection biases among the 93 histone peptides. In brief, there is no correlation between detection efficiency and molecular weight, hydrophobicity, basicity, or modification type. The same types of modifications may have very different effects on detection efficiencies depending on their positions within a peptide. We also observed antagonistic effects between modifications. In a study of mouse trophoblast stem cells, we utilized the detection efficiencies of the peptide library to correct for histone PTM/variant quantification. For most histone peptides examined, the corrected data did not change the biological conclusions but did alter the relative abundance of these peptides. For a low-abundant histone H2A variant, macroH2A, the corrected data led to a different conclusion than the

  17. Rapid Generation of a Nanocrystal-Labeled Peptide Library for Specific Identification of the Bacterium Clostrium Botulinum

    SciTech Connect

    Tok, J B

    2004-11-11

    Several peptide libraries containing up to 2 million unique peptide ligands have been synthesized. The peptides are attached onto a 80 micron resin and the length of these peptide ligands ranges from 5 to 9 amino acid residues. Using a novel calorimetric assay, the libraries were screened for binding to the ganglioside-binding domain of Clostridium Tetanus Toxin, a structural similar analog of the Clostridium Botulinum toxin. Several binding peptide sequences were identified, in which the detailed binding kinetics are currently underway using the Surface Plasmon Resonance (SPR) technique.

  18. 64Cu Labeled Sarcophagine Exendin-4 for MicroPET Imaging of Glucagon like Peptide-1 Receptor Expression

    PubMed Central

    Wu, Zhanhong; Liu, Shuanglong; Nair, Indu; Omori, Keiko; Scott, Stephen; Todorov, Ivan; Shively, John E.; Conti, Peter S.; Li, Zibo; Kandeel, Fouad

    2014-01-01

    The Glucagon-like peptide 1 receptor (GLP-1R) has become an important target for imaging due to its elevated expression profile in pancreatic islets, insulinoma, and the cardiovascular system. Because native GLP-1 is degraded rapidly by dipeptidyl peptidase-IV (DPP-IV), several studies have conjugated different chelators to a more stable analog of GLP-1 (such as exendin-4) as PET or SPECT imaging agents with various advantages and disadvantages. Based on the recently developed Sarcophagin chelator, here, we describe the construction of GLP-1R targeted PET probes containing monomeric and dimeric exendin-4 subunit. The in vitro binding affinity of BarMalSar-exendin-4 and Mal2Sar-(exendin-4)2 was evaluated in INS-1 cells, which over-express GLP-1R. Mal2Sar-(exendin-4)2 demonstrated around 3 times higher binding affinity compared with BaMalSar-exendin-4. After 64Cu labeling, microPET imaging of 64Cu-BaMalSar-exendin-4 and 64Cu-Mal2Sar-(exendin-4)2 were performed on subcutaneous INS-1 tumors, which were clearly visualized with both probes. The tumor uptake of 64Cu-Mal2Sar-(exendin-4)2 was significantly higher than that of 64Cu-BaMaSarl-exendin-4, which could be caused by polyvalency effect. The receptor specificity of these probes was confirmed by effective blocking of the uptake in both tumor and normal positive organs with 20-fold excess of unlabeled exendin-4. In conclusion, sarcophagine cage conjugated exendin-4 demonstrated persistent and specific uptake in INS-1 insulinoma model. Dimerization of exendin-4 could successfully lead to increased tumor uptake in vivo. Both 64Cu-BaMalSar-exendin-4 and 64Cu-Mal2Sar-(exendin-4)2 hold a great potential for GLP-1R targeted imaging. PMID:24955138

  19. The effects of shared peptides on protein quantitation in label-free proteomics by LC/MS/MS

    SciTech Connect

    Jin, Shuangshuang; Daly, Don S.; Springer, David L.; Miller, John H.

    2008-01-02

    Assessment of differential protein abundance from the observed properties of detected peptides is an essential part of protein profiling based on shotgun proteomics. However, the abundance observed for degenerate peptides may be due to contributions from multiple proteins that are affected differently by a given treatment. Excluding degenerate peptides eliminates this ambiguity but may significantly decrease the number of proteins for which abundance estimates can be obtained. Peptide degeneracy within a family of biologically related proteins does not cause ambiguity if family members have a common response to treatment. Based on this concept, we have developed an approach for including degenerate peptides in the analysis of differential protein abundance in protein profiling. Data from a recent proteomics study of lung tissue from mice exposed to lipopolysaccharide, cigarette smoke, and a combination of these agents is used to illustrate our method. Starting from data where about half of the protein identifications involved degenerate peptides, 82% of the affected proteins were grouped into families, based on FASTA annotation, with closure on peptide degeneracy. In many cases, a common abundance relative to control was sufficient to explain ion-current peak areas for peptides, both unique and degenerate, that identified biologically-related proteins in a peptide-degeneracy closure group. Based on these results, we propose that peptide-degeneracy closure groups provide a way to include abundance data for degenerate-peptides in quantitative protein profiling by high throughput mass spectrometry.

  20. Studies of peptide a- and b-type fragment ions using stable isotope labeling and integrated ion mobility/tandem mass spectrometry.

    PubMed

    Riba Garcia, Isabel; Giles, Kevin; Bateman, Robert H; Gaskell, Simon J

    2008-12-01

    The structures of peptide a- and b-type fragment ions were studied using synthetic peptides including a set of isomeric peptides, differing in the sequence location of an alanine residue labeled with (15)N and uniformly with (13)C. The pattern of isotope labeling of second-generation fragment ions derived via a(n) and b(n) ions (where n = 4 or 5) suggested that these intermediates existed in part as macrocyclic structures, where alternative sites of ring opening gave rise to different linear forms whose simple cleavage might give rise to the observed final products. Similar conclusions were derived from combined ion mobility/tandem MS analyses where different fragmentation patterns were observed for isomeric a- or b-type ions that display different ion mobilities. These analyses were facilitated by a new approach to the processing of ion mobility/tandem MS data, from which distinct and separate product ion spectra are derived from ions that are incompletely separated by ion mobility. Finally, an example is provided of evidence for a macrocyclic structure for b(n) ions where n = 8 or 9.

  1. 203Pb-Labeled Alpha-Melanocyte-Stimulating Hormone Peptide as an Imaging Probe for Melanoma Detection

    SciTech Connect

    Yubin, Miao; Figueroa, Said D.; Fisher, Darrell R.; Moore, Herbert A.; Testa, Richard F.; Hoffman, Timothy J.; Quinn, Thomas P.

    2008-05-01

    Abbreviations: a-MSH; alpha melanocyte stimulating hormone, DOTA; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, Re(Arg11)CCMSH; DOTA-[Cys3,4,10, D-Phe7, Arg11]a-MSH3-13, NDP; [Nle4,d-Phe7] a-MSH3-13. Abstract Peptide-targeted alpha therapy with 200 mCi of 212Pb-DOTA-Re(Arg11)CCMSH cured 45% of B16/F1 murine melanoma-bearing C57 mice in a 120-day study, highlighting its melanoma treatment potential. However, there is a need to develop an imaging surrogate for patient specific dosimetry and to monitor the tumor response to 212Pb-DOTA-Re(Arg11)CCMSH therapy. The purpose of this study was to evaluate the potential of 203Pb-DOTA-Re(Arg11)CCMSH as a matched-pair SPECT imaging agent for 212Pb-DOTA-Re(Arg11)CCMSH. Method: DOTA-Re(Arg11)CCMSH was labeled with 203Pb in 0.5 M NH4OAc buffer at pH 5.4. The internalization and efflux of 203Pb-DOTA-Re(Arg11)CCMSH were determined in B16/F1 melanoma cells. The pharmacokinetics of 203Pb-DOTA-Re(Arg11)CCMSH were examined in B16/F1 melanoma-bearing C57 mice. A micro-SPECT/CT imaging study was performed with 203Pb-DOTA-Re(Arg11)CCMSH in a B16/F1 melanoma-bearing C57 mouse at 2 h post-injection. Results: 203Pb-DOTA-Re(Arg11)CCMSH was easily prepared in NH4OAc buffer and completely separated from the excess non-radiolabeled peptide by RP-HPLC. 203Pb-DOTA-Re(Arg11)CCMSH displayed fast internalization and extended retention in B16/F1 cells. Approximately 73% of 203Pb-DOTA-Re(Arg11)CCMSH activity internalized after a 20-min incubation at 25C. After incubating the cells in culture media for 20 min, 78% of internalized activity remained in the cells. 203Pb-DOTA-Re(Arg11)CCMSH exhibited similar biodistribution pattern with 212Pb-DOTA-Re(Arg11)CCMSH in B16/F1 melanoma-bearing mice. 203Pb-DOTA-Re(Arg11)CCMSH exhibited the peak tumor uptake of 12.00 +/- 3.20 %ID/g at 1 h post-injection. The tumor uptake gradually decreased to 3.43 +/- 1.12 %ID/g at 48 h post-injection. 203Pb-DOTA-Re(Arg11)CCMSH exhibited the peak tumor to kidney

  2. Melanoma-targeting properties of (99m)technetium-labeled cyclic alpha-melanocyte-stimulating hormone peptide analogues.

    PubMed

    Chen, J; Cheng, Z; Hoffman, T J; Jurisson, S S; Quinn, T P

    2000-10-15

    Preliminary reports have demonstrated that (99m)technetium (Tc)-labeled cyclic [Cys(3,4,10), D-Phe7]alpha-MSH(3-13) (CCMSH) exhibits high tumor uptake and retention values in a murine melanoma mouse model. In this report, the tumor targeting mechanism of 99mTc-CCMSH was studied and compared with four other radiolabeled alpha-melanocyte stimulating hormone (alpha-MSH) peptide analogues: 125I-(Tyr2)-[Nle4, D-Phe7]alpha-MSH [125I-(Tyr2)-NDP]; 99mTc-CGCG-NDP; 99mTc-Gly11-CCMSH; and 99mTc-Nle11-CCMSH. In vitro receptor binding, internalization, and cellular retention of radiolabeled alpha-MSH analogues in B16/F1 murine cell line demonstrated that >70% of the receptor-bound radiolabeled analogues were internalized together with the receptor. Ninety % of the internalized 125I-(Tyr2)-NDP, whereas only 36% of internalized 99mTc-CCMSH, was released from the cells into the medium during a 4-h incubation at 37 degrees C. Two mouse models, C57 mice and severe combined immunodeficient (Scid) mice, inoculated s.c. with B16/F1 murine and TXM-13 human melanoma cells were used for the in vivo studies. Tumor uptake values of 11.32 and 2.39 [% injected dose (ID)/g] for 99mTc-CCMSH at 4 h after injection, resulted in an uptake ratio of tumor:blood of 39.0 and 11.5 in murine melanoma-C57 and human melanoma-Scid mouse models, respectively. Two strategies for decreasing the nonspecific kidney uptake of 99mTc-CCMSH, substitution of Lys11 in CCMSH with Gly11 or Nle11, and lysine coinjection, were evaluated. The biodistribution data for the modified peptides showed that Lys11 replacement dramatically decreased the kidney uptake, whereas the tumor uptakes of 99mTc-Nle11- and 99mTc-Gly11-CCMSH were significantly lower than that of 99mTc-CCMSH. Lysine coinjection significantly decreased the kidney uptake (e.g., from 14.6% ID/g to 4.5% ID/g at 4 h after injection in murine melanoma-C57 mice) without significantly changing the value of tumor uptake of 99mTc-CCMSH. In conclusion, the compact

  3. Preclinical Pharmacokinetic Studies of the Tritium Labelled D-Enantiomeric Peptide D3 Developed for the Treatment of Alzheimer´s Disease

    PubMed Central

    Jiang, Nan; Leithold, Leonie H. E.; Post, Julia; Ziehm, Tamar; Mauler, Jörg; Gremer, Lothar; Cremer, Markus; Schartmann, Elena; Shah, N. Jon; Kutzsche, Janine; Langen, Karl-Josef; Breitkreutz, Jörg; Willbold, Dieter; Willuweit, Antje

    2015-01-01

    Targeting toxic amyloid beta (Aβ) oligomers is currently a very attractive drug development strategy for treatment of Alzheimer´s disease. Using mirror-image phage display against Aβ1-42, we have previously identified the fully D-enantiomeric peptide D3, which is able to eliminate Aβ oligomers and has proven therapeutic potential in transgenic Alzheimer´s disease animal models. However, there is little information on the pharmacokinetic behaviour of D-enantiomeric peptides in general. Therefore, we conducted experiments with the tritium labelled D-peptide D3 (3H-D3) in mice with different administration routes to study its distribution in liver, kidney, brain, plasma and gastrointestinal tract, as well as its bioavailability by i.p. and p.o. administration. In addition, we investigated the metabolic stability in liver microsomes, mouse plasma, brain, liver and kidney homogenates, and estimated the plasma protein binding. Based on its high stability and long biological half-life, our pharmacokinetic results support the therapeutic potential of D-peptides in general, with D3 being a new promising drug candidate for Alzheimer´s disease treatment. PMID:26046986

  4. [3H]Azidodantrolene photoaffinity labeling, synthetic domain peptides and monoclonal antibody reactivity identify the dantrolene binding sequence on RyR1

    SciTech Connect

    Paul-Pletzer, Kalanethee; Yamamoto, Takeshi; Bhat, Manju B.; Ma, Jianjie; Ikemoto, Noriaki; Jimenez, Leslie S.; Morimoto, Hiromi; Williams, Philip G.; Parness, Jerome

    2002-06-14

    Dantrolene is a drug that suppresses intracellular Ca2+ release from sarcoplasmic reticulum in normal skeletal muscle and is used as a therapeutic agent in individuals susceptible to malignant hyperthermia. Though its precise mechanism of action has not been elucidated, we have identified the N-terminal region (amino acids 1-1400) of the skeletal muscle isoform of the ryanodine receptor (RyR1), the primary Ca2+ release channel in sarcoplasmic reticulum, as a molecular target for dantrolene using the photoaffinity analog [3H]azidodantrolene(1). Here, we demonstrate that heterologously expressed RyR1 retains its capacity to be specifically labeled with [3H]azidodantrolene,indicating that muscle specific factors are not required for this ligand-receptor interaction. Synthetic domain peptides of RyR1, previously shown to affect RyR1 function in vitro and in vivo, were exploited as potential drug binding site mimics and used in photoaffinity labeling experiments. Only DP1 and DP1-2, peptide s containing the amino acid sequence corresponding to RyR1 residues 590-609, were specifically labeled by [3H]azidodantrolene. A monoclonal anti-RyR1 antibody which recognizes RyR1 and its 1400 amino acid N-terminal fragment, recognizes DP1 and DP1-2 in both Western blots and immunoprecipitation assays, and specifically inhibits [3H]azidodantrolene photolabeling of RyR1 and its N-terminal fragment in sarcoplasmic reticulum. Our results indicate that synthetic domain peptides can mimic a native, ligand binding conformation in vitro, and that the dantrolene binding site and the epitope for the monoclonal antibody on RyR1 are equivalent and composed of amino-acids 590-609.

  5. Determination of bovine lactoferrin in dairy products by ultra-high performance liquid chromatography-tandem mass spectrometry based on tryptic signature peptides employing an isotope-labeled winged peptide as internal standard.

    PubMed

    Zhang, Jingshun; Lai, Shiyun; Cai, Zengxuan; Chen, Qi; Huang, Baifen; Ren, Yiping

    2014-06-01

    A new and sensitive determination method was developed for bovine lactoferrin in dairy products including infant formulas based on the signature peptide by ultra high-performance liquid chromatography and triple-quadrupole tandem mass spectrometry under the multiple reaction monitoring mode. The simple pretreatment procedures included the addition of a winged peptide containing the isotope-labeled signature peptide as internal standard, followed by an enzymatic digestion with trypsin. The signature peptide was chosen and identified from the tryptic hydrolyzates of bovine lactoferrin by ultra high-performance liquid chromatography and quadrupole-time-of-flight tandem mass spectrometry based on sequence database search. Analytes were separated on an ACQUITY UPLC BEH 300 C18 column and monitored by MS/MS in seven minutes. Quantitative result bias due to matrix effect and tryptic efficiency was corrected through the use of synthetic isotope-labeled standards. The limit of detection and limit of quantification were 0.3 mg/100 g and 1.0 mg/100 g, respectively. Bovine lactoferrin within the concentration range of 10-1000 nmol L(-1) showed a strong linear relationship with a linear correlation coefficient (r) of >0.998. The intra- and inter-day precision of the method were RSD<6.5% and RSD<7.1%, respectively. Excellent repeatability (RSD<6.4%) substantially supported the application of this method for the determination of bovine lactoferrin in dairy samples. The present method was successfully validated and applied to determination of bovine lactoferrin in dairy products including infant formulas.

  6. PET Imaging of Extracellular pH in Tumors with (64)Cu- and (18)F-Labeled pHLIP Peptides: A Structure-Activity Optimization Study.

    PubMed

    Demoin, Dustin Wayne; Wyatt, Linden C; Edwards, Kimberly J; Abdel-Atti, Dalya; Sarparanta, Mirkka; Pourat, Jacob; Longo, Valerie A; Carlin, Sean D; Engelman, Donald M; Andreev, Oleg A; Reshetnyak, Yana K; Viola-Villegas, Nerissa; Lewis, Jason S

    2016-09-21

    pH (low) insertion peptides (pHLIP peptides) target acidic extracellular environments in vivo due to pH-dependent cellular membrane insertion. Two variants (Var3 and Var7) and wild-type (WT) pHLIP peptides have shown promise for in vivo imaging of breast cancer. Two positron emitting radionuclides ((64)Cu and (18)F) were used to label the NOTA- and NO2A-derivatized Var3, Var7, and WT peptides for in vivo biodistribution studies in 4T1 orthotopic tumor-bearing BALB/c mice. All of the constructs were radiolabeled with (64)Cu or [(18)F]-AlF in good yield. The in vivo biodistribution of the 12 constructs in 4T1 orthotopic allografted female BALB/c mice indicated that NO2A-cysVar3, radiolabeled with either (18)F (4T1 uptake; 8.9 ± 1.7%ID/g at 4 h p.i.) or (64)Cu (4T1 uptake; 8.2 ± 0.9%ID/g at 4 h p.i. and 19.2 ± 1.8% ID/g at 24 h p.i.), shows the most promise for clinical translation. Additional studies to investigate other tumor models (melanoma, prostate, and brain tumor models) indicated the universality of tumor targeting of these tracers. From this study, future clinical translation will focus on (18)F- or (64)Cu-labeled NO2A-cysVar3. PMID:27396694

  7. PET Imaging of Extracellular pH in Tumors with 64Cu- and 18F-Labeled pHLIP Peptides: A Structure–Activity Optimization Study

    PubMed Central

    2016-01-01

    pH (low) insertion peptides (pHLIP peptides) target acidic extracellular environments in vivo due to pH-dependent cellular membrane insertion. Two variants (Var3 and Var7) and wild-type (WT) pHLIP peptides have shown promise for in vivo imaging of breast cancer. Two positron emitting radionuclides (64Cu and 18F) were used to label the NOTA- and NO2A-derivatized Var3, Var7, and WT peptides for in vivo biodistribution studies in 4T1 orthotopic tumor-bearing BALB/c mice. All of the constructs were radiolabeled with 64Cu or [18F]-AlF in good yield. The in vivo biodistribution of the 12 constructs in 4T1 orthotopic allografted female BALB/c mice indicated that NO2A-cysVar3, radiolabeled with either 18F (4T1 uptake; 8.9 ± 1.7%ID/g at 4 h p.i.) or 64Cu (4T1 uptake; 8.2 ± 0.9%ID/g at 4 h p.i. and 19.2 ± 1.8% ID/g at 24 h p.i.), shows the most promise for clinical translation. Additional studies to investigate other tumor models (melanoma, prostate, and brain tumor models) indicated the universality of tumor targeting of these tracers. From this study, future clinical translation will focus on 18F- or 64Cu-labeled NO2A-cysVar3. PMID:27396694

  8. "Click"-cyclized (68)Ga-labeled peptides for molecular imaging and therapy: synthesis and preliminary in vitro and in vivo evaluation in a melanoma model system.

    PubMed

    Martin, Molly E; Sue O'Dorisio, M; Leverich, Whitney M; Kloepping, Kyle C; Walsh, Susan A; Schultz, Michael K

    2013-01-01

    Cyclization techniques are used often to impart higher in vivo stability and binding affinity to peptide targeting vectors for molecular imaging and therapy. The two most often used techniques to impart these qualities are lactam bridge construction and disulfide bond formation. While these techniques have been demonstrated to be effective, orthogonal protection/deprotection steps can limit achievable product yields. In the work described in this chapter, new α-melanocyte stimulating hormone (α-MSH) peptide analogs were synthesized and cyclized by copper-catalyzed terminal azide-alkyne cycloaddition "click" chemistry techniques. The α-MSH peptide and its cognate receptor (melanocortin receptor subtype 1, MC1R) represent a well-characterized model system to examine the effect of the triazole linkage for peptide cyclization on receptor binding in vitro and in vivo. Four new DOTA-conjugated α-MSH analogs were cyclized and evaluated by in vitro competitive binding assays, serum stability testing, and in vivo imaging by positron emission tomography (PET) of tumor-bearing mice. These new DOTA-conjugated click-cyclized analogs exhibited selective high binding affinity (<2 nM) for MC1R on melanoma cells in vitro, high stability in human serum, and produced high-contrast PET/CT images of tumor xenografts. (68)Ga-labeled DOTA bioconjugates displayed rapid pharmacokinetics with receptor-mediated tumor accumulation of up to 16 ± 5% ID/g. The results indicate that the triazole ring is an effective bioisosteric replacement for the standard lactam bridge assemblage for peptide cyclization. Radiolabeling results confirm that Cu catalyst is sufficiently removed prior to DOTA chelator addition to enable insertion of radio metals or stable metals for molecular imaging and therapy. Thus, these click-chemistry-cyclized variants show promise as agents for melanocortin receptor-targeted imaging and radionuclide therapy.

  9. Membrane lysis by the antibacterial peptides cecropins B1 and B3: A spin-label electron spin resonance study on phospholipid bilayers.

    PubMed Central

    Hung, S C; Wang, W; Chan, S I; Chen, H M

    1999-01-01

    Custom antibacterial peptides, cecropins B1 (CB1) and B3 (CB3), were synthesized. These peptides have particular sequence characteristics, with CB1 having two amphipathic alpha-helical segments and CB3 having two hydrophobic alpha-helical segments. These differences were exploited for a study of their efficacy in breaking up liposomes, which had different combinations of phosphatidic acid (PA) and phosphatidylcholine (PC), and a study of their lipid binding ability. Binding and nonbinding lysis actions of CB1 and CB3 on liposomes were examined further by electron spin resonance (ESR). The spin-labeled lipids 5'SL-PC, 7'SL-PC, 10'SL-PC, 12'SL-PC, and 16'SL-PC were used as probes. The ESR spectra revealed larger outer hyperfine splittings (2A(max)) for CB1 when the interactions of CB1 and CB3 with liposomes were compared. These observations indicate a larger restriction of the motion of the spin-labeled chains in the presence of CB1. Plots of the effective order parameter at the various probe positions (chain flexibility gradient) versus the peptide-lipid ratio further suggested that the lysis action of CB1 is related to its capacity to bind to the lipid bilayers. In contrast, there is no evidence of binding for CB3. To augment these findings, four spin-labeled peptides, C8SL-CB1, C32SL-CB1, C5SL-CB3, and C30SL-CB3, were also examined for their binding to and their state of aggregation within the lipid bilayers. Association isotherms of the peptides were measured for liposomes containing two molar fractions of PA (0.25 and 0.75). The membrane binding of the CB1 peptides exhibited a cooperative behavior, whereas the association isotherm of CB3 revealed binding to the lipid only for beta = 0.75 liposomes. To further identify the location of CB1 in the lipid bilayers, measurements of the collision rate with chromium oxalate in solution were conducted. Results from ESR power saturation measurements suggested that the NH(2)-terminal alpha-helix of CB1 is located on the

  10. Differential mass spectrometry: a label-free LC-MS method for finding significant differences in complex peptide and protein mixtures.

    PubMed

    Wiener, Matthew C; Sachs, Jeffrey R; Deyanova, Ekaterina G; Yates, Nathan A

    2004-10-15

    Efficiently identifying and quantifying disease- or treatment-related changes in the abundance of proteins is an important area of research for the pharmaceutical industry. Here we describe an automated, label-free method for finding differences in complex mixtures using complete LC-MS data sets, rather than subsets of extracted peaks or features. The method selectively finds statistically significant differences in the intensity of both high-abundance and low-abundance ions, accounting for the variability of measured intensities and the fact that true differences will persist in time. The method was used to compare two complex peptide mixtures with known peptide differences. This controlled experiment allowed us to assess the validity of each difference found and so to analyze the method's sensitivity and specificity. The method detects both presence versus absence and a 2-fold change in peptide concentration near the limit of detection of the instrument used, where chromatographic peaks may not be sufficiently well defined to be detected in individual samples. The method is more sensitive and gives fewer false positives than subtractive methods that ignore signal variability. Differential mass spectrometry combined with targeted MS/MS analysis of only identified differences may save both computation time and human effort compared to shotgun proteomics approaches. PMID:15481957

  11. Collision-induced dissociation of diazirine-labeled peptide ions. Evidence for Brønsted-acid assisted elimination of nitrogen.

    PubMed

    Marek, Aleš; Tureček, František

    2014-05-01

    Gas-phase dissociations were investigated for several peptide ions containing the Gly-Leu* N-terminal motif where Leu* was a modified norleucine residue containing the photolabile diazirine ring. Collisional activation of gas-phase peptide cations resulted in facile N₂ elimination that competed with backbone dissociations. A free lysine ammonium group can act as a Brønsted acid to facilitate N₂ elimination. This dissociation was accompanied by insertion of a lysine proton in the side chain of the photoleucine residue, as established by deuterium labeling and gas-phase sequencing of the products. Electron structure calculations were used to provide structures and energies of reactants, intermediates, and transition states for Gly-Leu*-Gly-Gly-Lys amide ions that were combined with RRKM calculations of unimolecular rate constants. The calculations indicated that Brønsted acid-catalyzed eliminations were kinetically preferred over direct loss of N₂ from the diazirine ring. Mechanisms are proposed to explain the proton-initiated reactions and discuss the reaction products. The non-catalyzed diazirine ring cleavage and N₂ loss is proposed as a thermometer dissociation for peptide ion dissociations. PMID:24549894

  12. Collision-Induced Dissociation of Diazirine-Labeled Peptide Ions. Evidence for Brønsted-Acid Assisted Elimination of Nitrogen

    NASA Astrophysics Data System (ADS)

    Marek, Aleš; Tureček, František

    2014-05-01

    Gas-phase dissociations were investigated for several peptide ions containing the Gly-Leu* N-terminal motif where Leu* was a modified norleucine residue containing the photolabile diazirine ring. Collisional activation of gas-phase peptide cations resulted in facile N2 elimination that competed with backbone dissociations. A free lysine ammonium group can act as a Brønsted acid to facilitate N2 elimination. This dissociation was accompanied by insertion of a lysine proton in the side chain of the photoleucine residue, as established by deuterium labeling and gas-phase sequencing of the products. Electron structure calculations were used to provide structures and energies of reactants, intermediates, and transition states for Gly-Leu*-Gly-Gly-Lys amide ions that were combined with RRKM calculations of unimolecular rate constants. The calculations indicated that Brønsted acid-catalyzed eliminations were kinetically preferred over direct loss of N2 from the diazirine ring. Mechanisms are proposed to explain the proton-initiated reactions and discuss the reaction products. The non-catalyzed diazirine ring cleavage and N2 loss is proposed as a thermometer dissociation for peptide ion dissociations.

  13. Investigating the Effect of Ligand Amount and Injected Therapeutic Activity: A Simulation Study for 177Lu-Labeled PSMA-Targeting Peptides.

    PubMed

    Kletting, Peter; Schuchardt, Christiane; Kulkarni, Harshad R; Shahinfar, Mostafa; Singh, Aviral; Glatting, Gerhard; Baum, Richard P; Beer, Ambros J

    2016-01-01

    In molecular radiotherapy with 177Lu-labeled prostate specific membrane antigen (PSMA) peptides, kidney and/or salivary glands doses limit the activity which can be administered. The aim of this work was to investigate the effect of the ligand amount and injected activity on the tumor-to-normal tissue biologically effective dose (BED) ratio for 177Lu-labeled PSMA peptides. For this retrospective study, a recently developed physiologically based pharmacokinetic model was adapted for PSMA targeting peptides. General physiological parameters were taken from the literature. Individual parameters were fitted to planar gamma camera measurements (177Lu-PSMA I&T) of five patients with metastasizing prostate cancer. Based on the estimated parameters, the pharmacokinetics of tumor, salivary glands, kidneys, total body and red marrow was simulated and time-integrated activity coefficients were calculated for different peptide amounts. Based on these simulations, the absorbed doses and BEDs for normal tissue and tumor were calculated for all activities leading to a maximal tolerable kidney BED of 10 Gy2.5/cycle, a maximal salivary gland absorbed dose of 7.5 Gy/cycle and a maximal red marrow BED of 0.25 Gy15/cycle. The fits yielded coefficients of determination > 0.85, acceptable relative standard errors and low parameter correlations. All estimated parameters were in a physiologically reasonable range. The amounts (for 25-29 nmol) and pertaining activities leading to a maximal tumor dose, considering the defined maximal tolerable doses to organs of risk, were calculated to be 272±253 nmol (452±420 μg) and 7.3±5.1 GBq. Using the actually injected amount (235±155 μg) and the same maximal tolerable doses, the potential improvement for the tumor BED was 1-3 fold. The results suggest that currently given amounts for therapy are in the appropriate order of magnitude for many lesions. However, for lesions with high binding site density or lower perfusion, optimizing the peptide

  14. Investigating the Effect of Ligand Amount and Injected Therapeutic Activity: A Simulation Study for 177Lu-Labeled PSMA-Targeting Peptides

    PubMed Central

    Schuchardt, Christiane; Kulkarni, Harshad R.; Shahinfar, Mostafa; Singh, Aviral; Glatting, Gerhard; Baum, Richard P.; Beer, Ambros J.

    2016-01-01

    In molecular radiotherapy with 177Lu-labeled prostate specific membrane antigen (PSMA) peptides, kidney and/or salivary glands doses limit the activity which can be administered. The aim of this work was to investigate the effect of the ligand amount and injected activity on the tumor-to-normal tissue biologically effective dose (BED) ratio for 177Lu-labeled PSMA peptides. For this retrospective study, a recently developed physiologically based pharmacokinetic model was adapted for PSMA targeting peptides. General physiological parameters were taken from the literature. Individual parameters were fitted to planar gamma camera measurements (177Lu-PSMA I&T) of five patients with metastasizing prostate cancer. Based on the estimated parameters, the pharmacokinetics of tumor, salivary glands, kidneys, total body and red marrow was simulated and time-integrated activity coefficients were calculated for different peptide amounts. Based on these simulations, the absorbed doses and BEDs for normal tissue and tumor were calculated for all activities leading to a maximal tolerable kidney BED of 10 Gy2.5/cycle, a maximal salivary gland absorbed dose of 7.5 Gy/cycle and a maximal red marrow BED of 0.25 Gy15/cycle. The fits yielded coefficients of determination > 0.85, acceptable relative standard errors and low parameter correlations. All estimated parameters were in a physiologically reasonable range. The amounts (for 25−29 nmol) and pertaining activities leading to a maximal tumor dose, considering the defined maximal tolerable doses to organs of risk, were calculated to be 272±253 nmol (452±420 μg) and 7.3±5.1 GBq. Using the actually injected amount (235±155 μg) and the same maximal tolerable doses, the potential improvement for the tumor BED was 1–3 fold. The results suggest that currently given amounts for therapy are in the appropriate order of magnitude for many lesions. However, for lesions with high binding site density or lower perfusion, optimizing the

  15. Investigating the Effect of Ligand Amount and Injected Therapeutic Activity: A Simulation Study for 177Lu-Labeled PSMA-Targeting Peptides.

    PubMed

    Kletting, Peter; Schuchardt, Christiane; Kulkarni, Harshad R; Shahinfar, Mostafa; Singh, Aviral; Glatting, Gerhard; Baum, Richard P; Beer, Ambros J

    2016-01-01

    In molecular radiotherapy with 177Lu-labeled prostate specific membrane antigen (PSMA) peptides, kidney and/or salivary glands doses limit the activity which can be administered. The aim of this work was to investigate the effect of the ligand amount and injected activity on the tumor-to-normal tissue biologically effective dose (BED) ratio for 177Lu-labeled PSMA peptides. For this retrospective study, a recently developed physiologically based pharmacokinetic model was adapted for PSMA targeting peptides. General physiological parameters were taken from the literature. Individual parameters were fitted to planar gamma camera measurements (177Lu-PSMA I&T) of five patients with metastasizing prostate cancer. Based on the estimated parameters, the pharmacokinetics of tumor, salivary glands, kidneys, total body and red marrow was simulated and time-integrated activity coefficients were calculated for different peptide amounts. Based on these simulations, the absorbed doses and BEDs for normal tissue and tumor were calculated for all activities leading to a maximal tolerable kidney BED of 10 Gy2.5/cycle, a maximal salivary gland absorbed dose of 7.5 Gy/cycle and a maximal red marrow BED of 0.25 Gy15/cycle. The fits yielded coefficients of determination > 0.85, acceptable relative standard errors and low parameter correlations. All estimated parameters were in a physiologically reasonable range. The amounts (for 25-29 nmol) and pertaining activities leading to a maximal tumor dose, considering the defined maximal tolerable doses to organs of risk, were calculated to be 272±253 nmol (452±420 μg) and 7.3±5.1 GBq. Using the actually injected amount (235±155 μg) and the same maximal tolerable doses, the potential improvement for the tumor BED was 1-3 fold. The results suggest that currently given amounts for therapy are in the appropriate order of magnitude for many lesions. However, for lesions with high binding site density or lower perfusion, optimizing the peptide

  16. Affinity labeling of lysine-149 in the anion-binding exosite of human. alpha. -thrombin with an N sup. alpha. -(dinitrofluorobenzyl)hirudin C-terminal peptide

    SciTech Connect

    Bourdon, P.; Maraganore, J.M. ); Fenton, J.W. II )

    1990-07-10

    In order to define structural regions in thrombin that interact with hirudin, the N{sup {alpha}}-dinitrofluorobenzyl analogue of an undecapeptide was synthesized corresponding to residues 54-64 of hirudin (GDFEEIPEEY(O{sup 35}SO{sub 3})L (DNFB-({sup 35}S)Hir{sub 54-64})). DNFB-({sup 35}S)Hir{sub 54-64} was reacted at a 10-fold molar excess with human {alpha}-thrombin in phosphate-buffered saline at pH 7.4 and 23{degree}C for 18 h. Autoradiographs of the product in reducing SDS-polyacrylamide gels revealed a single {sup 35}S-labeled band of M{sub r} {approximately}32,500. The labeled product was coincident with a band on Coomassie Blue stained gels migrating slightly above an unlabeled thrombin band at M{sub r} {approximately}31,000. Incorporation of the {sup 35}S affinity reagent peptide was found markedly reduced when reaction with thrombin was performed in the presence of 5- and 20-fold molar excesses of unlabeled hirudin peptide, showing that a specific site was involved in complex formation. The human {alpha}-thrombin-DNFB-Hir{sub 54-64} complex was reduced, S-carboxymethylated, and treated with pepsin. Peptic fragments were separated by reverse-phase HPLC revealing two major peaks containing absorbance at 310 nm. Automated Edman degradation of the peptide fragments allowed identification of Lys-149 of human thrombin as the major site of DNFB-Hir{sub 54-64} derivatization. These data suggest that the anionic C-terminal tail of hirudin interacts with an anion-binding exosite in human thrombin removed 18-20 {angstrom} from the catalytic apparatus.

  17. Specific affinity-labeling of the nociceptin ORL1 receptor using a thiol-activated Cys(Npys)-containing peptide ligand.

    PubMed

    Matsushima, Ayami; Nishimura, Hirokazu; Matsuyama, Yutaka; Liu, Xiaohui; Costa, Tommaso; Shimohigashi, Yasuyuki

    2016-11-01

    We previously showed that an antagonist-based peptide ligand, H-Cys(Npys)-Arg-Tyr-Tyr-Arg- Ile-Lys-NH2 , captures the free thiol groups in the ligand-binding site of the nociceptin receptor ORL1. However, the exact receptor sites of this thiol-disulfide exchange reaction have not been uncovered, although such identification would help to clarify the ligand recognition site. Since the Cys→Ala substitution prevents the reaction, we performed the so-called Ala scanning for all the Cys residues in the transmembrane (TM) domains of the ORL1 receptor. Seven different mutant receptors were soundly expressed in the COS-7 cells and examined for their specific affinity labeling by a competitive binding assay using nociceptin and [(3) H]nociceptin. The results of in vitro Ala scanning analyses revealed that the labeled residues were Cys59 in TM1, Cys215 and Cys231 in TM5, and Cys310 in TM7. The present study has provided a novel method of Cys(Npys)-affinity labeling for identification of the ligand-binding sites in the ORL1 receptor. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 460-469, 2016.

  18. Labeling Primary Amine Groups in Peptides and Proteins with N-Hydroxysuccinimidyl Ester Modified Fe3O4@SiO2 Nanoparticles Containing Cleavable Disulfide-bond Linkers

    PubMed Central

    Patil, Ujwal S.; Qu, Haiou; Caruntu, Daniela; O’Connor, Charles J.; Sharma, Arjun; Cai, Yang; Tarr, Matthew A.

    2013-01-01

    The surface of superparamagnetic silica coated iron oxide (Fe3O4@SiO2) nanoparticles was functionalized with a disulfide bond linked N-hydroxysuccinimidyl (NHS) ester group in order to develop a method for labeling primary amines in peptides/proteins. The nanoparticle labeled proteins/peptides formed after NHS ester reaction with the primary amine groups were isolated using a magnet without any additional purification step. Nanoparticle moieties conjugated to peptides/proteins were then trimmed by cleavage at the disulfide linker with a reducing agent. The labeled peptides were analyzed by LC-MS/MS to determine their sequences and the sites of NHS ester labeling. This novel approach allowed characterization of lysine residues on the solvent accessible surface of native bovine serum albumin. Low cost, rapid magnetic separation, and specificity towards primary amine groups make NHS ester coated Fe3O4@SiO2 nanoparticles a potential labeling probe to study proteins on living cell surfaces. PMID:23909594

  19. Cleavable ester linked magnetic nanoparticles for labeling of solvent exposed primary amine groups of peptides/proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In order to study the solvent exposed lysine residues of peptides/proteins, we previously reported disulfide linked N-hydrosuccinimide ester modified silica coated iron oxide magnetic nanoparticles (NHS-SS-SiO2@Fe3O4 MNPs). The presence of a disulfide bond in the linker limits the use of disulfide r...

  20. Convenient preparation of deuterium-labeled analogs of peptides containing N-substituted glycines for a stable isotope dilution LC-MS quantitative analysis.

    PubMed

    Bąchor, Remigiusz; Dębowski, Dawid; Łęgowska, Anna; Stefanowicz, Piotr; Rolka, Krzysztof; Szewczuk, Zbigniew

    2015-11-01

    N-substituted glycines constitute mimics of natural amino acids that are of great interest in the peptide-based drug development. Peptoids-oligo(N-substituted glycines) have been recently demonstrated to be highly active peptidomimetics in biological systems, resistant to proteolytic degradation. We developed a method of the deuterium labeling of peptidomimetics containing N-substituted glycine residues via H/D exchange of their α-carbon hydrogen atoms. The labeling was shown to be easy, inexpensive, and without the use of derivatization reagents or the need for a further purification. The deuterons introduced at the α-carbon atoms do not undergo a back exchange under acidic conditions during liquid chromatography mass spectrometry (LC-MS) analysis. The LC-MS analysis of a mixture of isotopologues revealed a co-elution of deuterated and nondeuterated forms of the peptidomimetics, which may be useful in the quantitative isotope dilution analysis of peptoids and other derivatives of N-substituted glycines. PMID:26415697

  1. Fischer carbene mediated covalent grafting of a peptide nucleic acid on gold surfaces and IR optical detection of DNA hybridization with a transition metalcarbonyl label

    NASA Astrophysics Data System (ADS)

    Srivastava, Pratima; Ghasemi, Mahsa; Ray, Namrata; Sarkar, Amitabha; Kocabova, Jana; Lachmanova, Stepanka; Hromadova, Magdalena; Boujday, Souhir; Cauteruccio, Silvia; Thakare, Pramod; Licandro, Emanuela; Fosse, Céline; Salmain, Michèle

    2016-11-01

    Amine-reactive surfaces comprising N-hydroxysuccinimide ester groups as well as much more unusual Fischer alkoxymetallocarbene groups were generated on gold-coated surfaces via self-assembled monolayers of carboxy- and azido-terminated thiolates, respectively. These functions were further used to immobilize homothymine peptide nucleic acid (PNA) decamer in a covalent fashion involving the primary amine located at its N-terminus. These stepwise processes were monitored by polarization modulation reflection - absorption infrared spectroscopy (PM-RAIRS) that gave useful information on the molecular composition of the organic layers. PNA grafting and hybridization with complementary DNA strand were successfully transduced by quartz crystal microbalance (QCM) measurements. Unfortunately, attempts to transduce the hybridization optically by IR in a label-free fashion were inconclusive. Therefore we undertook to introduce an IR reporter group, namely a transition metalcarbonyl (TMC) entity at the 5‧ terminus of complementary DNA. Evidence for the formation of PNA-DNA heteroduplex was brought by the presence of ν(Ctbnd O) bands in the 2000 cm-1 region of the IR spectrum of the gold surface owing to the metalcarbonyl label.

  2. Combining Near-UV Photodissociation with Electron Transfer. Reduction of the Diazirine Ring in a Photomethionine-Labeled Peptide Ion.

    PubMed

    Shaffer, Christopher J; Marek, Aleš; Nguyen, Huong T H; Tureček, František

    2015-08-01

    Electron transfer dissociation of peptide ions with the diazirine-containing residue photomethionine (M*) results in side-chain dissociations by loss of C3H7N2 radicals in addition to standard backbone cleavages. The side-chain dissociations are particularly prominent upon activation of long-lived, charge-reduced, cation radicals (GM*GGR + 2H)(+•). Investigation of these cation radicals by near-UV photodissociation and collisional activation revealed different fragmentation products and mechanisms resulting from these ion activation modes. The dissociations observed for photomethionine were dramatically different from those previously reported for the lower homologue photoleucine; here, a difference by a single methylene group in the side chain had a large effect on the chemistries of the cation radicals upon ETD and further activation. ETD intermediates and products were probed by tandem 355-nm UV photodissociation-collision induced dissociation and found to contain chromophores that resulted from electron attachment to the diazirine ring. The nature of the newly formed chromophores and ion energetics and kinetics were investigated by electron structure calculations combining ab initio and density functional theory methods and Rice-Ramsperger-Kassel-Marcus (RRKM) theory. The dramatic difference between the dissociations of L* and M* containing peptide cation radicals is explained by electronic effects that play a role in stabilizing critical reaction intermediates and steer the dissociations into kinetically favored reaction channels. In addition, a new alternating UVPD-ETD-UVPD MS(4) experiment is introduced and utilized for ion structure elucidation.

  3. 125I-labeled peptide mapping of some heat-modifiable proteins of the gonococcal outer membrane.

    PubMed Central

    Swanson, J

    1980-01-01

    Gonococci from opaque colonies have cell wall outer membrane proteins that are lacking from organisms which form transparent colonies. These "colony opacity-associated" proteins are among a group of "minor" proteins that exhibit heat modification of their apparent subunit molecular sizes, are easily extracted by deoxycholate, have apparent subunit molecular weights varying from 24,000 to 29,000 and are exposed on the surfaces of gonococci. Other minor proteins found on gonococci are the "leukocyte association proteins," whose presence correlates with reactivities of gonococci with human neutrophils. Several of the colony opacity-associated proteins and leukocyte association proteins were subjected to 125I-peptide mapping of protein bands separated by polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate. The structural similarities and differences among these heat-modifiable surface proteins were studied, as well as their similarities with the major protein of the gonococcal outer membrane. A relatively high apparent degree of structural homology is found among the heat-modifiable proteins from different strains of opaque colony gonococcal forms. There is also some apparent structural homology for 125I-peptides of heat-modifiable versus major proteins of the gonococcal outer membrane. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 PMID:6769820

  4. Gas-Phase Hydrogen-Deuterium Exchange Labeling of Select Peptide Ion Conformer Types: a Per-Residue Kinetics Analysis

    NASA Astrophysics Data System (ADS)

    Khakinejad, Mahdiar; Kondalaji, Samaneh Ghassabi; Tafreshian, Amirmahdi; Valentine, Stephen J.

    2015-07-01

    The per-residue, gas-phase hydrogen deuterium exchange (HDX) kinetics for individual amino acid residues on selected ion conformer types of the model peptide KKDDDDDIIKIIK have been examined using ion mobility spectrometry (IMS) and HDX-tandem mass spectrometry (MS/MS) techniques. The [M + 4H]4+ ions exhibit two major conformer types with collision cross sections of 418 Å2 and 446 Å2; the [M + 3H]3+ ions also yield two different conformer types having collision cross sections of 340 Å2 and 367 Å2. Kinetics plots of HDX for individual amino acid residues reveal fast- and slow-exchanging hydrogens. The contributions of each amino acid residue to the overall conformer type rate constant have been estimated. For this peptide, N- and C-terminal K residues exhibit the greatest contributions for all ion conformer types. Interior D and I residues show decreased contributions. Several charge state trends are observed. On average, the D residues of the [M + 3H]3+ ions show faster HDX rate contributions compared with [M + 4H]4+ ions. In contrast the interior I8 and I9 residues show increased accessibility to exchange for the more elongated [M + 4H]4+ ion conformer type. The contribution of each residue to the overall uptake rate showed a good correlation with a residue hydrogen accessibility score model calculated using a distance from charge site and initial incorporation site for nominal structures obtained from molecular dynamic simulations (MDS).

  5. A Label-Free Electrochemical Impedance Cytosensor Based on Specific Peptide-Fused Phage Selected from Landscape Phage Library.

    PubMed

    Han, Lei; Liu, Pei; Petrenko, Valery A; Liu, Aihua

    2016-02-24

    One of the major challenges in the design of biosensors for cancer diagnosis is to introduce a low-cost and selective probe that can recognize cancer cells. In this paper, we combined the phage display technology and electrochemical impedance spectroscopy (EIS) to develop a label-free cytosensor for the detection of cancer cells, without complicated purification of recognition elements. Fabrication steps of the cytosensing interface were monitored by EIS. Due to the high specificity of the displayed octapeptides and avidity effect of their multicopy display on the phage scaffold, good biocompatibility of recombinant phage, the fibrous nanostructure of phage, and the inherent merits of EIS technology, the proposed cytosensor demonstrated a wide linear range (2.0 × 10(2) - 2.0 × 10(8) cells mL(-1)), a low limit of detection (79 cells mL(-1), S/N = 3), high specificity, good inter-and intra-assay reproducibility and satisfactory storage stability. This novel cytosensor designing strategy will open a new prospect for rapid and label-free electrochemical platform for tumor diagnosis.

  6. A Label-Free Electrochemical Impedance Cytosensor Based on Specific Peptide-Fused Phage Selected from Landscape Phage Library

    NASA Astrophysics Data System (ADS)

    Han, Lei; Liu, Pei; Petrenko, Valery A.; Liu, Aihua

    2016-02-01

    One of the major challenges in the design of biosensors for cancer diagnosis is to introduce a low-cost and selective probe that can recognize cancer cells. In this paper, we combined the phage display technology and electrochemical impedance spectroscopy (EIS) to develop a label-free cytosensor for the detection of cancer cells, without complicated purification of recognition elements. Fabrication steps of the cytosensing interface were monitored by EIS. Due to the high specificity of the displayed octapeptides and avidity effect of their multicopy display on the phage scaffold, good biocompatibility of recombinant phage, the fibrous nanostructure of phage, and the inherent merits of EIS technology, the proposed cytosensor demonstrated a wide linear range (2.0 × 102 ‑ 2.0 × 108 cells mL‑1), a low limit of detection (79 cells mL‑1, S/N = 3), high specificity, good inter-and intra-assay reproducibility and satisfactory storage stability. This novel cytosensor designing strategy will open a new prospect for rapid and label-free electrochemical platform for tumor diagnosis.

  7. A Label-Free Electrochemical Impedance Cytosensor Based on Specific Peptide-Fused Phage Selected from Landscape Phage Library

    PubMed Central

    Han, Lei; Liu, Pei; Petrenko, Valery A.; Liu, Aihua

    2016-01-01

    One of the major challenges in the design of biosensors for cancer diagnosis is to introduce a low-cost and selective probe that can recognize cancer cells. In this paper, we combined the phage display technology and electrochemical impedance spectroscopy (EIS) to develop a label-free cytosensor for the detection of cancer cells, without complicated purification of recognition elements. Fabrication steps of the cytosensing interface were monitored by EIS. Due to the high specificity of the displayed octapeptides and avidity effect of their multicopy display on the phage scaffold, good biocompatibility of recombinant phage, the fibrous nanostructure of phage, and the inherent merits of EIS technology, the proposed cytosensor demonstrated a wide linear range (2.0 × 102 − 2.0 × 108 cells mL−1), a low limit of detection (79 cells mL−1, S/N = 3), high specificity, good inter-and intra-assay reproducibility and satisfactory storage stability. This novel cytosensor designing strategy will open a new prospect for rapid and label-free electrochemical platform for tumor diagnosis. PMID:26908277

  8. A Label-Free Electrochemical Impedance Cytosensor Based on Specific Peptide-Fused Phage Selected from Landscape Phage Library.

    PubMed

    Han, Lei; Liu, Pei; Petrenko, Valery A; Liu, Aihua

    2016-01-01

    One of the major challenges in the design of biosensors for cancer diagnosis is to introduce a low-cost and selective probe that can recognize cancer cells. In this paper, we combined the phage display technology and electrochemical impedance spectroscopy (EIS) to develop a label-free cytosensor for the detection of cancer cells, without complicated purification of recognition elements. Fabrication steps of the cytosensing interface were monitored by EIS. Due to the high specificity of the displayed octapeptides and avidity effect of their multicopy display on the phage scaffold, good biocompatibility of recombinant phage, the fibrous nanostructure of phage, and the inherent merits of EIS technology, the proposed cytosensor demonstrated a wide linear range (2.0 × 10(2) - 2.0 × 10(8) cells mL(-1)), a low limit of detection (79 cells mL(-1), S/N = 3), high specificity, good inter-and intra-assay reproducibility and satisfactory storage stability. This novel cytosensor designing strategy will open a new prospect for rapid and label-free electrochemical platform for tumor diagnosis. PMID:26908277

  9. Isolation and sequencing of an active-site peptide from Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase after affinity labeling with 2-((Bromoacetyl)amino)pentitol 1,5-bisphosphate

    SciTech Connect

    Fraij, B.; Hartman, F.C.

    1983-01-01

    2-((Bromoacetyl)amino)pentitol 1,5-bisphosphate was reported to be a highly selective affinity label for ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum. The enzyme has now been inactivated with a /sup 14/C-labeled reagent in order to identify the target residue at the sequence level. Subsequent to inactivation, the enzyme was carboxymethylated with iodoacetate and then digested with trypsin. The only radioactive peptide in the digest was obtained at a high degree of purity by successive chromatography on DEAE-cellulose, SP-Sephadex, and Sephadex G-25. On the basis of amino acid analysis of the purified peptide, the derivatized residue was a methionyl sulfonium salt. Automated Edman degradation confirmed the purity of the labeled peptide and established its sequence as Leu-Gln-Gly-Ala-Ser-Gly-Ile-His-Thr-Gly-Thr-Met-Gly-Phe-Gly-Lys-Met-Glu-Gly-Glu-Ser-Ser-Asp-Arg. Cleavage of this peptide with cyanogen bromide showed that the reagent moiety was covalently attached to the second methionyl residue. Sequence homology with the carboxylase/oxygenase from spinach indicates that the lysyl residue immediately preceding the alkylated methionine corresponds to Lys-334, a residue previously implicated at the active site. 31 references, 4 figures, 3 tables.

  10. High affinity receptor labeling based on basic leucine zipper domain peptides conjugated with pH-sensitive fluorescent dye: Visualization of AMPA-type glutamate receptor endocytosis in living neurons.

    PubMed

    Hayashi, Ayako; Asanuma, Daisuke; Kamiya, Mako; Urano, Yasuteru; Okabe, Shigeo

    2016-01-01

    Techniques to visualize receptor trafficking in living neurons are important, but currently available methods are limited in their labeling efficiency, specificity and reliability. Here we report a method for receptor labeling with a basic leucine zipper domain peptide (ZIP) and a binding cassette specific to ZIP. Receptors are tagged with a ZIP-binding cassette at their extracellular domain. Tagged receptors expressed in cultured cells were labeled with exogenously applied fluorescently labeled ZIP with low background and high affinity. To test if ZIP labeling is useful in monitoring endocytosis and intracellular trafficking, we next conjugated ZIP with a pH-sensitive dye RhP-M (ZIP-RhP-M). ZIP binding to its binding cassette was pH-resistant and RhP-M fluorescence dramatically increased in acidic environment. Thus AMPA-type glutamate receptors (AMPARs) labeled by ZIP-RhP-M can report receptor endocytosis and subsequent intracellular trafficking. Application of ZIP-RhP-M to cultured hippocampal neurons expressing AMPARs tagged with a ZIP-binding cassette resulted in appearance of fluorescent puncta in PSD-95-positive large spines, suggesting local endocytosis and acidification of AMPARs in individual mature spines. This spine pool of AMPARs in acidic environment was distinct from the early endosomes labeled by transferrin uptake. These results suggest that receptor labeling by ZIP-RhP-M is a useful technique for monitoring endocytosis and intracellular trafficking. This article is part of the Special Issue entitled 'Synaptopathy--from Biology to Therapy'.

  11. Imaging the Met Receptor Tyrosine Kinase (Met) and Assessing Tumor Responses to a Met Tyrosine Kinase Inhibitor in Human Xenograft Mouse Models with a [99mTc] (AH-113018) or Cy 5** (AH-112543) Labeled Peptide.

    PubMed

    Jagoda, Elaine M; Bhattacharyya, Sibaprasad; Kalen, Joseph; Riffle, Lisa; Leeder, Avrum; Histed, Stephanie; Williams, Mark; Wong, Karen J; Xu, Biying; Szajek, Lawrence P; Elbuluk, Osama; Cecchi, Fabiola; Raffensperger, Kristen; Golla, Meghana; Bottaro, Donald P; Choyke, Peter

    2015-01-01

    Developing an imaging agent targeting the hepatocyte growth factor receptor protein (Met) status of cancerous lesions would aid in the diagnosis and monitoring of Met-targeted tyrosine kinase inhibitors (TKIs). A peptide targeting Met labeled with [(99m)Tc] had high affinity in vitro (Kd = 3.3 nM) and detected relative changes in Met in human cancer cell lines. In vivo [(99m)Tc]-Met peptide (AH-113018) was retained in Met-expressing tumors, and high-expressing Met tumors (MKN-45) were easily visualized and quantitated using single-photon emission computed tomography or optical imaging. In further studies, MKN-45 mouse xenografts treated with PHA 665752 (Met TKI) or vehicle were monitored weekly for tumor responses by [(99m)Tc]-Met peptide imaging and measurement of tumor volumes. Tumor uptake of [(99m)Tc]-Met peptide was significantly decreased as early as 1 week after PHA 665752 treatment, corresponding to decreases in tumor volumes. These results were comparable to Cy5**-Met peptide (AH-112543) fluorescence imaging using the same treatment model. [(99m)Tc] or Cy5**-Met peptide tumor uptake was further validated by histologic (necrosis, apoptosis) and immunoassay (total Met, p Met, and plasma shed Met) assessments in imaged and nonimaged cohorts. These data suggest that [(99m)Tc] or Cy5**-Met peptide imaging may have clinical diagnostic, prognostic, and therapeutic monitoring applications. PMID:26461980

  12. Allogeneic/xenogeneic transplantation of peptide-labeled mitochondria in Parkinson's disease: restoration of mitochondria functions and attenuation of 6-hydroxydopamine-induced neurotoxicity.

    PubMed

    Chang, Jui-Chih; Wu, Shey-Lin; Liu, Ko-Hung; Chen, Ya-Hui; Chuang, Chieh-Sen; Cheng, Fu-Chou; Su, Hong-Lin; Wei, Yau-Huei; Kuo, Shou-Jen; Liu, Chin-San

    2016-04-01

    Although restoration of mitochondrial function in mitochondrial diseases through peptide-mediated allogeneic mitochondrial delivery (PMD) has been demonstrated in vitro, the in vivo therapeutic efficacy of PMD in Parkinson's disease (PD) has yet to be determined. In this study, we compared the functionality of mitochondrial transfer with or without Pep-1 conjugation in neurotoxin (6-hydroxydopamine, 6-OHDA)-induced PC12 cells and PD rat models. We injected mitochondria into the medial forebrain bundle (MFB) of the PD rats after subjecting the nigrostriatal pathway to a unilateral 6-OHDA lesion for 21 days, and we verified the effectiveness of the mitochondrial graft in enhancing mitochondrial function in the soma of the substantia nigra (SN) neuron through mitochondrial transport dynamics in the nigrostriatal circuit. The result demonstrated that only PMD with allogeneic and xenogeneic sources significantly sustained mitochondrial function to resist the neurotoxin-induced oxidative stress and apoptotic death in the rat PC12 cells. The remaining cells exhibited a greater capability of neurite outgrowth. Furthermore, allogeneic and xenogeneic transplantation of peptide-labeled mitochondria after 3 months improved the locomotive activity in the PD rats. This increase was accompanied by a marked decrease in dopaminergic neuron loss in the substantia nigra pars compacta (SNc) and consistent enhancement of tyrosine hydroxylase-positive immunoreaction of dopaminergic neurons in the SNc and striatum. We also observed that in the SN dopaminergic neuron in the treated PD rats, mitochondrial complex I protein and mitochondrial dynamics were restored, thus ameliorating the oxidative DNA damage. Moreover, we determined signal translocation of graft allogeneic mitochondria from the MFB to the calbindin-positive SN neuron, which demonstrated the regulatory role of mitochondrial transport in alleviating 6-OHDA-induced degeneration of dopaminergic neurons. PMID:26730494

  13. Allogeneic/xenogeneic transplantation of peptide-labeled mitochondria in Parkinson's disease: restoration of mitochondria functions and attenuation of 6-hydroxydopamine-induced neurotoxicity.

    PubMed

    Chang, Jui-Chih; Wu, Shey-Lin; Liu, Ko-Hung; Chen, Ya-Hui; Chuang, Chieh-Sen; Cheng, Fu-Chou; Su, Hong-Lin; Wei, Yau-Huei; Kuo, Shou-Jen; Liu, Chin-San

    2016-04-01

    Although restoration of mitochondrial function in mitochondrial diseases through peptide-mediated allogeneic mitochondrial delivery (PMD) has been demonstrated in vitro, the in vivo therapeutic efficacy of PMD in Parkinson's disease (PD) has yet to be determined. In this study, we compared the functionality of mitochondrial transfer with or without Pep-1 conjugation in neurotoxin (6-hydroxydopamine, 6-OHDA)-induced PC12 cells and PD rat models. We injected mitochondria into the medial forebrain bundle (MFB) of the PD rats after subjecting the nigrostriatal pathway to a unilateral 6-OHDA lesion for 21 days, and we verified the effectiveness of the mitochondrial graft in enhancing mitochondrial function in the soma of the substantia nigra (SN) neuron through mitochondrial transport dynamics in the nigrostriatal circuit. The result demonstrated that only PMD with allogeneic and xenogeneic sources significantly sustained mitochondrial function to resist the neurotoxin-induced oxidative stress and apoptotic death in the rat PC12 cells. The remaining cells exhibited a greater capability of neurite outgrowth. Furthermore, allogeneic and xenogeneic transplantation of peptide-labeled mitochondria after 3 months improved the locomotive activity in the PD rats. This increase was accompanied by a marked decrease in dopaminergic neuron loss in the substantia nigra pars compacta (SNc) and consistent enhancement of tyrosine hydroxylase-positive immunoreaction of dopaminergic neurons in the SNc and striatum. We also observed that in the SN dopaminergic neuron in the treated PD rats, mitochondrial complex I protein and mitochondrial dynamics were restored, thus ameliorating the oxidative DNA damage. Moreover, we determined signal translocation of graft allogeneic mitochondria from the MFB to the calbindin-positive SN neuron, which demonstrated the regulatory role of mitochondrial transport in alleviating 6-OHDA-induced degeneration of dopaminergic neurons.

  14. Label-free DNA biosensor based on a peptide nucleic acid-functionalized microstructured optical fiber-Bragg grating

    NASA Astrophysics Data System (ADS)

    Candiani, Alessandro; Bertucci, Alessandro; Giannetti, Sara; Konstantaki, Maria; Manicardi, Alex; Pissadakis, Stavros; Cucinotta, Annamaria; Corradini, Roberto; Selleri, Stefano

    2013-05-01

    We describe a novel sensing approach based on a functionalized microstructured optical fiber-Bragg grating for specific DNA target sequences detection. The inner surface of a microstructured fiber, where a Bragg grating was previously inscribed, has been functionalized by covalent linking of a peptide nucleic acid probe targeting a DNA sequence bearing a single point mutation implicated in cystic fibrosis (CF) disease. A solution of an oligonucleotide (ON) corresponding to a tract of the CF gene containing the mutated DNA has been infiltrated inside the fiber capillaries and allowed to hybridize to the fiber surface according to the Watson-Crick pairing. In order to achieve signal amplification, ON-functionalized gold nanoparticles were then infiltrated and used in a sandwich-like assay. Experimental measurements show a clear shift of the reflected high order mode of a Bragg grating for a 100 nM DNA solution, and fluorescence measurements have confirmed the successful hybridization. Several experiments have been carried out on the same fiber using the identical concentration, showing the same modulation trend, suggesting the possibility of the reuse of the sensor. Measurements have also been made using a 100 nM mismatched DNA solution, containing a single nucleotide mutation and corresponding to the wild-type gene, and the results demonstrate the high selectivity of the sensor.

  15. Improved PET Imaging of uPAR Expression Using new 64Cu-labeled Cross-Bridged Peptide Ligands: Comparative in vitro and in vivo Studies

    PubMed Central

    Persson, Morten; Hosseini, Masood; Madsen, Jacob; Jørgensen, Thomas J. D.; Jensen, Knud J; Kjaer, Andreas; Ploug, Michael

    2013-01-01

    The correlation between uPAR expression, cancer cell invasion and metastases is now well-established and has prompted the development of a number of uPAR PET imaging agents, which could potentially identify cancer patients with invasive and metastatic lesions. In the present study, we synthesized and characterized two new cross-bridged 64Cu-labeled peptide conjugates for PET imaging of uPAR and performed a head-to-head comparison with the corresponding and more conventionally used DOTA conjugate. Based on in-source laser-induced reduction of chelated Cu(II) to Cu(I), we now demonstrate the following ranking with respect to the chemical inertness of their complexed Cu ions: DOTA-AE105 << CB-TE2A-AE105 < CB-TE2A-PA-AE105, which is correlated to their corresponding demetallation rate. No penalty in the uPAR receptor binding affinity of the targeting peptide was encountered by conjugation to either of the macrobicyclic chelators (IC50 ~ 5-10 nM) and high yields and radiochemical purities (>95%) were achieved in all cases by incubation at 95ºC. In vivo, they display identical tumor uptake after 1h, but differ significantly after 22 hrs, where the DOTA-AE105 uptake remains surprisingly high. Importantly, the more stable of the new uPAR PET tracers, 64Cu-CB-TE2A-PA-AE105, exhibits a significantly reduced liver uptake compared to 64Cu-DOTA-AE105 as well as 64Cu-CB-TE2A-AE105, (p<0.0001), emphasizing that our new in vitro stability measurements by mass spectrometry predicts in vivo stability in mice. Specificity of the best performing ligand, 64Cu-CB-TE2A-PA-AE105 was finally confirmed in vivo using a non-binding 64Cu-labeled peptide as control (64Cu-CB-TE2A-PA-AE105mut). This control PET-tracer revealed significantly reduced tumor uptake (p<0.0001), but identical hepatic uptake compared to its active counterpart (64Cu-CB-TE2A-PA-AE105) after 1h. In conclusion, our new approach using in-source laser-induced reduction of Cu(II)-chelated PET-ligands provides useful

  16. Characterization of a benzyladenine binding-site peptide isolated from a wheat cytokinin-binding protein: Sequence analysis and identification of a single affinity-labeled histidine residue by mass spectrometry

    SciTech Connect

    Brinegar, A.C.; Cooper, G.; Stevens, A.; Hauer, C.R.; Shabanowitz, J.; Hunt, D.F.; Fox, J.E. )

    1988-08-01

    A wheat embryo cytokinin-binding protein was covalently modified with the radiolabeled photoaffinity ligand 2-azido-N{sup 6}-({sup 14}C)benzyladenine. A single labeled peptide was obtained after proteolytic digestion and isolation by reversed-phase and anion-exchange HPLC. Sequencing by classical Edman degradation identified 11 of the 12 residues but failed to identify the labeled amino acid. Analysis by laser photodissociation Fourier-transform mass spectrometry of 10 pmol of the peptide independently confirmed the Edman data and also demonstrated that the histidine residue nearest the C terminus (underlined) was modified by the reagent in the sequence Ala-Phe-Leu-Gln-Pro-Ser-His-His{und His}-Asp-Ala-Asp-Glu.

  17. Fragmentation of doubly-protonated peptide ion populations labeled by H/D exchange with CD3OD

    NASA Astrophysics Data System (ADS)

    Herrmann, Kristin A.; Kuppannan, Krishna; Wysocki, Vicki H.

    2006-03-01

    Doubly-protonated bradykinin (RPPGFSPFR) and an angiotensin III analogue (RVYIFPF) were subjected to hydrogen/deuterium (H/D) exchange with CD3OD in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. A bimodal distribution of deuterium incorporation was present for bradykinin after H/D exchange for 90 s at a CD3OD pressure of 4 × 10-7 Torr, indicating the existence of at least two distinct populations. Bradykinin ion populations corresponding to 0-2 and 5-11 deuteriums (i.e., D0, D1, D2, D5, D6, D7, D8, D9, D10, and D11) were each monoisotopically selected and fragmented via sustained off-resonance irradiation (SORI) collision-induced dissociation (CID). The D0-D2 ion populations, which correspond to the slower exchanging population, consistently require lower SORI amplitude to achieve a similar precursor ion survival yield as the faster-reacting (D5-D11) populations. These results demonstrate that conformation/protonation motif has an effect on fragmentation efficiency for bradykinin. Also, the partitioning of the deuterium atoms into fragment ions suggests that the C-terminal arginine residue exchanges more rapidly than the N-terminal arginine. Total deuterium incorporation in the b1/y8 and b2/y7 ion pairs matches very closely the theoretical values for all ion populations studied, indicating that the ions of a complementary pair are likely formed during the same fragmentation event, or that no scrambling occurs upon SORI. Deuterium incorporation into the y1/a8 pseudo-ion pair does not closely match the expected theoretical values. The other peptide, doubly-protonated RVYIFPF, has a trimodal distribution of deuterium incorporation upon H/D exchange with CD3OD at a pressure of 1 × 10-7 Torr for 600 s, indicating at least three distinct ion populations. After 90 s of H/D exchange where at least two distinct populations are detected, the D0-D7 ion populations were monoisotopically selected and fragmented via SORI-CID over a range of SORI

  18. Characterizing the Epothilone Binding Site on β-Tubulin by Photoaffinity Labeling: Identification of β-Tubulin Peptides TARGSQQY and TSRGSQQY as Targets of an Epothilone Photoprobe for Polymerized Tubulin.

    PubMed

    Ranade, Adwait R; Higgins, LeeAnn; Markowski, Todd W; Glaser, Nicole; Kashin, Dmitry; Bai, Ruoli; Hong, Kwon Ho; Hamel, Ernest; Höfle, Gerhard; Georg, Gunda I

    2016-04-14

    Photoaffinity labeling with an epothilone A photoprobe led to the identification of the β-tubulin peptides TARGSQQY and TSRGSQQY as targets of the photoprobe for polymerized tubulin. These peptides represent residues 274-281 in different β-tubulin isotypes. Placing the carbene producing 21-diazo/triazolo moiety of the photoprobe in the vicinity of the TARGSQQY peptide in a homology model of TBB3 predicted a binding pose and conformation of the photoprobe that are very similar to the ones reported for 1) the high resolution cocrystal structure of epothilone A with an α,β-tubulin complex and for 2) a saturation transfer difference NMR and transferred NOESY NMR study of dimeric and polymerized tubulin. Our findings thus provide additional support for these models as physiologically the most relevant among several modes of binding that have been proposed for epothilone A in the taxane pocket of β-tubulin.

  19. Characterizing the Epothilone Binding Site on β-Tubulin by Photoaffinity Labeling: Identification of β-Tubulin Peptides TARGSQQY and TSRGSQQY as Targets of an Epothilone Photoprobe for Polymerized Tubulin.

    PubMed

    Ranade, Adwait R; Higgins, LeeAnn; Markowski, Todd W; Glaser, Nicole; Kashin, Dmitry; Bai, Ruoli; Hong, Kwon Ho; Hamel, Ernest; Höfle, Gerhard; Georg, Gunda I

    2016-04-14

    Photoaffinity labeling with an epothilone A photoprobe led to the identification of the β-tubulin peptides TARGSQQY and TSRGSQQY as targets of the photoprobe for polymerized tubulin. These peptides represent residues 274-281 in different β-tubulin isotypes. Placing the carbene producing 21-diazo/triazolo moiety of the photoprobe in the vicinity of the TARGSQQY peptide in a homology model of TBB3 predicted a binding pose and conformation of the photoprobe that are very similar to the ones reported for 1) the high resolution cocrystal structure of epothilone A with an α,β-tubulin complex and for 2) a saturation transfer difference NMR and transferred NOESY NMR study of dimeric and polymerized tubulin. Our findings thus provide additional support for these models as physiologically the most relevant among several modes of binding that have been proposed for epothilone A in the taxane pocket of β-tubulin. PMID:26986898

  20. Biological evaluation of (177)Lu-labeled DOTA-Ala(SO3H)-Aminooctanoyl-Gln-Trp-Ala-Val-N methyl Gly-His-Statine-Leu-NH2 for gastrin-releasing peptide receptor-positive prostate tumor targeting.

    PubMed

    Lim, Jae Cheong; Cho, Eun Ha; Kim, Jin Joo; Choi, Sang Mu; Lee, So young; Nam, Sung Soo; Park, Ul Jae; Park, Soo Hyun

    2015-02-01

    Bombesin binds with selectivity and high affinity to a Gastrin-releasing peptide receptor (GRPR), which is highly overexpressed in prostate cancer cells. The present study describes the in vitro and in vivo biological characteristics of DOTA-Ala(SO3H)-Aminooctanoyl-Gln-Trp-Ala-Val-N methyl Gly-His-Statine-Leu-NH2 (DOTA-sBBNA), an antagonist analogue of bombesin peptide for the targeting of GRPR. DOTA-sBBNA was synthesized and labeled with (177)Lu as previously published. A saturation assay on PC-3 human prostate cancer cells revealed that the Kd value of the radiolabeled peptide was 1.88 nM with a maximum binding capacity (Bmax) of 289.3 fmol/10(6) cells. The radio-peptide slowly internalized, and 24.4±0.5% of the total binding was internalized in 4hr. Biodistribution studies were conducted in healthy and PC-3 xenografted balb/c mice, which showed high uptake and retention of tumor-associated radioactivity in PC-3 xenografted mice. The tumor-to-blood ratio was 126.02±9.36 at 1.5hr p.i., and was increased to 216.33±61.58 at 24hr p.i., which means that the radiolabeled peptide was highly accumulated in a tumor and rapidly cleared from the blood pool. The GRPR is also over-expressed in Korean prostate cancer patients. These results suggest that this (177)Lu-labeled peptide has promising characteristics for application in nuclear medicine, namely for the diagnosis and treatment of GRPR over-expressing prostate tumors.

  1. Biological evaluation of (177)Lu-labeled DOTA-Ala(SO3H)-Aminooctanoyl-Gln-Trp-Ala-Val-N methyl Gly-His-Statine-Leu-NH2 for gastrin-releasing peptide receptor-positive prostate tumor targeting.

    PubMed

    Lim, Jae Cheong; Cho, Eun Ha; Kim, Jin Joo; Choi, Sang Mu; Lee, So young; Nam, Sung Soo; Park, Ul Jae; Park, Soo Hyun

    2015-02-01

    Bombesin binds with selectivity and high affinity to a Gastrin-releasing peptide receptor (GRPR), which is highly overexpressed in prostate cancer cells. The present study describes the in vitro and in vivo biological characteristics of DOTA-Ala(SO3H)-Aminooctanoyl-Gln-Trp-Ala-Val-N methyl Gly-His-Statine-Leu-NH2 (DOTA-sBBNA), an antagonist analogue of bombesin peptide for the targeting of GRPR. DOTA-sBBNA was synthesized and labeled with (177)Lu as previously published. A saturation assay on PC-3 human prostate cancer cells revealed that the Kd value of the radiolabeled peptide was 1.88 nM with a maximum binding capacity (Bmax) of 289.3 fmol/10(6) cells. The radio-peptide slowly internalized, and 24.4±0.5% of the total binding was internalized in 4hr. Biodistribution studies were conducted in healthy and PC-3 xenografted balb/c mice, which showed high uptake and retention of tumor-associated radioactivity in PC-3 xenografted mice. The tumor-to-blood ratio was 126.02±9.36 at 1.5hr p.i., and was increased to 216.33±61.58 at 24hr p.i., which means that the radiolabeled peptide was highly accumulated in a tumor and rapidly cleared from the blood pool. The GRPR is also over-expressed in Korean prostate cancer patients. These results suggest that this (177)Lu-labeled peptide has promising characteristics for application in nuclear medicine, namely for the diagnosis and treatment of GRPR over-expressing prostate tumors. PMID:25457455

  2. Feasibility of 68Ga-labeled Siglec-9 peptide for the imaging of acute lung inflammation: a pilot study in a porcine model of acute respiratory distress syndrome

    PubMed Central

    Retamal, Jaime; Sörensen, Jens; Lubberink, Mark; Suarez-Sipmann, Fernando; Borges, João Batista; Feinstein, Ricardo; Jalkanen, Sirpa; Antoni, Gunnar; Hedenstierna, Göran; Roivainen, Anne; Larsson, Anders; Velikyan, Irina

    2016-01-01

    There is an unmet need for noninvasive, specific and quantitative imaging of inherent inflammatory activity. Vascular adhesion protein-1 (VAP-1) translocates to the luminal surface of endothelial cells upon inflammatory challenge. We hypothesized that in a porcine model of acute respiratory distress syndrome (ARDS), positron emission tomography (PET) with sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) based imaging agent targeting VAP-1 would allow quantification of regional pulmonary inflammation. ARDS was induced by lung lavages and injurious mechanical ventilation. Hemodynamics, respiratory system compliance (Crs) and blood gases were monitored. Dynamic examination using [15O]water PET-CT (10 min) was followed by dynamic (90 min) and whole-body examination using VAP-1 targeting 68Ga-labeled 1,4,7,10-tetraaza cyclododecane-1,4,7-tris-acetic acid-10-ethylene glycol-conjugated Siglec-9 motif peptide ([68Ga]Ga-DOTA-Siglec-9). The animals received an anti-VAP-1 antibody for post-mortem immunohistochemistry assay of VAP-1 receptors. Tissue samples were collected post-mortem for the radioactivity uptake, histology and immunohistochemistry assessment. Marked reduction of oxygenation and Crs, and higher degree of inflammation were observed in ARDS animals. [68Ga]Ga-DOTA-Siglec-9 PET showed significant uptake in lungs, kidneys and urinary bladder. Normalization of the net uptake rate (Ki) for the tissue perfusion resulted in 4-fold higher uptake rate of [68Ga]Ga-DOTA-Siglec-9 in the ARDS lungs. Immunohistochemistry showed positive VAP-1 signal in the injured lungs. Detection of pulmonary inflammation associated with a porcine model of ARDS was possible with [68Ga]Ga-DOTA-Siglec-9 PET when using kinetic modeling and normalization for tissue perfusion. PMID:27069763

  3. Feasibility of (68)Ga-labeled Siglec-9 peptide for the imaging of acute lung inflammation: a pilot study in a porcine model of acute respiratory distress syndrome.

    PubMed

    Retamal, Jaime; Sörensen, Jens; Lubberink, Mark; Suarez-Sipmann, Fernando; Borges, João Batista; Feinstein, Ricardo; Jalkanen, Sirpa; Antoni, Gunnar; Hedenstierna, Göran; Roivainen, Anne; Larsson, Anders; Velikyan, Irina

    2016-01-01

    There is an unmet need for noninvasive, specific and quantitative imaging of inherent inflammatory activity. Vascular adhesion protein-1 (VAP-1) translocates to the luminal surface of endothelial cells upon inflammatory challenge. We hypothesized that in a porcine model of acute respiratory distress syndrome (ARDS), positron emission tomography (PET) with sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) based imaging agent targeting VAP-1 would allow quantification of regional pulmonary inflammation. ARDS was induced by lung lavages and injurious mechanical ventilation. Hemodynamics, respiratory system compliance (Crs) and blood gases were monitored. Dynamic examination using [(15)O]water PET-CT (10 min) was followed by dynamic (90 min) and whole-body examination using VAP-1 targeting (68)Ga-labeled 1,4,7,10-tetraaza cyclododecane-1,4,7-tris-acetic acid-10-ethylene glycol-conjugated Siglec-9 motif peptide ([(68)Ga]Ga-DOTA-Siglec-9). The animals received an anti-VAP-1 antibody for post-mortem immunohistochemistry assay of VAP-1 receptors. Tissue samples were collected post-mortem for the radioactivity uptake, histology and immunohistochemistry assessment. Marked reduction of oxygenation and Crs, and higher degree of inflammation were observed in ARDS animals. [(68)Ga]Ga-DOTA-Siglec-9 PET showed significant uptake in lungs, kidneys and urinary bladder. Normalization of the net uptake rate (Ki) for the tissue perfusion resulted in 4-fold higher uptake rate of [(68)Ga]Ga-DOTA-Siglec-9 in the ARDS lungs. Immunohistochemistry showed positive VAP-1 signal in the injured lungs. Detection of pulmonary inflammation associated with a porcine model of ARDS was possible with [(68)Ga]Ga-DOTA-Siglec-9 PET when using kinetic modeling and normalization for tissue perfusion. PMID:27069763

  4. Rapid label-free visual assay for the detection and quantification of viral RNA using peptide nucleic acid (PNA) and gold nanoparticles (AuNPs).

    PubMed

    Joshi, Vinay G; Chindera, Kantaraja; Singh, Arvind Kumar; Sahoo, Aditya P; Dighe, Vikas D; Thakuria, Dimpal; Tiwari, Ashok K; Kumar, Satish

    2013-09-17

    A rapid label-free visual assay for the detection of viral RNA using peptide nucleic acid (PNA) probes and gold nanoparticles (AuNPs) is presented in this study. Diagnosis is a crucial step for the molecular surveillance of diseases, and a rapid visual test with high specificity could play a vital role in the management of viral diseases. In this assay, the specific agglomerative behavior of PNA with gold nanoparticles was manipulated by its complementation with viral RNA. The assay was able to detect 5-10 ng of viral RNA from various biological samples, such as allantoic fluids, cell culture fluids and vaccines, in 100 μl of test solution. The developed assay was more sensitive than a hemagglutination (HA) test, a routine platform test for the detection of Newcastle disease virus (NDV), and the developed assay was able to visually detect NDV with as little as 0.25 HA units of virus. In terms of the specificity, the test could discriminate single nucleotide differences in the target RNA and hence could provide visual viral genotyping/pathotyping. This observation was confirmed by pathotyping different known isolates of NDV. Further, the PNA-induced colorimetric changes in the presence of the target RNA at different RNA to PNA ratios yielded a standard curve with a linear coefficient of R(2)=0.990, which was comparable to the value of R(2)=0.995 from real-time PCR experiments with the same viral RNA. Therefore, the viral RNA in a given samples could be quantified using a simple visual spectrophotometer available in any clinical laboratory. This assay may find application in diagnostic assays for other RNA viruses, which are well known to undergo mutations, thus presenting challenges for their molecular surveillance, genotyping and quantification.

  5. Peptide receptor radionuclide therapy of treatment-refractory metastatic thyroid cancer using 90Yttrium and 177Lutetium labeled somatostatin analogs: toxicity, response and survival analysis

    PubMed Central

    Budiawan, Hendra; Salavati, Ali; Kulkarni, Harshad R; Baum, Richard P

    2014-01-01

    The overall survival rate of non-radioiodine avid differentiated (follicular, papillary, medullary) thyroid carcinoma is significantly lower than for patients with iodine-avid lesions. The purpose of this study was to evaluate toxicity and efficacy (response and survival) of peptide receptor radionuclide therapy (PRRT) in non-radioiodine-avid or radioiodine therapy refractory thyroid cancer patients. Sixteen non-radioiodine-avid and/or radioiodine therapy refractory thyroid cancer patients, including follicular thyroid carcinoma (n = 4), medullary thyroid carcinoma (n = 8), Hürthle cell thyroid carcinoma (n = 3), and mixed carcinoma (n = 1) were treated with PRRT by using 90Yttrium and/or 177Lutetium labeled somatostatin analogs. 68Ga somatostatin receptor PET/CT was used to determine the somatostatin receptor density in the residual tumor/metastatic lesions and to assess the treatment response. Hematological profiles and renal function were periodically examined after treatment. By using fractionated regimen, only mild, reversible hematological toxicity (grade 1) or nephrotoxicity (grade 1) were seen. Response assessment (using EORTC criteria) was performed in 11 patients treated with 2 or more (maximum 5) cycles of PRRT and showed disease stabilization in 4 (36.4%) patients. Two patients (18.2%) showed partial remission, in the remaining 5 patients (45.5%) disease remained progressive. Kaplan-Meier analysis resulted in a mean survival after the first PRRT of 4.2 years (95% CI, range 2.9-5.5) and median progression free survival of 25 months (inter-quartiles: 12-43). In non-radioiodine-avid/radioiodine therapy refractory thyroid cancer patients, PRRT is a promising therapeutic option with minimal toxicity, good response rate and excellent survival benefits. PMID:24380044

  6. Therapeutic Efficacy of a {sup 188}Re-Labeled {alpha}-Melanocyte-Stimulating Hormone Peptide Analog in Murine and Human Melanoma-Bearing Mouse Models

    SciTech Connect

    Miao, Yubin; Owen, Nellie K.; Fisher, Darrell R.; Hoffman, Timothy J.; Quinn, Thomas P.

    2005-01-01

    The purpose of this study was to examine the therapeutic efficacy of {sup 188}Re-(Arg{sup 11})CCMSH in the B16/F1 murine melanoma and TXM13 human melanoma bearing mouse models. Method: (Arg11)CCMSH was synthesized and labeled with {sup 188}Re to form {sup 188}Re-(Agr{sup 11})CCMSH. B16/F1 melanoma tumor bearing mice were administrated with 200 Ci, 600 Ci and 2x400 Ci of {sup 188}Re-(Arg{sup 11})CCMSH via the tail vein, respectively. TXM13 melanoma tumor hearing mice were separately injected with 600 Ci, 2x400 Ci and 1000 Ci of 100Re-(Arg{sup 11})CCMSH through the tail vein. Two groups of 10 mice bearing either B16/F1 or TXM13 tumors were injected with saline as untreated controls. Results: In contrast to the untreated control group, {sup 188}Re(Arg11)CCMSH yielded rapid and lasting therapeutic effects in the treatment groups with either B16/F1 or TXM13 tumors. The tumor growth rate was reduced and the survival rate was prolonged in the treatment groups. Treatment with 2x400 Ci of {sup 188}Re-Arg{sup 11}CCMSH significantly extended the mean life of B16/F1 tumor mice (p<0.05), while the mean life of TXm13 tumor mice was significantly prolonged after treatment with 600 Ci and 1000 Ci doses of {sup 188}Re-(Arg{sup 11})CCMSH (p<0.05 High-dose {sup 188}Re-(Arg{sup 11}))CCMSH produced no observed normal-tissue toxicity. Conclusions: The therapy study results revealed that {sup 188}Re-Arg11 CCMSH yielded significant therapeutic effects in both B16/F1 murine melanoma and TXM13 human melanoma bearing mouse models. {sup 188}Re-(Arg{sup 11})CCMSH appears to be a promising radiolabeled peptide for targeted radionuclide therapy of melanoma.

  7. Post-translational heterocyclic backbone modifications in the 43-peptide antibiotic microcin B17. Structure elucidation and NMR study of a 13C,15N-labelled gyrase inhibitor.

    PubMed

    Bayer, A; Freund, S; Jung, G

    1995-12-01

    Microcin B17 (McB17), the first known gyrase inhibitor of peptidic nature, is produced by ribosomal synthesis and post-translational modification of the 69-residue precursor protein by an Escherichia coli strain. To elucidate the chemical structure of the mature 43-residue peptide antibiotic, fermentation and purification protocols were established and optimized which allowed the isolation and purification of substantial amounts of highly pure McB17 (non-labelled, 15N-labelled and 13C/15N-labelled peptide. By ultraviolet-absorption spectroscopy. HPLC-electrospray mass spectrometry and GC-mass spectrometry, amino acid analysis, protein sequencing, and, in particular, multidimensional NMR, we could demonstrate and unequivocally prove that the enzymic modification of the precursor backbone at Gly-Cys and Gly-Ser segments leads to the formation of 2-aminomethylthiazole-4-carboxylic acid and 2-aminomethyloxazole-4-carboxylic acid, respectively. In addition, two bicyclic modifications 2-(2-aminomethyloxazolyl)thiazole-4-carboxylic acid and 2-(2-aminomethylthiazolyl)oxazole-4-carboxylic acid were found that consist of directly linked thiazole and oxazole rings derived from one Gly-Ser-Cys and one Gly-Cys-Ser segment. Analogous to the thiazole and oxazole rings found in antitumor peptides of microbial and marine origin, these heteroaromatic ring systems of McB17 presumably play an important role in its gyrase-inhibiting activity, e.g. interacting with the DNA to trap the covalent protein-DNA intermediate of the breakage-reunion reaction of the gyrase.

  8. 13C-13C and 15N-13C correlation spectroscopy of membrane-associated and uniformly labeled human immunodeficiency virus and influenza fusion peptides: Amino acid-type assignments and evidence for multiple conformations

    NASA Astrophysics Data System (ADS)

    Bodner, Michele L.; Gabrys, Charles M.; Struppe, Jochem O.; Weliky, David P.

    2008-02-01

    Many viruses which cause disease including human immunodeficiency virus (HIV) and influenza are "enveloped" by a membrane and infection of a host cell begins with joining or "fusion" of the viral and target cell membranes. Fusion is catalyzed by viral proteins in the viral membrane. For HIV and for the influenza virus, these fusion proteins contain an ˜20-residue apolar "fusion peptide" that binds to target cell membranes and plays a critical role in fusion. For this study, the HIV fusion peptide (HFP) and influenza virus fusion peptide (IFP) were chemically synthesized with uniform C13, N15 labeling over large contiguous regions of amino acids. Two-dimensional C13-C13 and N15-C13 spectra were obtained for the membrane-bound fusion peptides and an amino acid-type C13 assignment was obtained for the labeled residues in HFP and IFP. The membrane used for the HFP sample had a lipid headgroup and cholesterol composition comparable to that of host cells of the virus, and the C13 chemical shifts were more consistent with β strand conformation than with helical conformation. The membrane used for the IFP sample did not contain cholesterol, and the chemical shifts of the dominant peaks were more consistent with helical conformation than with β strand conformation. There were additional peaks in the IFP spectrum whose shifts were not consistent with helical conformation. An unambiguous C13 and N15 assignment was obtained in an HFP sample with more selective labeling, and two shifts were identified for the Leu-9 CO, Gly-10 N, and Gly-10 Cα nuclei. These sets of two shifts may indicate two β strand registries such as parallel and antiparallel. Although most spectra were obtained on a 9.4T instrument, one C13-C13 correlation spectrum was obtained on a 16.4T instrument and was better resolved than the comparable 9.4T spectrum. More selective labeling and higher field may, therefore, be approaches to obtaining unambiguous assignments for membrane-associated fusion peptides.

  9. Order of amino acids in C-terminal cysteine-containing peptide-based chelators influences cellular processing and biodistribution of 99mTc-labeled recombinant Affibody molecules.

    PubMed

    Altai, Mohamed; Wållberg, Helena; Orlova, Anna; Rosestedt, Maria; Hosseinimehr, Seyed Jalal; Tolmachev, Vladimir; Ståhl, Stefan

    2012-05-01

    Affibody molecules constitute a novel class of molecular display selected affinity proteins based on non-immunoglobulin scaffold. Preclinical investigations and pilot clinical data have demonstrated that Affibody molecules provide high contrast imaging of tumor-associated molecular targets shortly after injection. The use of cysteine-containing peptide-based chelators at the C-terminus of recombinant Affibody molecules enabled site-specific labeling with the radionuclide 99mTc. Earlier studies have demonstrated that position, composition and the order of amino acids in peptide-based chelators influence labeling stability, cellular processing and biodistribution of Affibody molecules. To investigate the influence of the amino acid order, a series of anti-HER2 Affibody molecules, containing GSGC, GEGC and GKGC chelators have been prepared and characterized. The affinity to HER2, cellular processing of 99mTc-labeled Affibody molecules and their biodistribution were investigated. These properties were compared with that of the previously studied 99mTc-labeled Affibody molecules containing GGSC, GGEC and GGKC chelators. All variants displayed picomolar affinities to HER2. The substitution of a single amino acid in the chelator had an appreciable influence on the cellular processing of 99mTc. The biodistribution of all 99mTc-labeled Affibody molecules was in general comparable, with the main difference in uptake and retention of radioactivity in excretory organs. The hepatic accumulation of radioactivity was higher for the lysine-containing chelators and the renal retention of 99mTc was significantly affected by the amino acid composition of chelators. The order of amino acids influenced renal uptake of some conjugates at 1 h after injection, but the difference decreased at later time points. Such information can be helpful for the development of other scaffold protein-based imaging and therapeutic radiolabeled conjugates.

  10. Peptide-level Robust Ridge Regression Improves Estimation, Sensitivity, and Specificity in Data-dependent Quantitative Label-free Shotgun Proteomics.

    PubMed

    Goeminne, Ludger J E; Gevaert, Kris; Clement, Lieven

    2016-02-01

    Peptide intensities from mass spectra are increasingly used for relative quantitation of proteins in complex samples. However, numerous issues inherent to the mass spectrometry workflow turn quantitative proteomic data analysis into a crucial challenge. We and others have shown that modeling at the peptide level outperforms classical summarization-based approaches, which typically also discard a lot of proteins at the data preprocessing step. Peptide-based linear regression models, however, still suffer from unbalanced datasets due to missing peptide intensities, outlying peptide intensities and overfitting. Here, we further improve upon peptide-based models by three modular extensions: ridge regression, improved variance estimation by borrowing information across proteins with empirical Bayes and M-estimation with Huber weights. We illustrate our method on the CPTAC spike-in study and on a study comparing wild-type and ArgP knock-out Francisella tularensis proteomes. We show that the fold change estimates of our robust approach are more precise and more accurate than those from state-of-the-art summarization-based methods and peptide-based regression models, which leads to an improved sensitivity and specificity. We also demonstrate that ionization competition effects come already into play at very low spike-in concentrations and confirm that analyses with peptide-based regression methods on peptide intensity values aggregated by charge state and modification status (e.g. MaxQuant's peptides.txt file) are slightly superior to analyses on raw peptide intensity values (e.g. MaxQuant's evidence.txt file).

  11. Data on biodistribution and radiation absorbed dose profile of a novel (64)Cu-labeled high affinity cell-specific peptide for positron emission tomography imaging of tumor vasculature.

    PubMed

    Merrill, Joseph R; Krajewski, Krzysztof; Yuan, Hong; Frank, Jonathan E; Lalush, David S; Patterson, Cam; Veleva, Anka N

    2016-06-01

    New peptide-based diagnostic and therapeutic approaches hold promise for highly selective targeting of cancer leading to more precise and effective diagnostic and therapeutic modalities. An important feature of these approaches is to reach the tumor tissue while limiting or minimizing the dose to normal organs. In this context, efforts to design and engineer materials with optimal in vivo targeting and clearance properties are important. This Data In Brief article reports on biodistribution and radiation absorbed dose profile of a novel high affinity radiopeptide specific for bone marrow-derived tumor vasculature. Background information on the design, preparation, and in vivo characterization of this peptide-based targeted radiodiagnostic is described in the article "Synthesis and comparative evaluation of novel 64Cu-labeled high affinity cell-specific peptides for positron emission tomography of tumor vasculature" (Merrill et al., 2016) [1]. Here we report biodistribution measurements in mice and calculate the radiation absorbed doses to normal organs using a modified Medical Internal Radiation Dosimetry (MIRD) methodology that accounts for physical and geometric factors and cross-organ beta doses. PMID:27014735

  12. Purification of labeled cyanogen bromide peptides of the alpha polypeptide from sodium ion and potassium ion activated adenosinetriphosphatase modified with N-(/sup 3/H)ethylmaleimide

    SciTech Connect

    Le, D.T.

    1986-05-06

    Sodium ion and potassium ion activated adenosinetriphosphatase, isolated from canine kidney, was reacted with N-(/sup 3/H)ethylmaleimide while it was poised in three different conformations, ostensibly E2-P, E2, and E1, respectively. These assignments were made from a consideration of the particular concentrations of ligands in the respective alkylation mixtures. After a 30-min reaction, the remaining enzymatic activity was found to vary among these three different samples from 90 to 30% of that of unalkylated controls. In all cases, the alpha polypeptide was purified and subjected to digestion with cyanogen bromide, and in each digest the same two distinct radioactive peptides were identified and purified by gel filtration on a column of Sephadex LH-60. The incorporation of N-(/sup 3/H)ethylmaleimide into one of these two peptides correlated closely with enzymatic inactivation, while the incorporation into the other was most extensive when the portion of the active site to which ATP binds was unoccupied. Alkylation of the residue within the latter peptide, however, does not result in inactivation of the enzyme. Both peptides were further purified by high-pressure liquid chromatography, and their amino-terminal sequences were determined by manual dansyl Edman or solid-phase techniques. The peptide containing the sulfhydryl protected by ATP has, as its amino terminus, the lysine that reacts exclusively with fluoresceinyl 5'-isothiocyanate.

  13. Absolute Quantification of Prion Protein (90-231) Using Stable Isotope-Labeled Chymotryptic Peptide Standards in a LC-MRM AQUA Workflow

    NASA Astrophysics Data System (ADS)

    Sturm, Robert; Sheynkman, Gloria; Booth, Clarissa; Smith, Lloyd M.; Pedersen, Joel A.; Li, Lingjun

    2012-09-01

    Substantial evidence indicates that the disease-associated conformer of the prion protein (PrPTSE) constitutes the etiologic agent in prion diseases. These diseases affect multiple mammalian species. PrPTSE has the ability to convert the conformation of the normal prion protein (PrPC) into a β-sheet rich form resistant to proteinase K digestion. Common immunological techniques lack the sensitivity to detect PrPTSE at subfemtomole levels, whereas animal bioassays, cell culture, and in vitro conversion assays offer higher sensitivity but lack the high-throughput the immunological assays offer. Mass spectrometry is an attractive alternative to the above assays as it offers high-throughput, direct measurement of a protein's signature peptide, often with subfemtomole sensitivities. Although a liquid chromatography-multiple reaction monitoring (LC-MRM) method has been reported for PrPTSE, the chemical composition and lack of amino acid sequence conservation of the signature peptide may compromise its accuracy and make it difficult to apply to multiple species. Here, we demonstrate that an alternative protease (chymotrypsin) can produce signature peptides suitable for a LC-MRM absolute quantification (AQUA) experiment. The new method offers several advantages, including: (1) a chymotryptic signature peptide lacking chemically active residues (Cys, Met) that can confound assay accuracy; (2) low attomole limits of detection and quantitation (LOD and LOQ); and (3) a signature peptide retaining the same amino acid sequence across most mammals naturally susceptible to prion infection as well as important laboratory models. To the authors' knowledge, this is the first report on the use of a non-tryptic peptide in a LC-MRM AQUA workflow.

  14. Substitution of the Lys linker with the β-Ala linker dramatically decreased the renal uptake of 99mTc-labeled Arg-X-Asp-conjugated and X-Ala-Asp-conjugated α-melanocyte stimulating hormone peptides.

    PubMed

    Flook, Adam M; Yang, Jianquan; Miao, Yubin

    2014-11-13

    The purpose of this study was to examine whether the substitution of the Lys linker with the β-Ala could reduce the renal uptake of (99m)Tc-labeled Arg-X-Asp-conjugated and X-Ala-Asp-conjugated α-melanocyte stimulating hormone (α-MSH) peptides. RSD-β-Ala-(Arg(11))CCMSH (1) {c[Arg-Ser-Asp-dTyr-Asp]-β-Ala-Cys-Cys-Glu-His-dPhe-Arg-Trp-Cys-Arg-Pro-Val-NH2}, RTD-β-Ala-(Arg(11))CCMSH (2), RVD-β-Ala-(Arg(11))CCMSH (3), RAD-β-Ala-(Arg(11))CCMSH (4), NAD-β-Ala-(Arg(11))CCMSH (5), and EAD-β-Ala-(Arg(11))CCMSH (6) peptides were synthesized and evaluated for their melanocortin 1 (MC1) receptor binding affinities in B16/F1 melanoma cells. The biodistribution of their (99m)Tc-conjugates were determined in B16/F1 melanoma-bearing C57 mice. The substitution of the Lys linker with β-Ala linker dramatically reduced the renal uptake of all six (99m)Tc-peptides. (99m)Tc-4 exhibited the highest melanoma uptake (15.66 ± 6.19% ID/g) and the lowest kidney uptake (20.18 ± 3.86% ID/g) among these (99m)Tc-peptides at 2 h postinjection. The B16/F1 melanoma lesions could be clearly visualized by single photon emission computed tomography (SPECT)/CT using (99m)Tc-4 as an imaging probe.

  15. 2-(4-bromoacetamido)anilino-2-deoxypentitol 1,5-bisphosphate, a new affinity label for ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum: determination of reaction parameters and characterization of an active site peptide

    SciTech Connect

    Herndon, C.S.; Hartman, F.C.

    1984-03-10

    Reductive amination of ribulose-P/sub 2/ with p-phenylenediamine in the presence of sodium cyanoborohydride yielded an epimeric mixture which was resolved by chromatography. Subsequent bromoacetylation of the isolated amino bisphosphates gave reagents A and B (ribo and arabino epimers of 2-(4-bromoacetamido)anilino-2-deoxypentitol 1.5-bisphosphate) which were competitive inhibitors of the carboxylase with K/sub i/ values of 705 and 104 ..mu..M, respectively. Reagent A exhibited no time-dependent effects on the carboxylase in either the deactivated or activated state. Incubation of the enzyme with reagent B in the presence of the essential activators CO/sub 2/ and Mg/sup 2 +/, however, resulted in an irreversible, time-dependent loss of activity, with a K/sub inact/ of 125 ..mu..M and a minimal half-time of 7.3 min. Covalent incorporation of (/sup 14/C)reagent B was directly proportional to the loss of activity, with total inactivation correlating with an incorporation of 1.1 mol of reagent/mol of subunit. Inclusion of the competitive inhibitor 2-carboxyribitol 1,5-bisphosphate protected against inactivation with a concomitant reduction in incorporation. Neither reagent affected the activity of spinach carboxylase. Fractionation of (/sup 14/C)reagent B-modified enzyme on DEAE-cellulose, subsequent to carboxymethylation and tryptic digestion, revealed two major radioactive peaks of approximately equal area. Digestion of each peak with alkaline phosphatase and rechromatography on DEAE-cellulose resulted in pure peptides I and II. The peptides were identical except in the site of labeling: peptide I contained a modified cysteinyl residue while peptide II contained a modified histidyl residue. 60 references, 7 figures, tables.

  16. Novel 1:1 labeling and purification process for C-terminal thioester and single cysteine recombinant proteins using generic peptidic toolbox reagents.

    PubMed

    Portal, Christophe F; Seifert, Jan-Marcus; Buehler, Christof; Meisner-Kober, Nicole-Claudia; Auer, Manfred

    2014-07-16

    We developed a versatile set of chemical labeling reagents which allow dye ligation to the C-terminus of a protein or a single internal cysteine and target purification in a simple two-step process. This simple process results in a fully 1:1 labeled conjugate suitable for all quantitative fluorescence spectroscopy and imaging experiments. We refer to a "generic labeling toolbox" because of the flexibility to choose one of many available dyes, spacers of different lengths and compositions which increase the target solubility, a variety of affinity purification tags, and different cleavage chemistries to release the 1:1 labeled proteins. Studying protein function in vitro or in the context of live cells and organisms is of vital importance in biological research. Although label free detection technologies gain increasing interest in molecular recognition science, fluorescence spectroscopy is still the most often used detection technique for assays and screens both in academic as well as in industrial groups. For generations, fluorescence spectroscopists have labeled their proteins of interest with small fluorescent dyes by random chemical linking on the proteins' exposed lysines and cysteines. Chemical reactions with a certain excess of activated esters or maleimides of longer wavelength dyes hardly ever result in quantitative labeling of the target protein. Most of the time, more than one exposed amino acid side chain reacts. This results in a mixture of dye-protein complexes of different labeling stoichiometries and labeling sites. Only mass spectrometry allows resolving the precise chemical composition of the conjugates. In "classical" ensemble averaging fluorescent experiments, these labeled proteins are still useful, and quantification of, e.g., ligand binding experiments, is achieved via knowledge of the overall protein concentration and a fluorescent signal change which is proportional to the amount of complex formed. With the development of fluorescence

  17. An Open Label Clinical Trial of a Peptide Treatment Serum and Supporting Regimen Designed to Improve the Appearance of Aging Facial Skin.

    PubMed

    Draelos, Zoe Diana; Kononov, Tatiana; Fox, Theresa

    2016-09-01

    A 14-week single-center clinical usage study was conducted to test the efficacy of a peptide treatment serum and supporting skincare regimen in 29 women with mild to moderately photodamaged facial skin. The peptide treatment serum contained gamma-aminobutyric acid (GABA) and various peptides with neurotransmitter inhibiting and cell signaling properties. It was hypothesized that the peptide treatment serum would ameliorate eye and facial expression lines including crow's feet and forehead lines. The efficacy of the supporting skincare regimen was also evaluated. An expert investigator examined the subjects at rest and at maximum smile. Additionally, the subjects completed self-assessment questionnaires. At week 14, the expert investigator found a statistically significant improvement in facial lines, facial wrinkles, eye lines, and eye wrinkles at rest when compared to baseline results. The expert investigator also found statistically significant improvement at week 14 in facial lines, eye lines, and eye wrinkles when compared to baseline results at maximum smile. In addition, there was continued highly statistically significant improvement in smoothness, softness, firmness, radiance, luminosity, and overall appearance at rest when compared to baseline results at the 14-week time point. The test regimen was well perceived by the subjects for efficacy and product attributes. The products were well tolerated with no adverse events.

    J Drugs Dermatol. 2016;15(9):1100-1106. PMID:27602972

  18. The in vivo disposition and in vitro transmembrane transport of two model radiometabolites of DOTA-conjugated receptor-specific peptides labelled with (177) Lu.

    PubMed

    Volková, Marie; Mandíková, Jana; Bárta, Pavel; Navrátilová, Lucie; Lázníčková, Alice; Trejtnar, František

    2015-01-01

    In vivo metabolism of the radiolabelled receptor-specific peptides has been described; however, information regarding the pharmacokinetic behaviour of the degradation products within the body is very scarce. The present study was designed to obtain new knowledge on the disposition and elimination of low-molecular radiometabolites of receptor-specific peptides in the organism and to reveal the potential involvement of selected membrane transport mechanisms in the cellular uptake of radiometabolites, especially in the kidney. The study compared pharmacokinetics of two radiometabolites: a final metabolite of somatostatin analogues, (177)Lu-DOTA-DPhe, and a tripeptide metabolite of (177)Lu-DOTA-minigastrin 11, (177)Lu-DOTA-DGlu-Ala-Tyr. Their pharmacokinetics was compared with that of respective parent (177)Lu-radiopeptide. Both radiometabolites exhibited relative rapid clearing from most body tissues in rats in vivo along with predominant renal excretion. The long-term renal retention of the smaller radiometabolite (177)Lu-DOTA-DPhe was lower than that of (177)Lu-DOTA-DGlu-Ala-Tyr. An uptake of (177)Lu-DOTA-DPhe by human renal influx transporter organic cation transporter 2 was found in vitro in a cellular model. The study brings the first experimental data on the in vivo pharmacokinetics of radiometabolites of receptor-specific somatostatin and gastrin analogues. The found results may indicate a negative correlation between the degree of decomposition of the parent peptide chain and the renal retention of the metabolite. PMID:26526343

  19. Trypsin-catalyzed oxygen-18 labeling for quantitative proteomics

    SciTech Connect

    Qian, Weijun; Petritis, Brianne O.; Nicora, Carrie D.; Smith, Richard D.

    2011-07-01

    Stable isotope labeling based on relative peptide/protein abundance measurements is commonly applied for quantitative proteomics. Recently, trypsin-catalyzed oxygen-18 labeling has grown in popularity due to its simplicity, cost-effectiveness, and its ability to universally label peptides with high sample recovery. In (18)O labeling, both C-terminal carboxyl group atoms of tryptic peptides can be enzymatically exchanged with (18)O, thus providing the labeled peptide with a 4 Da mass shift from the (16)O-labeled sample. Peptide (18)O labeling is ideally suited for generating a labeled "universal" reference sample used for obtaining accurate and reproducible quantitative measurements across large number of samples in quantitative discovery proteomics.

  20. Peptide targeted copper-64 radiopharmaceuticals.

    PubMed

    Ma, Michelle T; Donnelly, Paul S

    2011-01-01

    Peptide targeted ⁶⁴Cu-labelled diagnostic agents for positron emission tomography are viable candidates for molecular imaging of cancer. In a clinical setting, optimal image quality relies on selective tumor uptake of the ⁶⁴Cu-labelled radiotracer. The three components of the radiotracer construct--the chelate group, linker and targeting peptide--all influence the biodistribution of the ⁶⁴Cu-labelled radiotracer in vivo. Low or moderate Cu complex stability in vivo results in transmetallation of ⁶⁴Cu to endogenous proteins, giving rise to high background activity. The length and the nature of the linker group affect the affinity of the ⁶⁴Cu-labelled radiotracer for the target receptor. Variations in the peptide sequence can impact on the metabolic stability and therefore the bioavailability and tumor retention of the ⁶⁴Cu-labelled radiotracer in vivo. Lastly, the hydrophilicity of the construct can influence radiotracer metabolism and clearance pathways. Recent advances in the field of peptide targeted ⁶⁴Cu-labelled radiopharmaceuticals involve GRPR-targeted and αvβ3 integrin receptor-targeted constructs. These constructs are based on the bombesin peptide sequence and the RGD recognition motif respectively. These examples are reviewed as case studies in the optimisation of ⁶⁴Cu radiotracer design.

  1. 99mTc Labeled Glucagon-Like Peptide-1-Analogue (99mTc-GLP1) Scintigraphy in the Management of Patients with Occult Insulinoma

    PubMed Central

    Sowa-Staszczak, Anna; Trofimiuk-Müldner, Małgorzata; Stefańska, Agnieszka; Tomaszuk, Monika; Buziak-Bereza, Monika; Gilis-Januszewska, Aleksandra; Jabrocka-Hybel, Agata; Głowa, Bogusław; Małecki, Maciej; Bednarczuk, Tomasz; Kamiński, Grzegorz; Kowalska, Aldona; Mikołajczak, Renata; Janota, Barbara; Hubalewska-Dydejczyk, Alicja

    2016-01-01

    Introduction The aim of this study was to assess the utility of [Lys40(Ahx-HYNIC-99mTc/EDDA)NH2]-exendin-4 scintigraphy in the management of patients with hypoglycemia, particularly in the detection of occult insulinoma. Materials and Methods Forty patients with hypoglycemia and increased/confusing results of serum insulin and C-peptide concentration and negative/inconclusive results of other imaging examinations were enrolled in the study. In all patients GLP-1 receptor imaging was performed to localise potential pancreatic lesions. Results Positive results of GLP-1 scintigraphy were observed in 28 patients. In 18 patients postsurgical histopathological examination confirmed diagnosis of insulinoma. Two patients had contraindications to the surgery, one patient did not want to be operated. One patient, who presented with postprandial hypoglycemia, with positive result of GLP-1 imaging was not qualified for surgery and is in the observational group. Eight patients were lost for follow up, among them 6 patients with positive GLP-1 scintigraphy result. One patient with negative scintigraphy was diagnosed with malignant insulinoma. In two patients with negative scintigraphy Munchausen syndrome was diagnosed (patients were taking insulin). Other seven patients with negative results of 99mTcGLP-1 scintigraphy and postprandial hypoglycemia with C-peptide and insulin levels within the limits of normal ranges are in the observational group. We would like to mention that 99mTc-GLP1-SPECT/CT was also performed in 3 pts with nesidioblastosis (revealing diffuse tracer uptake in two and a focal lesion in one case) and in two patients with malignant insulinoma (with the a focal uptake in the localization of a removed pancreatic headin one case and negative GLP-1 1 scintigraphy in the other patient). Conclusions 99mTc-GLP1-SPECT/CT could be helpful examination in the management of patients with hypoglycemia enabling proper localization of the pancreatic lesion and effective

  2. Peptide receptor radionuclide therapy of Merkel cell carcinoma using (177)lutetium-labeled somatostatin analogs in combination with radiosensitizing chemotherapy: a potential novel treatment based on molecular pathology.

    PubMed

    Salavati, Ali; Prasad, Vikas; Schneider, Claus-Peter; Herbst, Rudolf; Baum, Richard Paul

    2012-05-01

    Few studies have been published on the safety and feasibility of synchronous use of peptide receptor radionuclide therapy (PRRNT), as source of internal radiation therapy, in combination with chemotherapy. In this study we reported a 53-year-old man with stage IV Merkel cell carcinoma (MCC), who underwent synchronous internal radiation therapy and chemotherapy. Based on presumable poor prognosis with chemotherapy only, functional similarities of MCC with other neuroendocrine tumors and available evidence of effectiveness and safety of synchronous use of external beam radiation therapy and chemotherapy in treatment of high-risk MCC patients, our interdisciplinary neuroendocrine tumor board recommended him to add PRRNT to his ongoing chemotherapy. He received 2 courses of (177)Lu-DOTATATE(1, 4, 7, 10-Tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid-1-D-Phe1-Tyr3-Thr8-octreotide) in combination with ongoing 8 cycles of liposomal doxorubicin based on standard protocols. Response to therapy was evaluated by (18)F-FDG and (68)gallium-somatostatin-receptor PET/CT. There was an impressive improvement of the clinical symptoms. However, follow-up PET/CT studies showed mixed pattern of response. Synchronous use of PRRNT and radiosensitizing chemotherapy seems safe and feasible in high risk MCC patients, however, further prospective studies and clinical trials are warranted to provide reliable evidence of possible pitfalls and effectiveness of PRRNT and (68)Ga-somatostatin-receptor PET/CT in the management of MCC.

  3. Evaluation of copper-64 labeled AmBaSar conjugated cyclic RGD peptide for improved microPET imaging of integrin alphavbeta3 expression.

    PubMed

    Cai, Hancheng; Li, Zibo; Huang, Chiun-Wei; Shahinian, Anthony H; Wang, Hui; Park, Ryan; Conti, Peter S

    2010-08-18

    Recently, we have developed a new cage-like bifunctional chelator 4-((8-amino-3,6,10,13,16,19-hexaazabicyclo [6.6.6] icosane-1-ylamino) methyl) benzoic acid (AmBaSar) for copper-64 labeling and synthesized the positron emission tomography (PET) tracer (64)Cu-AmBaSar-RGD. In this study, we further evaluate the biological property of this new AmBaSar chelator by using (64)Cu-AmBaSar-RGD as the model compound. In vitro and in vivo stability, lipophilicity, cell binding and uptake, microPET imaging, receptor blocking experiments, and biodistribution studies of (64)Cu-AmBaSar-RGD were investigated, and the results were directly compared with the established radiotracer (64)Cu-DOTA-RGD. The (64)Cu-AmBaSar-RGD was obtained with high radiochemical yield (> or =95%) and purity (> or =99%) under mild conditions (pH 5.0-5.5 and 23-37 degrees C) in less than 30 min. For in vitro studies, the radiochemical purity of (64)Cu-AmBaSar-RGD was more than 97% in PBS or FBS and 95% in mouse serum after 24 h of incubation. The log P value of (64)Cu-AmBaSar-RGD was -2.44 +/- 0.12. For in vivo studies, (64)Cu-AmBaSar-RGD and (64)Cu-DOTA-RGD have demonstrated comparable tumor uptake at selected time points on the basis of microPET imaging. The integrin alpha(v)beta(3) receptor specificity was confirmed by blocking experiments for both tracers. Compared with (64)Cu-DOTA-RGD, (64)Cu-AmBaSar-RGD demonstrated much lower liver accumulation in both microPET imaging and biodistribution studies. Metabolic studies also directly supported the observation that (64)Cu-AmBaSar-RGD was more stable in vivo than (64)Cu-DOTA-RGD. In summary, the in vitro and in vivo evaluations of the (64)Cu-AmBaSar-RGD have demonstrated its improved Cu-chelation stability compared with that of the established tracer (64)Cu-DOTA-RGD. The AmBaSar chelator will also have general applications for (64)Cu labeling of various bioactive molecules in high radiochemical yield and high in vivo stability.

  4. Precise quantitation of 136 urinary proteins by LC/MRM-MS using stable isotope labeled peptides as internal standards for biomarker discovery and/or verification studies.

    PubMed

    Percy, Andrew J; Yang, Juncong; Hardie, Darryl B; Chambers, Andrew G; Tamura-Wells, Jessica; Borchers, Christoph H

    2015-06-15

    Spurred on by the growing demand for panels of validated disease biomarkers, increasing efforts have focused on advancing qualitative and quantitative tools for more highly multiplexed and sensitive analyses of a multitude of analytes in various human biofluids. In quantitative proteomics, evolving strategies involve the use of the targeted multiple reaction monitoring (MRM) mode of mass spectrometry (MS) with stable isotope-labeled standards (SIS) used for internal normalization. Using that preferred approach with non-invasive urine samples, we have systematically advanced and rigorously assessed the methodology toward the precise quantitation of the largest, multiplexed panel of candidate protein biomarkers in human urine to date. The concentrations of the 136 proteins span >5 orders of magnitude (from 8.6 μg/mL to 25 pg/mL), with average CVs of 8.6% over process triplicate. Detailed here is our quantitative method, the analysis strategy, a feasibility application to prostate cancer samples, and a discussion of the utility of this method in translational studies.

  5. Site-directed isotope labeling and ATR-FTIR difference spectroscopy of bacteriorhodopsin: the peptide carbonyl group of Tyr 185 is structurally active during the bR-->N transition.

    PubMed

    Ludlam, C F; Sonar, S; Lee, C P; Coleman, M; Herzfeld, J; RajBhandary, U L; Rothschild, K J

    1995-01-10

    The largest secondary structural change occurs in the bacteriorhodopsin (bR) photocycle during the M-->N transition. In this work site-directed isotope labeling (SDIL) and attenuated total reflection Fourier transform infrared (ATR-FTIR) difference spectroscopy were used to investigate this conformational change. L-Tyrosine containing a 13C isotope at the carbonyl carbon was selectively incorporated at Tyr 57, Tyr 147, and Tyr 185 by SDIL. This involves the cell-free expression of bR in the presence of Escherichia coli suppressor tRNA(CUATyr) aminoacylated with L-[1-13C]Tyr. ATR-FTIR difference spectroscopy reveals that of the 11 tyrosines, only the peptide carbonyl group of Tyr 185 undergoes a significant structural change during the bR-->N transition. Along with other spectroscopic evidence, this result suggests that the Tyr 185-Pro 186 region of the protein is structurally active and may function as a hinge which facilitates the tilt of the cytoplasmic portion of the F-helix in bacteriorhodopsin during the M-->N transition.

  6. Peptides and proteins

    SciTech Connect

    Bachovchin, W.W.; Unkefer, C.J.

    1994-12-01

    Advances in magnetic resonance and vibrational spectroscopy make it possible to derive detailed structural information about biomolecular structures in solution. These techniques are critically dependent on the availability of labeled compounds. For example, NMR techniques used today to derive peptide and protein structures require uniformity {sup 13}C-and {sup 15}N-labeled samples that are derived biosynthetically from (U-6-{sup 13}C) glucose. These experiments are possible now because, during the 1970s, the National Stable Isotope Resource developed algal methods for producing (U-6-{sup 13}C) glucose. If NMR techniques are to be used to study larger proteins, we will need sophisticated labelling patterns in amino acids that employ a combination of {sup 2}H, {sup 13}C, and {sup 15}N labeling. The availability of these specifically labeled amino acids requires a renewed investment in new methods for chemical synthesis of labeled amino acids. The development of new magnetic resonance or vibrational techniques to elucidate biomolecular structure will be seriously impeded if we do not see rapid progress in labeling technology. Investment in labeling chemistry is as important as investment in the development of advanced spectroscopic tools.

  7. Peptide nanotubes.

    PubMed

    Hamley, Ian W

    2014-07-01

    The self-assembly of different classes of peptide, including cyclic peptides, amyloid peptides and surfactant-like peptides into nanotube structures is reviewed. The modes of self-assembly are discussed. Additionally, applications in bionanotechnology and synthetic materials science are summarized.

  8. 13 C solid-state NMR study of the 13 C-labeled peptide, (E)8 GGLGGQGAG(A)6 GGAGQGGYGG as a model for the local structure of Nephila clavipes dragline silk (MaSp1) before and after spinning.

    PubMed

    Yazawa, Koji; Yamaguchi, Erika; Knight, David; Asakura, Tetsuo

    2012-06-01

    We prepared the water soluble model peptide, (E)(8) GGLGGQGAG(A)(6) GGAGQGGYGG, to throw light on the local structure of spidroin 1 (MaSpl) protein in spider dragline silk of Nephila clavipes before and after spinning. Solution (13) C NMR showed that the conformation of the peptide in aqueous solution was essentially random coil. Solid-state NMR was used to follow conformation-dependent (13) C chemical shifts in (13) C selectively labeled versions of the peptide. The peptide lyophilized from an aqueous solution at neutral pH (hereafter referred to as "without acid treatment)"was used to mimic the state of the spidroin stored in the spider's silk gland while the peptide precipitated from the acidic solution ("with acid treatment") was used to simulate the role of acid treatment in inducing conformation change in the natural spinning process. In without acid treatment, the fraction of random coil conformation was lowest in the N-terminal region (residues 15-18) when compared with the C-terminus. The conformational change produced by the acid treatment occurred in the sequence, G(15) AG(A)(6) GGAG(27), interposed between pairs of Gly residues pairs, Gly(12,13), and Gly(29,30). The acid treated peptide showed a remarkable decrease in the fraction of random coil conformation from A(20) to A(23) in the poly-Ala region when compared with the peptide without acid treatment. These observations taken together suggest that the peptide can be used as a model for studying the localization of the conformation change in spider silk fibroin in the natural spinning and the role of acid treatment in this process.

  9. Contrast-matched small-angle X-ray scattering from a heavy-atom-labeled protein in structure determination: application to a lead-substituted calmodulin-peptide complex.

    PubMed

    Grishaev, Alexander; Anthis, Nicholas J; Clore, G Marius

    2012-09-12

    The information content in 1-D solution X-ray scattering profiles is generally restricted to low-resolution shape and size information that, on its own, cannot lead to unique 3-D structures of biological macromolecules comparable to all-atom models derived from X-ray crystallography or NMR spectroscopy. Here we show that contrast-matched X-ray scattering data collected on a protein incorporating specific heavy-atom labels in 65% aqueous sucrose buffer can dramatically enhance the power of conventional small- and wide-angle X-ray scattering (SAXS/WAXS) measurements. Under contrast-matching conditions the protein is effectively invisible and the main contribution to the X-ray scattering intensity arises from the heavy atoms, allowing direct extraction of pairwise distances between them. In combination with conventional aqueous SAXS/WAXS data, supplemented by NMR-derived residual dipolar couplings (RDCs) measured in a weakly aligning medium, we show that it is possible to position protein domains relative to one another within a precision of 1 Å. We demonstrate this approach with respect to the determination of domain positions in a complex between calmodulin, in which the four Ca(2+) ions have been substituted by Pb(2+), and a target peptide. The uniqueness of the resulting solution is established by an exhaustive search over all models compatible with the experimental data, and could not have been achieved using aqueous SAXS and RDC data alone. Moreover, we show that the correct structural solution can be recovered using only contrast-matched SAXS and aqueous SAXS/WAXS data. PMID:22908850

  10. Contrast-Matched Small-Angle X-ray Scattering from a Heavy-Atom-Labeled Protein in Structure Determination: Application to a Lead-Substituted Calmodulin-Peptide Complex

    SciTech Connect

    Grishaev, Alexander; Anthis, Nicholas J; Clore, G Marius

    2012-10-29

    The information content in 1-D solution X-ray scattering profiles is generally restricted to low-resolution shape and size information that, on its own, cannot lead to unique 3-D structures of biological macromolecules comparable to all-atom models derived from X-ray crystallography or NMR spectroscopy. Here we show that contrast-matched X-ray scattering data collected on a protein incorporating specific heavy-atom labels in 65% aqueous sucrose buffer can dramatically enhance the power of conventional small- and wide-angle X-ray scattering (SAXS/WAXS) measurements. Under contrast-matching conditions the protein is effectively invisible and the main contribution to the X-ray scattering intensity arises from the heavy atoms, allowing direct extraction of pairwise distances between them. In combination with conventional aqueous SAXS/WAXS data, supplemented by NMR-derived residual dipolar couplings (RDCs) measured in a weakly aligning medium, we show that it is possible to position protein domains relative to one another within a precision of 1 Å. We demonstrate this approach with respect to the determination of domain positions in a complex between calmodulin, in which the four Ca2+ ions have been substituted by Pb2+, and a target peptide. The uniqueness of the resulting solution is established by an exhaustive search over all models compatible with the experimental data, and could not have been achieved using aqueous SAXS and RDC data alone. Moreover, we show that the correct structural solution can be recovered using only contrast-matched SAXS and aqueous SAXS/WAXS data.

  11. Isoelectric focusing of proteins and peptides

    NASA Technical Reports Server (NTRS)

    Egen, N.

    1979-01-01

    Egg-white solution was chosen as the reference solution in order to assess the effects of operational parameters (voltage, flow rate, ampholine pH range and concentration, and protein concentration) of the RIEF apparatus on protein resolution. Topics of discussion include: (1) comparison of RIEF apparatus to conventional IEF techniques (column and PAG) with respect to resolution and throughput; (2) peptide and protein separation (AHF, Thymosin - Fraction 5, vasoactive peptide, L-asparaginase and ACP); and (3) detection of peptides - dansyl derivatives of amino acids and peptides, post-focusing fluorescent labeling of amino acids, peptides and proteins, and ampholine extraction from focused gels.

  12. Nutrition Labeling

    NASA Astrophysics Data System (ADS)

    Metzger, Lloyd E.

    Nutrition labeling regulations differ in countries around the world. The focus of this chapter is on nutrition labeling regulations in the USA, as specified by the Food and Drug Administration (FDA) and the Food Safety and Inspection Service (FSIS) of the United States Department of Agriculture (USDA). A major reason for analyzing the chemical components of foods in the USA is nutrition labeling regulations. Nutrition label information is not only legally required in many countries, but also is of increasing importance to consumers as they focus more on health and wellness.

  13. Antimicrobial peptides

    PubMed Central

    2014-01-01

    With increasing antibiotics resistance, there is an urgent need for novel infection therapeutics. Since antimicrobial peptides provide opportunities for this, identification and optimization of such peptides have attracted much interest during recent years. Here, a brief overview of antimicrobial peptides is provided, with focus placed on how selected hydrophobic modifications of antimicrobial peptides can be employed to combat also more demanding pathogens, including multi-resistant strains, without conferring unacceptable toxicity. PMID:24758244

  14. Ion Mobility Separation of Peptide Isotopomers

    NASA Astrophysics Data System (ADS)

    Kaszycki, Julia L.; Bowman, Andrew P.; Shvartsburg, Alexandre A.

    2016-05-01

    Differential or field asymmetric waveform ion mobility spectrometry (FAIMS) operating at high electric fields fully resolves isotopic isomers for a peptide with labeled residues. The naturally present isotopes, alone and together with targeted labels, also cause spectral shifts that approximately add for multiple heavy atoms. Separation qualitatively depends on the gas composition. These findings may enable novel strategies in proteomic and metabolomic analyses using stable isotope labeling.

  15. Mimicking of Arginine by Functionalized N(ω)-Carbamoylated Arginine As a New Broadly Applicable Approach to Labeled Bioactive Peptides: High Affinity Angiotensin, Neuropeptide Y, Neuropeptide FF, and Neurotensin Receptor Ligands As Examples.

    PubMed

    Keller, Max; Kuhn, Kilian K; Einsiedel, Jürgen; Hübner, Harald; Biselli, Sabrina; Mollereau, Catherine; Wifling, David; Svobodová, Jaroslava; Bernhardt, Günther; Cabrele, Chiara; Vanderheyden, Patrick M L; Gmeiner, Peter; Buschauer, Armin

    2016-03-10

    Derivatization of biologically active peptides by conjugation with fluorophores or radionuclide-bearing moieties is an effective and commonly used approach to prepare molecular tools and diagnostic agents. Whereas lysine, cysteine, and N-terminal amino acids have been mostly used for peptide conjugation, we describe a new, widely applicable approach to peptide conjugation based on the nonclassical bioisosteric replacement of the guanidine group in arginine by a functionalized carbamoylguanidine moiety. Four arginine-containing peptide receptor ligands (angiotensin II, neurotensin(8-13), an analogue of the C-terminal pentapeptide of neuropeptide Y, and a neuropeptide FF analogue) were subject of this proof-of-concept study. The N(ω)-carbamoylated arginines, bearing spacers with a terminal amino group, were incorporated into the peptides by standard Fmoc solid phase peptide synthesis. The synthesized chemically stable peptide derivatives showed high receptor affinities with Ki values in the low nanomolar range, even when bulky fluorophores had been attached. Two new tritiated tracers for angiotensin and neurotensin receptors are described.

  16. Analysis of peptide uptake and location of root hair-promoting peptide accumulation in plant roots.

    PubMed

    Matsumiya, Yoshiki; Taniguchi, Rikiya; Kubo, Motoki

    2012-03-01

    Peptide uptake by plant roots from degraded soybean-meal products was analyzed in Brassica rapa and Solanum lycopersicum. B. rapa absorbed about 40% of the initial water volume, whereas peptide concentration was decreased by 75% after 24 h. Analysis by reversed-phase HPLC showed that number of peptides was absorbed by the roots during soaking in degraded soybean-meal products for 24 h. Carboxyfluorescein-labeled root hair-promoting peptide was synthesized, and its localization, movement, and accumulation in roots were investigated. The peptide appeared to be absorbed by root hairs and then moved to trichoblasts. Furthermore, the peptide was moved from trichoblasts to atrichoblasts after 24 h. The peptide was accumulated in epidermal cells, suggesting that the peptide may have a function in both trichoblasts and atrichoblasts.

  17. Cleavage and resynthesis of peptide cross bridges in Escherichia coli murein.

    PubMed Central

    Goodell, E W; Schwarz, U

    1983-01-01

    In Escherichia coli, peptide cross bridges in the murein undergo turnover after they are synthesized. Peptide cross bridges formed in the presence of [3H]diaminopimelic acid were found to lose 3H label from their donor peptides after the [3H]diaminopimelic acid was removed from the growth medium. There was a corresponding increase in the amount of 3H label in acceptor peptides so that the total amount of label in the peptide cross bridges remained constant. Our explanation of this observation is that the cross bridges are cleaved by the cell, and the original 3H-labeled donor peptides are incorporated into new cross bridges. Since these 3H-labeled peptides are now only tetrapeptides, they can only be used as acceptors when new cross bridges are formed. PMID:6352673

  18. ARSENITE BINDING TO SYNTHETIC PEPTIDES: THE EFFECT OF INCREASING LENGTH BETWEEN TWO CYSTEINES

    EPA Science Inventory

    Binding of trivalent arsenicals to peptides and proteins can alter peptide/protein structure and enzyme function and thereby contribute to arsenic toxicity and carcinogenicity. We utilized radioactive 73As- labeled arsenite and vacuum filtration methodology to determine the bindi...

  19. Protein quantification using a cleavable reporter peptide.

    PubMed

    Duriez, Elodie; Trevisiol, Stephane; Domon, Bruno

    2015-02-01

    Peptide and protein quantification based on isotope dilution and mass spectrometry analysis are widely employed for the measurement of biomarkers and in system biology applications. The accuracy and reliability of such quantitative assays depend on the quality of the stable-isotope labeled standards. Although the quantification using stable-isotope labeled peptides is precise, the accuracy of the results can be severely biased by the purity of the internal standards, their stability and formulation, and the determination of their concentration. Here we describe a rapid and cost-efficient method to recalibrate stable isotope labeled peptides in a single LC-MS analysis. The method is based on the equimolar release of a protein reference peptide (used as surrogate for the protein of interest) and a universal reporter peptide during the trypsinization of a concatenated polypeptide standard. The quality and accuracy of data generated with such concatenated polypeptide standards are highlighted by the quantification of two clinically important proteins in urine samples and compared with results obtained with conventional stable isotope labeled reference peptides. Furthermore, the application of the UCRP standards in complex samples is described.

  20. Protein quantification using a cleavable reporter peptide.

    PubMed

    Duriez, Elodie; Trevisiol, Stephane; Domon, Bruno

    2015-02-01

    Peptide and protein quantification based on isotope dilution and mass spectrometry analysis are widely employed for the measurement of biomarkers and in system biology applications. The accuracy and reliability of such quantitative assays depend on the quality of the stable-isotope labeled standards. Although the quantification using stable-isotope labeled peptides is precise, the accuracy of the results can be severely biased by the purity of the internal standards, their stability and formulation, and the determination of their concentration. Here we describe a rapid and cost-efficient method to recalibrate stable isotope labeled peptides in a single LC-MS analysis. The method is based on the equimolar release of a protein reference peptide (used as surrogate for the protein of interest) and a universal reporter peptide during the trypsinization of a concatenated polypeptide standard. The quality and accuracy of data generated with such concatenated polypeptide standards are highlighted by the quantification of two clinically important proteins in urine samples and compared with results obtained with conventional stable isotope labeled reference peptides. Furthermore, the application of the UCRP standards in complex samples is described. PMID:25411902

  1. Localization of site-specific probes on the Ca-ATPase of sarcoplasmic reticulum using fluorescence energy transfer.

    PubMed

    Squier, T C; Bigelow, D J; Garcia de Ancos, J; Inesi, G

    1987-04-01

    Highly reactive sulfhydryls, previously labeled with an iodoacetamide spin label on the Ca-ATPase of sarcoplasmic reticulum, were labeled with the fluorescent probe, 5-(2-[iodoacetyl)amino)ethyl)aminonaphthalene-1-sulfonic acid (IAEDANS), without loss of enzymatic activity. We have selectively measured the apparent distance of the more reactive site, relative to other site-specific probes at both the nucleotide and the high affinity calcium binding sites. Fluorescence energy transfer efficiencies from the donor IAEDANS to two acceptors: fluorescein 5'-isothiocyanate or 2',3'-O-(2,4,3-trinitrophenyl)adenosine monophosphate, situated at or near the nucleotide site, were measured using fluorescence lifetimes and yields. Fluorescence on polyacrylamide gels shows that the IAEDANS and fluorescein 5'-isothiocyanate labels are both associated with the B tryptic fragment. The energy transfer measurements are consistent with distances of 56 and 68 A between IAEDANS and these respective binding sites. On the other hand, energy transfer measurements using the lanthanide, praseodymium (Pr3+), as an acceptor indicate that IAEDANS is located 16-18 A from the binding site(s) of this calcium analog. Pr3+ is shown to be a good analog for calcium binding to the high affinity sites on the enzyme since it competitively displaces calcium, as evidenced by the reversal of the specific calcium-dependent intrinsic fluorescent signal and inactivation of ATPase activity, over the same narrow range in Pr3+ concentration where energy transfer is observed. Our observations suggest that the portion of the B fragment spanning the cytoplasmic portion of the ATPase is folded onto the A fragment, bringing the IAEDANS label in close proximity to the high affinity calcium binding domain.

  2. [Study on oral absorption enhancers of astragalus polysaccharides].

    PubMed

    Chen, Xiao-Yun; Tan, Xiao-Bin; Sun, E; Liu, Dan; Jia, Xiao-Bin; Zhang, Zhen-Hai

    2014-04-01

    Astragalus polysaccharides was lounded to 4-(2-aminoethylphenol), followed by labeling the APS-Tyr with fluorescein-5-isothiocyanate (FITC) at the secondary amino group. The absorption enhancement effects of low molecular weight chitosan and protamine on astragalus polysaccharides were evaluated via Caco-2 cell culture model. The results show that the fluorecent labeling compound has good stability and high sensitivity. On the other hand low molecular weight chitosan and protamine also can promoted absorption of the astragalus polysaccharides without any cytotoxity, and the absorption increase was more significant with increasing the amount of low molecular weight chitosan and protamine. At the same time, the low molecular weight chitosan has slightly better effect. The transepithelial electric resistance (TEER) of Caco-2 cells show that absorption enhancers could improve its membrane transport permeability by opening tight junctions between cells and increasing the cell membrane fluidity.

  3. Peptide identification

    DOEpatents

    Jarman, Kristin H [Richland, WA; Cannon, William R [Richland, WA; Jarman, Kenneth D [Richland, WA; Heredia-Langner, Alejandro [Richland, WA

    2011-07-12

    Peptides are identified from a list of candidates using collision-induced dissociation tandem mass spectrometry data. A probabilistic model for the occurrence of spectral peaks corresponding to frequently observed partial peptide fragment ions is applied. As part of the identification procedure, a probability score is produced that indicates the likelihood of any given candidate being the correct match. The statistical significance of the score is known without necessarily having reference to the actual identity of the peptide. In one form of the invention, a genetic algorithm is applied to candidate peptides using an objective function that takes into account the number of shifted peaks appearing in the candidate spectrum relative to the test spectrum.

  4. Synthetic peptides.

    PubMed

    Francis, M J

    1996-01-01

    Efforts to produce more stable and defined vaccines have concentrated on studying, in detail, the immune response to many infectious diseases in order to identify the antigenic sites on the pathogens that are involved in stimulating protective immumty. Armed with this knowledge, it is possible to mimic such sites by producing short chains of amino acids (peptides) and to use these as the basis for novel vaccines. The earliest documented work on peptide immunization is actually for a plant virus, tobacco mosaic virus. In 1963, Anderer (1) demonstrated that rabbit antibodies to an isolated hexapeptide fragment from the virus-coat protein coupled to bovine serum albumm would neutralize the infectious vn-us in culture. Two years later, he used a synthetically produced copy of the same peptide to confirm this observation. This was pioneering work, and it was over 10 years before the next example of a peptide that elicited antivirus antibody appeared following work by Sela and his colleagues (2) on a virus, MS2 bacteriophage, which infects bacteria. The emergence of more accessible techniques for sequencing proteins in 1977, coupled with the ability to synthesize readily peptides already developed in 1963, heralded a decade of intensive research into experimental peptide vaccines. The first demonstration that peptides could elicit protective immunity in vivo, in addition to neutralizing activity in vitro, was obtained using a peptide from the VP1 coat protein of foot-and-mouth disease virus (FMDV) in 1982, with the guinea pig as a laboratory animal model (3, 4). PMID:21359696

  5. Understanding Lipid Recognition by Protein-Mimicking Cyclic Peptides.

    PubMed

    Hosseini, Azade S; Zheng, Hong; Gao, Jianmin

    2014-10-21

    This paper describes our investigation of the structural determinants of a designed cyclic peptide (cLac, cyclic peptide mimicking lactadherin)(1) for phosphatidylserine (PS) recognition. A highly efficient strategy that takes advantage of the native chemical ligation (NCL) chemistry has been developed for the synthesis and labeling of cyclic peptides in general. Ala scanning of the cLac peptide revealed a sophisticated model for PS binding, in which the peptide scaffold assembles multiple polar residues to balance the desolvation and electrostatic interactions (salt bridge and hydrogen bonding) to achieve lipid selectivity. The results suggest that cLac effectively mimics the membrane binding mechanism of the parent protein lactadherin.

  6. A double fluorescence staining protocol to determine the cross-sectional area of myofibers using image analysis

    NASA Technical Reports Server (NTRS)

    Mozdziak, P. E.; Fassel, T. A.; Schultz, E.; Greaser, M. L.; Cassens, R. G.

    1996-01-01

    A double fluorescence staining protocol was developed to facilitate computer based image analysis. Myofibers from experimentally treated (irradiated) and control growing turkey skeletal muscle were labeled with the anti-myosin antibody MF-20 and detected using fluorescein-5-isothiocyanate (FITC). Extracellular material was stained with concanavalin A (ConA)-Texas red. The cross-sectional area of the myofibers was determined by calculating the number of pixels (0.83 mu m(2)) overlying each myofiber after subtracting the ConA-Texas red image from the MF-20-FITC image for each region of interest. As expected, myofibers in the irradiated muscle were smaller (P < 0.05) than those in the non-irradiated muscle. This double fluorescence staining protocol combined with image analysis is accurate and less labor-intensive than classical procedures for determining the cross-sectional area of myofibers.

  7. Affinity labeling of the ribosomal P site in Drosophila melanogaster

    SciTech Connect

    North, D.

    1987-01-01

    Several recent studies have probed the peptidyl transferase region of the Drosophila ribosome via the use of reactive site specific analogues (affinity labels). P site proteins adjacent to the 3' end of the amino acid bearing tRNA strand were labeled with modified tRNA fragments. Drugs affecting the binding of these agents were used to further clarify the nature of the region. The nascent peptide region of the P site was not labeled in previous experiments. To label that region radioactive Bromoacetylphenylalanyl-tRNA (BrAcphe-tRNA) was synthesized. The alpha-bromoacetyl group of this analogue is potentially reactive with nucleophiles present in either proteins or RNAs. Charged tRNAs and tRNA analogues bearing a peptide bond on the N-terminus of their amino acid are recognized as having affinity for the ribosomal P site. Specific labeling of the P site by BrAcphe-tRNA was confirmed by its ability to radioactively label proteins indirectly. As many as 8 ribosomal proteins may be labeled under these conditions, however, the majority of the bound label is associated with 3 large subunit proteins and 2 small subunit proteins. Overlaps between the proteins labeled by BrAcphe-tRNA and those labeled by other affinity labels are examined and a model of the peptidyl transferase region of Drosophila ribosomes is presented.

  8. A general and efficient approach for NMR studies of peptide dynamics in class I MHC peptide binding grooves.

    PubMed

    Insaidoo, Francis K; Zajicek, Jaroslav; Baker, Brian M

    2009-10-20

    T-Cell receptor recognition of peptides bound by major histocompatibility complex (MHC) proteins initiates a cellular immune response. Dynamics of peptides within MHC binding grooves can influence TCR recognition, yet NMR studies which could address this rigorously have been hindered by the expense of isotopically labeled peptides and the large size of peptide-MHC complexes. Here we describe a methodology for characterizing peptide dynamics within MHC binding grooves via NMR, using a biosynthetic approach for producing labeled peptide. With the Tax(11-19) peptide bound to the human class I MHC HLA-A*0201, we demonstrate that peptide generated in this manner can be well characterized in MHC binding grooves by NMR, providing opportunities to more precisely study the role of peptide dynamics in TCR recognition. Demonstrating the utility of such studies, the data with the Tax(11-19) peptide indicate the presence of slow conformational exchange in the peptide, supporting an "induced-fit" style TCR binding mechanism.

  9. A general and efficient approach for NMR studies of peptide dynamics in class I MHC peptide binding grooves.

    PubMed

    Insaidoo, Francis K; Zajicek, Jaroslav; Baker, Brian M

    2009-10-20

    T-Cell receptor recognition of peptides bound by major histocompatibility complex (MHC) proteins initiates a cellular immune response. Dynamics of peptides within MHC binding grooves can influence TCR recognition, yet NMR studies which could address this rigorously have been hindered by the expense of isotopically labeled peptides and the large size of peptide-MHC complexes. Here we describe a methodology for characterizing peptide dynamics within MHC binding grooves via NMR, using a biosynthetic approach for producing labeled peptide. With the Tax(11-19) peptide bound to the human class I MHC HLA-A*0201, we demonstrate that peptide generated in this manner can be well characterized in MHC binding grooves by NMR, providing opportunities to more precisely study the role of peptide dynamics in TCR recognition. Demonstrating the utility of such studies, the data with the Tax(11-19) peptide indicate the presence of slow conformational exchange in the peptide, supporting an "induced-fit" style TCR binding mechanism. PMID:19772349

  10. DISSOCIATION OF ARSENITE-PEPTIDE COMPLEXES: TRIPHASIC NATURE, RATE CONSTANTS, HALF LIVES AND BIOLOGICAL IMPORTANCE

    EPA Science Inventory

    We determined the number and the dissociation rate constants of different complexes formed from arsenite and two peptides containing either one (RV AVGNDYASGYHYGV for peptide 20) or three cysteines (LE AWQGK VEGTEHLYSMK K for peptide 10) via radioactive 73As labeled arsenite and ...

  11. Histidine-iridium(III) coordination-based peptide luminogenic cyclization and cyclo-RGD peptides for cancer-cell targeting.

    PubMed

    Ma, Xiaochuan; Jia, Junli; Cao, Rui; Wang, Xiaobo; Fei, Hao

    2014-12-24

    In the field of peptide drug discovery, structural constraining and fluorescent labeling are two sought-after techniques important for both basic research and pharmaceutical development. In this work, we describe an easy-to-use approach for simultaneous peptide cyclization and luminescent labeling based on iridium(III)-histidine coordination (Ir-HH cyclization). Using a series of model peptides with histidine flanking each terminus, the binding activity and reaction kinetics of Ir-HH cyclization of different ring sizes were characterized. In the series, Ir-HAnH (n = 2, 3) with moderate ring sizes provides appropriate flexibility and proper distance between histidines for cyclic formation, which leads to the best binding affinity and structural stability in physiological conditions, as compared to other Ir-HH-cyclized peptides with smaller (n = 0, 1) or larger (n = 4, 5) ring sizes. Ir-HRGDH, an Ir-HH-cyclized peptide containing integrin targeting motif Arg-Gly-Asp (RGD), showed better targeting affinity than its linear form and enhanced membrane permeability in comparison with fluorescein-labeled cyclic RGDyK peptide. Cell death inducing peptide KLA-linked Ir-HRGDH (Ir-HRGDH-KLA) showed dramatically enhanced cytotoxicity and high selectivity for cancer cells versus noncancer cells. These data demonstrate that the method conveniently combines structural constraining of peptides with luminescent imaging capabilities, which facilitates functional and intracellular characterization of potential peptide-based drug leads, thus introducing a new tool to meet emerging needs in medicinal research.

  12. Antimicrobial peptides.

    PubMed

    Zhang, Ling-Juan; Gallo, Richard L

    2016-01-11

    Antimicrobial peptides and proteins (AMPs) are a diverse class of naturally occurring molecules that are produced as a first line of defense by all multicellular organisms. These proteins can have broad activity to directly kill bacteria, yeasts, fungi, viruses and even cancer cells. Insects and plants primarily deploy AMPs as an antibiotic to protect against potential pathogenic microbes, but microbes also produce AMPs to defend their environmental niche. In higher eukaryotic organisms, AMPs can also be referred to as 'host defense peptides', emphasizing their additional immunomodulatory activities. These activities are diverse, specific to the type of AMP, and include a variety of cytokine and growth factor-like effects that are relevant to normal immune homeostasis. In some instances, the inappropriate expression of AMPs can also induce autoimmune diseases, thus further highlighting the importance of understanding these molecules and their complex activities. This Primer will provide an update of our current understanding of AMPs. PMID:26766224

  13. Antimicrobial Peptides

    PubMed Central

    Bahar, Ali Adem; Ren, Dacheng

    2013-01-01

    The rapid increase in drug-resistant infections has presented a serious challenge to antimicrobial therapies. The failure of the most potent antibiotics to kill “superbugs” emphasizes the urgent need to develop other control agents. Here we review the history and new development of antimicrobial peptides (AMPs), a growing class of natural and synthetic peptides with a wide spectrum of targets including viruses, bacteria, fungi, and parasites. We summarize the major types of AMPs, their modes of action, and the common mechanisms of AMP resistance. In addition, we discuss the principles for designing effective AMPs and the potential of using AMPs to control biofilms (multicellular structures of bacteria embedded in extracellular matrixes) and persister cells (dormant phenotypic variants of bacterial cells that are highly tolerant to antibiotics). PMID:24287494

  14. Antimicrobial peptides.

    PubMed

    Bahar, Ali Adem; Ren, Dacheng

    2013-11-28

    The rapid increase in drug-resistant infections has presented a serious challenge to antimicrobial therapies. The failure of the most potent antibiotics to kill "superbugs" emphasizes the urgent need to develop other control agents. Here we review the history and new development of antimicrobial peptides (AMPs), a growing class of natural and synthetic peptides with a wide spectrum of targets including viruses, bacteria, fungi, and parasites. We summarize the major types of AMPs, their modes of action, and the common mechanisms of AMP resistance. In addition, we discuss the principles for designing effective AMPs and the potential of using AMPs to control biofilms (multicellular structures of bacteria embedded in extracellular matrixes) and persister cells (dormant phenotypic variants of bacterial cells that are highly tolerant to antibiotics).

  15. The active centre of rabbit muscle triose phosphate isomerase. The site that is labelled by glycidol phosphate.

    PubMed

    Miller, J C; Waley, S G

    1971-06-01

    1. Glycidol (2,3-epoxypropanol) phosphate is a specific irreversible inhibitor of rabbit muscle triose phosphate isomerase (EC 5.3.1.1); the site of attachment has now been studied. 2. The labelled enzyme was digested with pepsin and a modified peptide isolated. The sequence of the peptide is: Ala-Tyr-Glu-Pro-Val-Trp. 3. It is the glutamic acid residue in this peptide that is labelled: the peptide is thus a gamma-glutamyl ester derived from glycerol phosphoric acid. The same site is labelled by a mixture of glycidol and inorganic phosphate. 4. Kinetic and stereochemical features of these reactions are discussed.

  16. Peptide linkers for the assembly of semiconductor quantum dot bioconjugates

    NASA Astrophysics Data System (ADS)

    Boeneman, Kelly; Mei, Bing C.; Deschamps, Jeffrey R.; Delehanty, James B.; Mattoussi, Hedi; Medintz, Igor

    2009-02-01

    The use of semiconductor luminescent quantum dots for the labeling of biomolecules is rapidly expanding, however it still requires facile methods to attach functional globular proteins to biologically optimized quantum dots. Here we discuss the development of controlled variable length peptidyl linkers to attach biomolecules to poly(ethylene) glycol (PEG) coated quantum dots for both in vitro and in vivo applications. The peptides chosen, β-sheets and alpha helices are appended to polyhistidine sequences and this allows for control of the ratio of peptide bioconjugated to QD and the distance from QD to the biomolecule. Recombinant DNA engineering, bacterial peptide expression and Ni-NTA purification of histidine labeled peptides are utilized to create the linkers. Peptide length is confirmed by in vitro fluorescent resonance energy transfer (FRET).

  17. 99mTc: Labeling Chemistry and Labeled Compounds

    NASA Astrophysics Data System (ADS)

    Alberto, R.; Abram, U.

    This chapter reviews the radiopharmaceutical chemistry of technetium related to the synthesis of perfusion agents and to the labeling of receptor-binding biomolecules. To understand the limitations of technetium chemistry imposed by future application of the complexes in nuclear medicine, an introductory section analyzes the compulsory requirements to be considered when facing the incentive of introducing a novel radiopharmaceutical into the market. Requirements from chemistry, routine application, and market are discussed. In a subsequent section, commercially available 99mTc-based radiopharmaceuticals are treated. It covers the complexes in use for imaging the most important target organs such as heart, brain, or kidney. The commercially available radiopharmaceuticals fulfill the requirements outlined earlier and are discussed with this background. In a following section, the properties and perspectives of the different generations of radiopharmaceuticals are described in a general way, covering characteristics for perfusion agents and for receptor-specific molecules. Technetium chemistry for the synthesis of perfusion agents and the different labeling approaches for target-specific biomolecules are summarized. The review comprises a general introduction to the common approaches currently in use, employing the N x S4-x , [3+1] and 2-hydrazino-nicotinicacid (HYNIC) method as well as more recent strategies such as the carbonyl and the TcN approach. Direct labeling without the need of a bifunctional chelator is briefly reviewed as well. More particularly, recent developments in the labeling of concrete targeting molecules, the second generation of radiopharmaceuticals, is then discussed and prominent examples with antibodies/peptides, neuroreceptor targeting small molecules, myocardial imaging agents, vitamins, thymidine, and complexes relevant to multidrug resistance are given. In addition, a new approach toward peptide drug development is described. The section

  18. C-Peptide Test

    MedlinePlus

    ... C-peptide is a useful marker of insulin production. The following are some purposes of C-peptide ... it nearly impossible to directly evaluate endogenous insulin production. In these cases, C-peptide measurement is a ...

  19. A simple and effective method for producing nonrandom peptide libraries using cotton as a carrier in continuous flow peptide synthesizers.

    PubMed

    Mutulis, Felikss; Tysk, Marcus; Mutule, Ilze; Wikberg, Jarl E S

    2003-01-01

    A method has been developed for generating nonrandom peptide libraries on cotton. Disks of cotton fabric were chemically modified to enable peptide synthesis. Incorporation of a 6-aminocaproic acid residue handle on the cellulose turned out to be advantageous. Disks were labeled with silver ink, stacked one on top of another in a continuous flow peptide synthesizer column, and simultaneously subjected to automated synthesis procedures. Depending on the sequences to be synthesized, the automatic synthesis procedure was stopped, and the disks were removed from the column, sorted, and reapplied to subsequent synthesis steps. In this way, individual peptides could be easily prepared in milligram quantities on each of the cotton disks.

  20. Novel pH-Sensitive Cyclic Peptides.

    PubMed

    Weerakkody, Dhammika; Moshnikova, Anna; El-Sayed, Naglaa Salem; Adochite, Ramona-Cosmina; Slaybaugh, Gregory; Golijanin, Jovana; Tiwari, Rakesh K; Andreev, Oleg A; Parang, Keykavous; Reshetnyak, Yana K

    2016-01-01

    A series of cyclic peptides containing a number of tryptophan (W) and glutamic acid (E) residues were synthesized and evaluated as pH-sensitive agents for targeting of acidic tissue and pH-dependent cytoplasmic delivery of molecules. Biophysical studies revealed the molecular mechanism of peptides action and localization within the lipid bilayer of the membrane at high and low pHs. The symmetric, c[(WE)4WC], and asymmetric, c[E4W5C], cyclic peptides translocated amanitin, a polar cargo molecule of similar size, across the lipid bilayer and induced cell death in a pH- and concentration-dependent manner. Fluorescently-labelled peptides were evaluated for targeting of acidic 4T1 mammary tumors in mice. The highest tumor to muscle ratio (5.6) was established for asymmetric cyclic peptide, c[E4W5C], at 24 hours after intravenous administration. pH-insensitive cyclic peptide c[R4W5C], where glutamic acid residues (E) were replaced by positively charged arginine residues (R), did not exhibit tumor targeting. We have introduced a novel class of cyclic peptides, which can be utilized as a new pH-sensitive tool in investigation or targeting of acidic tissue. PMID:27515582

  1. Novel pH-Sensitive Cyclic Peptides

    PubMed Central

    Weerakkody, Dhammika; Moshnikova, Anna; El-Sayed, Naglaa Salem; Adochite, Ramona-Cosmina; Slaybaugh, Gregory; Golijanin, Jovana; Tiwari, Rakesh K.; Andreev, Oleg A.; Parang, Keykavous; Reshetnyak, Yana K.

    2016-01-01

    A series of cyclic peptides containing a number of tryptophan (W) and glutamic acid (E) residues were synthesized and evaluated as pH-sensitive agents for targeting of acidic tissue and pH-dependent cytoplasmic delivery of molecules. Biophysical studies revealed the molecular mechanism of peptides action and localization within the lipid bilayer of the membrane at high and low pHs. The symmetric, c[(WE)4WC], and asymmetric, c[E4W5C], cyclic peptides translocated amanitin, a polar cargo molecule of similar size, across the lipid bilayer and induced cell death in a pH- and concentration-dependent manner. Fluorescently-labelled peptides were evaluated for targeting of acidic 4T1 mammary tumors in mice. The highest tumor to muscle ratio (5.6) was established for asymmetric cyclic peptide, c[E4W5C], at 24 hours after intravenous administration. pH-insensitive cyclic peptide c[R4W5C], where glutamic acid residues (E) were replaced by positively charged arginine residues (R), did not exhibit tumor targeting. We have introduced a novel class of cyclic peptides, which can be utilized as a new pH-sensitive tool in investigation or targeting of acidic tissue. PMID:27515582

  2. Induced Pluripotent Stem Cell Labeling Using Quantum Dots

    PubMed Central

    Yukawa, Hiroshi; Suzuki, Kaoru; Kano, Yuki; Yamada, Tatsuya; Kaji, Noritada; Ishikawa, Tetsuya; Baba, Yoshinobu

    2013-01-01

    Induced pluripotent stem (iPS) cells have received remarkable attention as the cell sources for clinical applications of regenerative medicine including stem cell therapy. Additionally, labeling technology is in high demand for tracing transplanted cells used in stem cell therapy. In this study, we used quantum dots (QDs), which have distinct fluorescence abilities in comparison with traditional probes, as the labeling materials and investigated whether iPS cells could be labeled with QDs with no cytotoxicity. iPS cells could not be labeled with QDs alone but required the use of cell-penetrating peptides such as octaarginine (R8). No significant cytotoxicity to iPS cells was confirmed by up to 8 nM QDs, and the iPS cells labeled with QDs maintained their undifferentiated state and pluripotency. These data suggest that QDs can be used for fluorescence labeling of iPS cells. PMID:26858884

  3. Measuring peptide translocation into large unilamellar vesicles.

    PubMed

    Spinella, Sara A; Nelson, Rachel B; Elmore, Donald E

    2012-01-27

    There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors(1). Many of these are referred to as cell-penetrating peptides, which are frequently noted for their potential as drug delivery vectors(1-3). Moreover, there is increasing interest in antimicrobial peptides that operate via non-membrane lytic mechanisms(4,5), particularly those that cross bacterial membranes without causing cell lysis and kill cells by interfering with intracellular processes(6,7). In fact, authors have increasingly pointed out the relationship between cell-penetrating and antimicrobial peptides(1,8). A firm understanding of the process of membrane translocation and the relationship between peptide structure and its ability to translocate requires effective, reproducible assays for translocation. Several groups have proposed methods to measure translocation into large unilamellar lipid vesicles (LUVs)(9-13). LUVs serve as useful models for bacterial and eukaryotic cell membranes and are frequently used in peptide fluorescent studies(14,15). Here, we describe our application of the method first developed by Matsuzaki and co-workers to consider antimicrobial peptides, such as magainin and buforin II(16,17). In addition to providing our protocol for this method, we also present a straightforward approach to data analysis that quantifies translocation ability using this assay. The advantages of this translocation assay compared to others are that it has the potential to provide information about the rate of membrane translocation and does not require the addition of a fluorescent label, which can alter peptide properties(18), to tryptophan-containing peptides. Briefly, translocation ability into lipid vesicles is measured as a function of the Foster Resonance Energy Transfer (FRET) between native tryptophan residues and dansyl phosphatidylethanolamine when proteins are associated with the external LUV membrane (Figure 1). Cell

  4. Copper-64 labelling of triazacyclononane-triphosphinate chelators.

    PubMed

    Simeček, Jakub; Wester, Hans-Jürgen; Notni, Johannes

    2012-12-01

    The 1,4,7-triazacyclononane-1,4,7-tris(methylenephosphinic acid) chelators TRAP and NOPO are complexing copper-64 with similar efficiency as 1,4,7-triazacyclononane-triacetic acid (NOTA). The kinetic stability of Cu-64-labelled TRAP-peptides is sufficient for PET imaging at early time points (1-2 h post injection). For labelling of TRAP conjugates, Cu-64 can be recommended as an alternative to Ga-68 to achieve higher resolution of PET images.

  5. [SYNTHETIC PEPTIDE VACCINES].

    PubMed

    Sergeyev, O V; Barinsky, I F

    2016-01-01

    An update on the development and trials of synthetic peptide vaccines is reviewed. The review considers the successful examples of specific protection as a result of immunization with synthetic peptides using various protocols. The importance of conformation for the immunogenicity of the peptide is pointed out. An alternative strategy of the protection of the organism against the infection using synthetic peptides is suggested.

  6. Secondary structure propensity and chirality of the amyloidophilic peptide p5 and its analogues impacts ligand binding - In vitro characterization

    DOE PAGESBeta

    Wall, Jonathan S.; Williams, Angela; Wooliver, Craig; Martin, Emily B.; Cheng, Xiaolin; Heidel, R. Eric; Kennel, Stephen J.

    2016-08-11

    Here, polybasic helical peptides, such as peptide p5, bind human amyloid extracts and synthetic amyloid fibrils. When radio labeled, peptide p5 has been shown to specifically bind amyloid in vivo thereby allowing imaging of the disease. Structural requirements for heparin and amyloid binding have been studied using analogues of p5 that modify helicity and chirality.

  7. Mental Labels and Tattoos

    ERIC Educational Resources Information Center

    Hyatt, I. Ralph

    1977-01-01

    Discusses the ease with which mental labels become imprinted in our system, six basic axioms for maintaining negative mental tattoos, and psychological processes for eliminating mental tattoos and labels. (RK)

  8. A Peptide-Based Method for 13C Metabolic Flux Analysis in Microbial Communities

    PubMed Central

    Ghosh, Amit; Nilmeier, Jerome; Weaver, Daniel; Adams, Paul D.; Keasling, Jay D.; Mukhopadhyay, Aindrila; Petzold, Christopher J.; Martín, Héctor García

    2014-01-01

    The study of intracellular metabolic fluxes and inter-species metabolite exchange for microbial communities is of crucial importance to understand and predict their behaviour. The most authoritative method of measuring intracellular fluxes, 13C Metabolic Flux Analysis (13C MFA), uses the labeling pattern obtained from metabolites (typically amino acids) during 13C labeling experiments to derive intracellular fluxes. However, these metabolite labeling patterns cannot easily be obtained for each of the members of the community. Here we propose a new type of 13C MFA that infers fluxes based on peptide labeling, instead of amino acid labeling. The advantage of this method resides in the fact that the peptide sequence can be used to identify the microbial species it originates from and, simultaneously, the peptide labeling can be used to infer intracellular metabolic fluxes. Peptide identity and labeling patterns can be obtained in a high-throughput manner from modern proteomics techniques. We show that, using this method, it is theoretically possible to recover intracellular metabolic fluxes in the same way as through the standard amino acid based 13C MFA, and quantify the amount of information lost as a consequence of using peptides instead of amino acids. We show that by using a relatively small number of peptides we can counter this information loss. We computationally tested this method with a well-characterized simple microbial community consisting of two species. PMID:25188426

  9. Peptide quantitation with methyl iodide isotopic tags and mass spectrometry.

    PubMed

    Blagojevic, Voislav; Zhidkov, Nickholas; Tharmaratnam, Samuel; Pham, Van Thong; Kaplan, Harvey; Bohme, Diethard K

    2010-06-01

    A novel method is presented for the quantitation of peptides based on their methylation by in vacuo chemical reaction with methyl iodide. Samples of two small peptides, hexaglycine and pentaalanine, were labeled with CH(3)I and CD(3)I, representing the "unknown" and "standard" respectively, and then subjected to a series of tests using mass spectrometry to ascertain the suitability of the isotopic labels for peptide quantitation. The experiments show methyl iodide to be a very quantitative label, exhibiting a linear relationship in concentration over the dynamic range of the mass spectrometer used in the analysis (up to 4 orders of magnitude) both as pure samples and in a complex mixture of peptides. The tendency of trimethylated peptides to preferentially form a(2) fragment ions in MS(2) produces a significant increase in sensitivity, especially when the mass spectrometer is used in the MRM mode. Tests were also performed to verify the stability of the label against H/D exchange and its suitability for long-term storage, showing little degradation while in solution and during subsequent chemical processing.

  10. Brain natriutetic peptide test

    MedlinePlus

    ... medlineplus.gov/ency/article/007509.htm Brain natriuretic peptide test To use the sharing features on this page, please enable JavaScript. Brain natriuretic peptide (BNP) test is a blood test that measures ...

  11. Vasoactive intestinal peptide test

    MedlinePlus

    ... medlineplus.gov/ency/article/003508.htm Vasoactive intestinal peptide test To use the sharing features on this page, please enable JavaScript. Vasoactive intestinal peptide (VIP) is a test that measures the amount ...

  12. Preparation of Tyr-C-peptide from genetically altered human insulin precursor.

    PubMed

    Sun, H; Tang, J; Hu, M

    1995-12-01

    C-peptide radioimmunoassay (C-peptide RIA) is widely used in determination of pancreatic B-cell secretion activity. 125I labeled Tyr-C-peptide is indispensable in C-peptide RIA kit. Herein we discuss a way of obtaining recombinant Tyr-C-peptide. Arg32Tyr human pro-insulin mutant (R32Y-proinsulin) gene was constructed by site-directed mutagenesis and overexpressed in Escherichia coli. Purified R32Y-proinsulin was converted to insulin and Tyr-C-peptide by trypsin and carboxypeptidase B codigestion. Tyr-C-peptide was isolated through reverse-phase HPLC (RP-HPLC) and identified by C-peptide RIA and amino acid analysis.

  13. Preparation of Tyr-C-peptide from genetically altered human insulin presursor

    SciTech Connect

    Huaiyu Sun; Jianguo Tang; Meihao Hu

    1995-12-01

    C-peptide radiommunoassay (C-peptide RIA) is widely used in determination of pancreatic B-cell secretion activity. {sup 125}I labeled Tyr-C-peptide is indispensable in C-peptide RIA kit. Herein we discuss a way of obtaining recombinant Tyr-C-peptide. Arg32Tyr human proinsulin mutant (R32Y-proinsulin) gene was constructed by site-directed mutagenesis and overexpressed in Escherichia coli. Purified R32Y-proinsulin was converted to insulin and Tyr-C-peptide by trypsin and carboxypeptidase B codigestion. Tyr-C-peptide was isolated through reverse-phase HPLC (RP-HPLC) and identified by C-peptide RIA and amino acid analysis. 12 refs., 5 figs., 1 tab.

  14. [SYNTHETIC PEPTIDE VACCINES].

    PubMed

    Sergeyev, O V; Barinsky, I F

    2016-01-01

    An update on the development and trials of synthetic peptide vaccines is reviewed. The review considers the successful examples of specific protection as a result of immunization with synthetic peptides using various protocols. The importance of conformation for the immunogenicity of the peptide is pointed out. An alternative strategy of the protection of the organism against the infection using synthetic peptides is suggested. PMID:27145593

  15. A common precursor to two major crab neurosecretory peptides.

    PubMed

    Stuenkel, E L

    1986-01-01

    The biosynthesis of proteins by the X-organ sinus gland (XOSG) neurosecretory system of the crab, Cardisoma carnifex was studied using the pulse-chase technique. Analysis of radioactive proteins following 2D-PAGE showed that during pulse incubations of less than or equal to 30 min a single predominant 14Kd prohormone was synthesized. With chase less than or equal to 3 hr the primary 14Kd protein was found to undergo differential and/or multiple post-translational modifications prior to its proteolytic cleavage. Increasing the chase to greater than 3 hr showed a shift in labeling from the 14Kd forms to 3 separate 6Kd proteins. Two of the 6Kd proteins were identified as crustacean hyperglycemic peptides (CHH). Similarity in protein labeling using [3H]leucine and [35S]cysteine suggest a second major peptide group, the H peptide, known to lack cysteine, is also contained within the 14Kd precursor. Peptide mapping of the 14Kd proteins and of unlabeled CHH and peptide H provide substantive evidence for this biosynthetic scheme. Thus, both the CHH and H peptide groups, which together constitute greater than 90% of the XOSG peptide content, in this species, arise from a common 14Kd precursor molecule.

  16. Antimicrobial Peptides in 2014

    PubMed Central

    Wang, Guangshun; Mishra, Biswajit; Lau, Kyle; Lushnikova, Tamara; Golla, Radha; Wang, Xiuqing

    2015-01-01

    This article highlights new members, novel mechanisms of action, new functions, and interesting applications of antimicrobial peptides reported in 2014. As of December 2014, over 100 new peptides were registered into the Antimicrobial Peptide Database, increasing the total number of entries to 2493. Unique antimicrobial peptides have been identified from marine bacteria, fungi, and plants. Environmental conditions clearly influence peptide activity or function. Human α-defensin HD-6 is only antimicrobial under reduced conditions. The pH-dependent oligomerization of human cathelicidin LL-37 is linked to double-stranded RNA delivery to endosomes, where the acidic pH triggers the dissociation of the peptide aggregate to release its cargo. Proline-rich peptides, previously known to bind to heat shock proteins, are shown to inhibit protein synthesis. A model antimicrobial peptide is demonstrated to have multiple hits on bacteria, including surface protein delocalization. While cell surface modification to decrease cationic peptide binding is a recognized resistance mechanism for pathogenic bacteria, it is also used as a survival strategy for commensal bacteria. The year 2014 also witnessed continued efforts in exploiting potential applications of antimicrobial peptides. We highlight 3D structure-based design of peptide antimicrobials and vaccines, surface coating, delivery systems, and microbial detection devices involving antimicrobial peptides. The 2014 results also support that combination therapy is preferred over monotherapy in treating biofilms. PMID:25806720

  17. PH dependent adhesive peptides

    DOEpatents

    Tomich, John; Iwamoto, Takeo; Shen, Xinchun; Sun, Xiuzhi Susan

    2010-06-29

    A novel peptide adhesive motif is described that requires no receptor or cross-links to achieve maximal adhesive strength. Several peptides with different degrees of adhesive strength have been designed and synthesized using solid phase chemistries. All peptides contain a common hydrophobic core sequence flanked by positively or negatively charged amino acids sequences.

  18. Effects of histatin 5 and derived peptides on Candida albicans.

    PubMed Central

    Ruissen, A L; Groenink, J; Helmerhorst, E J; Walgreen-Weterings, E; Van't Hof, W; Veerman, E C; Nieuw Amerongen, A V

    2001-01-01

    Three anti-microbial peptides were compared with respect to their killing activity against Candida albicans and their ability to disturb its cellular and internal membranes. Histatin 5 is an anti-fungal peptide occurring naturally in human saliva, while dhvar4 and dhvar5 are variants of its active domain, with increased anti-microbial activity. dhvar4 has increased amphipathicity compared with histatin 5, whereas dhvar5 has amphipathicity comparable with that of histatin 5. All three peptides caused depolarization of the cytoplasmic and/or mitochondrial membrane, indicating membranolytic activity. For the variant peptides both depolarization and killing occurred at a faster rate. With FITC-labelled peptides, no association with the cytoplasmic membrane was observed, contradicting the formation of permanent transmembrane multimeric peptide pores. Instead, the peptides were internalized and act on internal membranes, as demonstrated with mitochondrion- and vacuole-specific markers. In comparison with histatin 5, the variant peptides showed a more destructive effect on mitochondria. Entry of the peptides and subsequent killing were dependent on the metabolic state of the cells. Blocking of the mitochondrial activity led to complete protection against histatin 5 activity, whereas that of dhvar4 was hardly affected and that of dhvar5 was affected only intermediately. PMID:11368762

  19. Bar Code Labels

    NASA Technical Reports Server (NTRS)

    1988-01-01

    American Bar Codes, Inc. developed special bar code labels for inventory control of space shuttle parts and other space system components. ABC labels are made in a company-developed anodizing aluminum process and consecutively marketed with bar code symbology and human readable numbers. They offer extreme abrasion resistance and indefinite resistance to ultraviolet radiation, capable of withstanding 700 degree temperatures without deterioration and up to 1400 degrees with special designs. They offer high resistance to salt spray, cleaning fluids and mild acids. ABC is now producing these bar code labels commercially or industrial customers who also need labels to resist harsh environments.

  20. Computational peptide vaccinology.

    PubMed

    Söllner, Johannes

    2015-01-01

    Immunoinformatics focuses on modeling immune responses for better understanding of the immune system and in many cases for proposing agents able to modify the immune system. The most classical of these agents are vaccines derived from living organisms such as smallpox or polio. More modern vaccines comprise recombinant proteins, protein domains, and in some cases peptides. Generating a vaccine from peptides however requires technologies and concepts very different from classical vaccinology. Immunoinformatics therefore provides the computational tools to propose peptides suitable for formulation into vaccines. This chapter introduces the essential biological concepts affecting design and efficacy of peptide vaccines and discusses current methods and workflows applied to design successful peptide vaccines using computers.

  1. Analysis of illegal peptide biopharmaceuticals frequently encountered by controlling agencies.

    PubMed

    Vanhee, Celine; Janvier, Steven; Desmedt, Bart; Moens, Goedele; Deconinck, Eric; De Beer, Jacques O; Courselle, Patricia

    2015-09-01

    derivatization or the use of expensive labelled peptides. This quantification method was successfully validated for a representative subset of 10 different peptides by using the "total error" approach in accordance with the validation requirements of ISO-17025.

  2. Analysis of illegal peptide biopharmaceuticals frequently encountered by controlling agencies.

    PubMed

    Vanhee, Celine; Janvier, Steven; Desmedt, Bart; Moens, Goedele; Deconinck, Eric; De Beer, Jacques O; Courselle, Patricia

    2015-09-01

    derivatization or the use of expensive labelled peptides. This quantification method was successfully validated for a representative subset of 10 different peptides by using the "total error" approach in accordance with the validation requirements of ISO-17025. PMID:26003685

  3. A Study into the Collision-induced Dissociation (CID) Behavior of Cross-Linked Peptides*

    PubMed Central

    Giese, Sven H.; Fischer, Lutz; Rappsilber, Juri

    2016-01-01

    Cross-linking/mass spectrometry resolves protein–protein interactions or protein folds by help of distance constraints. Cross-linkers with specific properties such as isotope-labeled or collision-induced dissociation (CID)-cleavable cross-linkers are in frequent use to simplify the identification of cross-linked peptides. Here, we analyzed the mass spectrometric behavior of 910 unique cross-linked peptides in high-resolution MS1 and MS2 from published data and validate the observation by a ninefold larger set from currently unpublished data to explore if detailed understanding of their fragmentation behavior would allow computational delivery of information that otherwise would be obtained via isotope labels or CID cleavage of cross-linkers. Isotope-labeled cross-linkers reveal cross-linked and linear fragments in fragmentation spectra. We show that fragment mass and charge alone provide this information, alleviating the need for isotope-labeling for this purpose. Isotope-labeled cross-linkers also indicate cross-linker-containing, albeit not specifically cross-linked, peptides in MS1. We observed that acquisition can be guided to better than twofold enrich cross-linked peptides with minimal losses based on peptide mass and charge alone. By help of CID-cleavable cross-linkers, individual spectra with only linear fragments can be recorded for each peptide in a cross-link. We show that cross-linked fragments of ordinary cross-linked peptides can be linearized computationally and that a simplified subspectrum can be extracted that is enriched in information on one of the two linked peptides. This allows identifying candidates for this peptide in a simplified database search as we propose in a search strategy here. We conclude that the specific behavior of cross-linked peptides in mass spectrometers can be exploited to relax the requirements on cross-linkers. PMID:26719564

  4. Spanish labeling guide.

    PubMed

    Juliá, A M; García, S V; Breckinridge, M F

    1983-01-01

    A systematic reference of English-Spanish prescription label translations is presented. The purpose of the reference list (which is the most comprehensive published to date) is to enable a pharmacist to write precise, accurate label directions in Spanish for any patient who cannot read English.

  5. Peptide storage: are you getting the best return on your investment? Defining optimal storage conditions for proteomics samples.

    PubMed

    Kraut, Alexandra; Marcellin, Marlène; Adrait, Annie; Kuhn, Lauriane; Louwagie, Mathilde; Kieffer-Jaquinod, Sylvie; Lebert, Dorothée; Masselon, Christophe D; Dupuis, Alain; Bruley, Christophe; Jaquinod, Michel; Garin, Jérôme; Gallagher-Gambarelli, Maighread

    2009-07-01

    To comply with current proteomics guidelines, it is often necessary to analyze the same peptide samples several times. Between analyses, the sample must be stored in such a way as to conserve its intrinsic properties, without losing either peptides or signal intensity. This article describes two studies designed to define the optimal storage conditions for peptide samples between analyses. With the use of a label-free strategy, peptide conservation was compared over a 28-day period in three different recipients: standard plastic tubes, glass tubes, and low-adsorption plastic tubes. The results of this study showed that standard plastic tubes are unsuitable for peptide storage over the period studied. Glass tubes were found to perform better than standard plastic, but optimal peptide recovery was achieved using low-adsorption plastic tubes. The peptides showing poor recovery following storage were mainly hydrophobic in nature. The differences in peptide recovery between glass and low-adsorption plastic tubes were further studied using isotopically labeled proteins. This study allowed accurate comparison of peptide recovery between the two tube types within the same LC-MS run. The results of the label-free study were confirmed. Further, it was possible to demonstrate that peptide recovery in low-adsorption plastic tubes was optimal whatever the peptide concentration stored.

  6. Spin-labeled polyribonucleotides.

    PubMed Central

    Petrov, A I; Sukhorukov, B I

    1980-01-01

    Poly (U), poly (C) and poly (A) were spin labeled with N-(2,2,5,5-tetramethyl-3-carbonylpyrroline-1-oxyl)-imidazole. This spin label interacts selectively with 2' OH ribose groups of polynucleotides and does not modify the nucleic acid bases. The extent of spin labeling is not dependent upon the nature of the base and is entirely determined by rigidity of the secondary structure of the polynucleotide. The extent of modification for poly (U), poly (C) and poly (A) was 4.2, 1.7 and 1.5 per cent, respectively, the secondary structure of the polynucleotides being practically unchanged. Some physico-chemical properties of the spin-labeled polynucleotides were investigated by ESR spectroscopy. Rotational correlation times of the spin label and activation energy of its motion were calculated. PMID:6253911

  7. Label fusion strategy selection.

    PubMed

    Robitaille, Nicolas; Duchesne, Simon

    2012-01-01

    Label fusion is used in medical image segmentation to combine several different labels of the same entity into a single discrete label, potentially more accurate, with respect to the exact, sought segmentation, than the best input element. Using simulated data, we compared three existing label fusion techniques-STAPLE, Voting, and Shape-Based Averaging (SBA)-and observed that none could be considered superior depending on the dissimilarity between the input elements. We thus developed an empirical, hybrid technique called SVS, which selects the most appropriate technique to apply based on this dissimilarity. We evaluated the label fusion strategies on two- and three-dimensional simulated data and showed that SVS is superior to any of the three existing methods examined. On real data, we used SVS to perform fusions of 10 segmentations of the hippocampus and amygdala in 78 subjects from the ICBM dataset. SVS selected SBA in almost all cases, which was the most appropriate method overall. PMID:22518113

  8. Chemical labeling of carbohydrates by oxidation and sodium borohydride reduction.

    PubMed

    Fukuda, M

    2001-05-01

    This unit describes a collection of methods for chemical labeling of carbohydrates (free oligosaccharides or oligosaccharides conjugated to proteins, peptides, or lipids) by oxidation followed by reduction or by direct reduction. Oligosaccharides can be labeled with either radioisotopes or nonradioactive fluorescent molecules. These labelings allow one to follow the oligosaccharides during chromatography and in cells if labeled by fluorescent molecules. Selective oxidation with mild periodate followed by reduction with tritiated sodium borohydride results in selective radiolabeling of sialic acid residues on oligosaccharides or glycoproteins. Alternatively, treatment of samples with galactose oxidase results in oxidation of galactose or N-acetylgalactosamine residues at nonreducing termini, rendering these residues susceptible to labeling with NaB[3H]4. Oxidized glycoconjugates can also be labeled using the fluorescent probe lucifer yellow CH. Free oligosaccharides can be labeled by reduction with NaB[3H]4. An additional protocol describes the release and simultaneous labeling of O-glycan oligosaccharides by alkaline beta-elimination in the presence of NaB[3H]4.

  9. Plant peptide hormone signalling.

    PubMed

    Motomitsu, Ayane; Sawa, Shinichiro; Ishida, Takashi

    2015-01-01

    The ligand-receptor-based cell-to-cell communication system is one of the most important molecular bases for the establishment of complex multicellular organisms. Plants have evolved highly complex intercellular communication systems. Historical studies have identified several molecules, designated phytohormones, that function in these processes. Recent advances in molecular biological analyses have identified phytohormone receptors and signalling mediators, and have led to the discovery of numerous peptide-based signalling molecules. Subsequent analyses have revealed the involvement in and contribution of these peptides to multiple aspects of the plant life cycle, including development and environmental responses, similar to the functions of canonical phytohormones. On the basis of this knowledge, the view that these peptide hormones are pivotal regulators in plants is becoming increasingly accepted. Peptide hormones are transcribed from the genome and translated into peptides. However, these peptides generally undergo further post-translational modifications to enable them to exert their function. Peptide hormones are expressed in and secreted from specific cells or tissues. Apoplastic peptides are perceived by specialized receptors that are located at the surface of target cells. Peptide hormone-receptor complexes activate intracellular signalling through downstream molecules, including kinases and transcription factors, which then trigger cellular events. In this chapter we provide a comprehensive summary of the biological functions of peptide hormones, focusing on how they mature and the ways in which they modulate plant functions.

  10. Plant peptide hormone signalling.

    PubMed

    Motomitsu, Ayane; Sawa, Shinichiro; Ishida, Takashi

    2015-01-01

    The ligand-receptor-based cell-to-cell communication system is one of the most important molecular bases for the establishment of complex multicellular organisms. Plants have evolved highly complex intercellular communication systems. Historical studies have identified several molecules, designated phytohormones, that function in these processes. Recent advances in molecular biological analyses have identified phytohormone receptors and signalling mediators, and have led to the discovery of numerous peptide-based signalling molecules. Subsequent analyses have revealed the involvement in and contribution of these peptides to multiple aspects of the plant life cycle, including development and environmental responses, similar to the functions of canonical phytohormones. On the basis of this knowledge, the view that these peptide hormones are pivotal regulators in plants is becoming increasingly accepted. Peptide hormones are transcribed from the genome and translated into peptides. However, these peptides generally undergo further post-translational modifications to enable them to exert their function. Peptide hormones are expressed in and secreted from specific cells or tissues. Apoplastic peptides are perceived by specialized receptors that are located at the surface of target cells. Peptide hormone-receptor complexes activate intracellular signalling through downstream molecules, including kinases and transcription factors, which then trigger cellular events. In this chapter we provide a comprehensive summary of the biological functions of peptide hormones, focusing on how they mature and the ways in which they modulate plant functions. PMID:26374891

  11. Cross-strand coupling of a beta-hairpin peptide stabilized with an Aib-Gly turn studied using isotope-edited IR spectroscopy.

    PubMed

    Huang, Rong; Setnicka, Vladimir; Etienne, Marcus A; Kim, Joohyun; Kubelka, Jan; Hammer, Robert P; Keiderling, Timothy A

    2007-11-01

    Isotope-edited IR spectroscopy was used to study a series of singly and doubly 13C=O-labeled beta-hairpin peptides stabilized by an Aib-Gly turn sequence. The double-labeled peptides have amide I' IR spectra that show different degrees of vibrational coupling between the 13C-labeled amides due to variations in the local geometry of the peptide structure. The single-labeled peptides provide controls to determine frequencies characteristic of the diagonal force field (FF) contributions at each position for the uncoupled 13C=O modes. Separation of diagonal FF and coupling effects on the spectra are used to explain the cross-strand labeled spectral patterns. DFT calculations based on an idealized model beta-hairpin peptide correctly predict the vibrational coupling patterns. Extending these model results by consideration of frayed ends and the hairpin conformational flexibility yields an alternate interpretation of details of the spectra. Temperature-dependent isotopically labeled IR spectra reveal differences in the thermal stabilities of the individual isotopically labeled sites. This is the first example of using an IR-based isotopic labeling technique to differentiate structural transitions at specific sites along the peptide backbone in model beta-hairpin peptides.

  12. Application of synthetic peptides for detection of anti-citrullinated peptide antibodies.

    PubMed

    Trier, Nicole Hartwig; Holm, Bettina Eide; Slot, Ole; Locht, Henning; Lindegaard, Hanne; Svendsen, Anders; Nielsen, Christoffer Tandrup; Jacobsen, Søren; Theander, Elke; Houen, Gunnar

    2016-02-01

    Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA) and represent an important tool for the serological diagnosis of RA. In this study, we describe ACPA reactivity to overlapping citrullinated Epstein-Barr virus nuclear antigen-1 (EBNA-1)-derived peptides and analyze their potential as substrates for ACPA detection by streptavidin capture enzyme-linked immunosorbent assay. Using systematically overlapping peptides, containing a 10 amino acid overlap, labelled with biotin C-terminally or N-terminally, sera from 160 individuals (RA sera (n=60), healthy controls (n=40), systemic lupus erythematosus (n=20), Sjögren's syndrome (n=40)) were screened for antibody reactivity. Antibodies to a panel of five citrullinated EBNA-1 peptides were found in 67% of RA sera, exclusively of the IgG isotype, while 53% of the patient sera reacted with a single peptide, ARGGSRERARGRGRG-Cit-GEKR, accounting for more than half of the ACPA reactivity alone. Moreover, these antibodies were detected in 10% of CCP2-negative RA sera. In addition, 47% of the RA sera reacted with two or three citrullinated EBNA-1 peptides from the selected peptide panel. Furthermore, a negative correlation between the biotin attachment site and the location of citrulline in the peptides was found, i.e. the closer the citrulline was located to biotin, the lower the antibody reactivity. Our data suggest that citrullinated EBNA-1 peptides may be considered a substrate for the detection of ACPAs and that the presence of Epstein-Barr virus may play a role in the induction of these autoantibodies.

  13. Probing Protein Structure by Amino Acid-Specific Covalent Labeling and Mass Spectrometry

    PubMed Central

    Mendoza, Vanessa Leah; Vachet, Richard W.

    2009-01-01

    For many years, amino acid-specific covalent labeling has been a valuable tool to study protein structure and protein interactions, especially for systems that are difficult to study by other means. These covalent labeling methods typically map protein structure and interactions by measuring the differential reactivity of amino acid side chains. The reactivity of amino acids in proteins generally depends on the accessibility of the side chain to the reagent, the inherent reactivity of the label and the reactivity of the amino acid side chain. Peptide mass mapping with ESI- or MALDI-MS and peptide sequencing with tandem MS are typically employed to identify modification sites to provide site-specific structural information. In this review, we describe the reagents that are most commonly used in these residue-specific modification reactions, details about the proper use of these covalent labeling reagents, and information about the specific biochemical problems that have been addressed with covalent labeling strategies. PMID:19016300

  14. Insights into the Mechanism of Peptide Cyclodehydrations Achieved Through the Chemoenzymatic Generation of Amide Derivatives

    PubMed Central

    Dunbar, Kyle L.; Mitchell, Douglas A.

    2013-01-01

    Current strategies for generating peptides and proteins bearing amide carbonyl derivatives rely on solid-phase peptide synthesis for amide functionalization. Although such strategies have been successfully implemented, technical limitations restrict both the length and sequence of the synthetic fragments. Herein we report the repurposing of a thiazole/oxazole-modified microcin (TOMM) cyclodehydratase to site-specifically install amide backbone labels onto diverse peptide substrates, a method we refer to as azoline-mediated peptide backbone labeling (AMPL). This convenient chemoenzymatic strategy can generate both thioamides and amides with isotopically labeled oxygen atoms. Moreover, we demonstrate the first leader peptide-independent activity of a TOMM synthetase, circumventing the requirement that sequences of interest be fused to a leader peptide for modification. Through bioinformatics-guided site-directed mutagenesis, we also convert a strictly dehydrogenase-dependent TOMM azole synthetase into an azoline synthetase. This vastly expands the spectrum of substrates modifiable by AMPL by allowing any in vitro reconstituted TOMM synthetase to be employed. To demonstrate the utility of AMPL for mechanistic enzymology studies, an 18O-labeled substrate was generated to provide direct evidence that cyclodehydrations in TOMMs occur through the phosphorylation of the carbonyl oxygen preceding the cyclized residue. Furthermore, we demonstrate that AMPL is a useful tool for establishing the location of azolines both on in vitro modified peptides and azoline-containing natural products. PMID:23721104

  15. How to Read Drug Labels

    MedlinePlus

    ... and alternative medicine Healthy Aging How to read drug labels Printer-friendly version How to Read Drug ... read drug labels How to read a prescription drug label View a text version of this picture. ...

  16. Use of Cation Exchange Chromatography for Human C-peptide Isotope Dilution – Mass Spectrometric Assay

    PubMed Central

    Stoyanov, Alexander V.; Rohlfing, Curt L.; Connolly, Shawn; Roberts, Matthew L.; Nauser, Christopher L.; Little, Randie R.

    2016-01-01

    An application of ion exchange chromatography for C-peptide analysis is described here. At the stage of C-peptide isolation, a strong cation exchanger (SP HP or MonoS) was used to purify the analyte from ballast proteins and peptides. The conditions of ion-exchange chromatographic separations were optimized using theoretical modeling of the net surface electric charge of the peptide as a function of pH. The purified and concentrated sample was further subjected to LC-MS/MS. In order to improve the reliability of analysis, two fragment ions were monitored simultaneously both for native C-peptide and internal standard, isotopically labeled C-peptides analogues (fragments with m/z of 927.7 and 147.2). Using ion-exchange chromatography, it became possible to process larger sample volumes, important for testing patients with very low C peptide levels, compared to currently used solid phase extraction methods. PMID:22098929

  17. Capacitive label reader

    DOEpatents

    Arlowe, H.D.

    1983-07-15

    A capacitive label reader includes an outer ring transmitting portion, an inner ring transmitting portion, and a plurality of insulated receiving portions. A label is the mirror-image of the reader except that identifying portions corresponding to the receiving portions are insulated from only one of two coupling elements. Positive and negative pulses applied, respectively, to the two transmitting rings biased a CMOS shift register positively to either a 1 or 0 condition. The output of the CMOS may be read as an indication of the label.

  18. Peptide Based Radiopharmaceuticals: Specific Construct Approach

    SciTech Connect

    Som, P; Rhodes, B A; Sharma, S S

    1997-10-21

    The objective of this project was to develop receptor based peptides for diagnostic imaging and therapy. A series of peptides related to cell adhesion molecules (CAM) and immune regulation were designed for radiolabeling with 99mTc and evaluated in animal models as potential diagnostic imaging agents for various disease conditions such as thrombus (clot), acute kidney failure, and inflection/inflammation imaging. The peptides for this project were designed by the industrial partner, Palatin Technologies, (formerly Rhomed, Inc.) using various peptide design approaches including a newly developed rational computer assisted drug design (CADD) approach termed MIDAS (Metal ion Induced Distinctive Array of Structures). In this approach, the biological function domain and the 99mTc complexing domain are fused together so that structurally these domains are indistinguishable. This approach allows construction of conformationally rigid metallo-peptide molecules (similar to cyclic peptides) that are metabolically stable in-vivo. All the newly designed peptides were screened in various in vitro receptor binding and functional assays to identify a lead compound. The lead compounds were formulated in a one-step 99mTc labeling kit form which were studied by BNL for detailed in-vivo imaging using various animals models of human disease. Two main peptides usingMIDAS approach evolved and were investigated: RGD peptide for acute renal failure and an immunomodulatory peptide derived from tuftsin (RMT-1) for infection/inflammation imaging. Various RGD based metallopeptides were designed, synthesized and assayed for their efficacy in inhibiting ADP-induced human platelet aggregation. Most of these peptides displayed biological activity in the 1-100 µM range. Based on previous work by others, RGD-I and RGD-II were evaluated in animal models of acute renal failure. These earlier studies showed that after acute ischemic injury the renal cortex displays

  19. Antimicrobial Peptides in Reptiles

    PubMed Central

    van Hoek, Monique L.

    2014-01-01

    Reptiles are among the oldest known amniotes and are highly diverse in their morphology and ecological niches. These animals have an evolutionarily ancient innate-immune system that is of great interest to scientists trying to identify new and useful antimicrobial peptides. Significant work in the last decade in the fields of biochemistry, proteomics and genomics has begun to reveal the complexity of reptilian antimicrobial peptides. Here, the current knowledge about antimicrobial peptides in reptiles is reviewed, with specific examples in each of the four orders: Testudines (turtles and tortosises), Sphenodontia (tuataras), Squamata (snakes and lizards), and Crocodilia (crocodilans). Examples are presented of the major classes of antimicrobial peptides expressed by reptiles including defensins, cathelicidins, liver-expressed peptides (hepcidin and LEAP-2), lysozyme, crotamine, and others. Some of these peptides have been identified and tested for their antibacterial or antiviral activity; others are only predicted as possible genes from genomic sequencing. Bioinformatic analysis of the reptile genomes is presented, revealing many predicted candidate antimicrobial peptides genes across this diverse class. The study of how these ancient creatures use antimicrobial peptides within their innate immune systems may reveal new understandings of our mammalian innate immune system and may also provide new and powerful antimicrobial peptides as scaffolds for potential therapeutic development. PMID:24918867

  20. Metal-tag labeling coupled with multiple reaction monitoring-mass spectrometry for absolute quantitation of proteins.

    PubMed

    Wang, Xueying; Wang, Xin; Qin, Weijie; Lin, Hongjun; Wang, Jifeng; Wei, Junying; Zhang, Yangjun; Qian, Xiaohong

    2013-09-21

    Mass spectrometry-based quantitative proteomics, consisting of relative and absolute parts, has been used to discover and validate proteins with key functions related to physiological and pathological processes. Currently, stable isotope dilution-multiple reaction monitoring-mass spectrometry (SID-MRM-MS) is the most commonly used method for the absolute determination of proteins in a biological sample. A prerequisite for this method is obtaining internal standards with isotope labels. Although many approaches have been developed for the labeling and preparation of internal peptides, expensive stable isotope labeling coupled with SID-MRM-MS has limited the application and development of an absolute quantitative method. Recently, a low-cost strategy using metal-tag labeling and MS has been developed for relative quantification of peptides or proteins. The introduction of labeling using metal tags has the merits of allowing multiple labeling and enlarging the mass shift to overcome the overlap of adjacent isotope clusters. However, most papers described MRM-MS for protein absolute quantification based on the metal in its peptides labelled with metal by inductively coupled plasma mass spectrometry (ICP MS) but not on its peptides labelled with metal. In this work, a novel approach based on metal-tag labeling coupled with MRM-MS was established for the absolute quantification of peptides or proteins. The principle of the method is that a bifunctional chelator, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid bearing an N-hydroxysuccinimide ester (DOTA-NHS ester), is used to modify the N-termini of signature peptides from a target protein, and the modified peptides then chelate a certain metal, such as thulium, to form metal-tagged peptides (Tm-DOTA-P). Internal peptides are chemically synthesized and labeled with another metal, such as terbium (Tb-DOTA-P), as the internal standard. Both the Tb-DOTA- and Tm-DOTA-labeled peptides in samples can be analysed via

  1. Chemical Derivatization of Peptide Carboxyl Groups for Highly Efficient Electron Transfer Dissociation

    NASA Astrophysics Data System (ADS)

    Frey, Brian L.; Ladror, Daniel T.; Sondalle, Samuel B.; Krusemark, Casey J.; Jue, April L.; Coon, Joshua J.; Smith, Lloyd M.

    2013-11-01

    The carboxyl groups of tryptic peptides were derivatized with a tertiary or quaternary amine labeling reagent to generate more highly charged peptide ions that fragment efficiently by electron transfer dissociation (ETD). All peptide carboxyl groups—aspartic and glutamic acid side-chains as well as C-termini—were derivatized with an average reaction efficiency of 99 %. This nearly complete labeling avoids making complex peptide mixtures even more complex because of partially-labeled products, and it allows the use of static modifications during database searching. Alkyl tertiary amines were found to be the optimal labeling reagent among the four types tested. Charge states are substantially higher for derivatized peptides: a modified tryptic digest of bovine serum albumin (BSA) generates ~90% of its precursor ions with z > 2, compared with less than 40 % for the unmodified sample. The increased charge density of modified peptide ions yields highly efficient ETD fragmentation, leading to many additional peptide identifications and higher sequence coverage (e.g., 70 % for modified versus only 43 % for unmodified BSA). The utility of this labeling strategy was demonstrated on a tryptic digest of ribosomal proteins isolated from yeast cells. Peptide derivatization of this sample produced an increase in the number of identified proteins, a >50 % increase in the sequence coverage of these proteins, and a doubling of the number of peptide spectral matches. This carboxyl derivatization strategy greatly improves proteome coverage obtained from ETD-MS/MS of tryptic digests, and we anticipate that it will also enhance identification and localization of post-translational modifications.

  2. Chemical derivatization of peptide carboxyl groups for highly efficient electron transfer dissociation

    PubMed Central

    Frey, Brian L.; Ladror, Daniel T.; Sondalle, Samuel B.; Krusemark, Casey J.; Jue, April L.; Coon, Joshua J.; Smith, Lloyd M.

    2013-01-01

    The carboxyl groups of tryptic peptides were derivatized with a tertiary or quaternary amine labeling reagent to generate more highly charged peptide ions that fragment efficiently by electron transfer dissociation (ETD). All peptide carboxyl groups—aspartic and glutamic acid side-chains as well as C-termini—were derivatized with an average reaction efficiency of 99%. This nearly complete labeling avoids making complex peptide mixtures even more complex due to partially-labeled products, and it allows the use of static modifications during database searching. Alkyl tertiary amines were found to be the optimal labeling reagent among the four types tested. Charge states are substantially higher for derivatized peptides: a modified tryptic digest of bovine serum albumin (BSA) generates ∼90% of its precursor ions with z > 2, compared to less than 40% for the unmodified sample. The increased charge density of modified peptide ions yields highly efficient ETD fragmentation, leading to many additional peptide identifications and higher sequence coverage (e.g. 70% for modified versus only 43% for unmodified BSA). The utility of this labeling strategy was demonstrated on a tryptic digest of ribosomal proteins isolated from yeast cells. Peptide derivatization of this sample produced an increase in the number of identified proteins, a >50% increase in the sequence coverage of these proteins, and a doubling of the number of peptide spectral matches. This carboxyl derivatization strategy greatly improves proteome coverage obtained from ETD-MS/MS of tryptic digests, and we anticipate that it will also enhance identification and localization of post-translational modifications. PMID:23918461

  3. Development of peptide and protein based radiopharmaceuticals.

    PubMed

    Wynendaele, Evelien; Bracke, Nathalie; Stalmans, Sofie; De Spiegeleer, Bart

    2014-01-01

    Radiolabelled peptides and proteins have recently gained great interest as theranostics, due to their numerous and considerable advantages over small (organic) molecules. Developmental procedures of these radiolabelled biomolecules start with the radiolabelling process, greatly defined by the amino acid composition of the molecule and the radionuclide used. Depending on the radionuclide selection, radiolabelling starting materials are whether or not essential for efficient radiolabelling, resulting in direct or indirect radioiodination, radiometal-chelate coupling, indirect radiofluorination or (3)H/(14)C-labelling. Before preclinical investigations are performed, quality control analyses of the synthesized radiopharmaceutical are recommended to eliminate false positive or negative functionality results, e.g. changed receptor binding properties due to (radiolabelled) impurities. Therefore, radionuclidic, radiochemical and chemical purity are investigated, next to the general peptide attributes as described in the European and the United States Pharmacopeia. Moreover, in vitro and in vivo stability characteristics of the peptides and proteins also need to be explored, seen their strong sensitivity to proteinases and peptidases, together with radiolysis and trans-chelation phenomena of the radiopharmaceuticals. In vitro biomedical characterization of the radiolabelled peptides and proteins is performed by saturation, kinetic and competition binding assays, analyzing KD, Bmax, kon, koff and internalization properties, taking into account the chemical and metabolic stability and adsorption events inherent to peptides and proteins. In vivo biodistribution can be adapted by linker, chelate or radionuclide modifications, minimizing normal tissue (e.g. kidney and liver) radiation, and resulting in favorable dosimetry analyses. Finally, clinical trials are initiated, eventually leading to the marketing of radiolabelled peptides and proteins for PET/SPECT-imaging and therapy

  4. Dual-purpose linker for alpha helix stabilization and imaging agent conjugation to glucagon-like peptide-1 receptor ligands.

    PubMed

    Zhang, Liang; Navaratna, Tejas; Liao, Jianshan; Thurber, Greg M

    2015-02-18

    Peptides display many characteristics of efficient imaging agents such as rapid targeting, fast background clearance, and low non-specific cellular uptake. However, poor stability, low affinity, and loss of binding after labeling often preclude their use in vivo. Using glucagon-like peptide-1 receptor (GLP-1R) ligands exendin and GLP-1 as a model system, we designed a novel α-helix-stabilizing linker to simultaneously address these limitations. The stabilized and labeled peptides showed an increase in helicity, improved protease resistance, negligible loss or an improvement in binding affinity, and excellent in vivo targeting. The ease of incorporating azidohomoalanine in peptides and efficient reaction with the dialkyne linker enable this technique to potentially be used as a general method for labeling α helices. This strategy should be useful for imaging beta cells in diabetes research and in developing and testing other peptide targeting agents. PMID:25594741

  5. Preliminary study on the inhibition of nuclear internalization of Tat peptides by conjugation with a receptor-specific peptide and fluorescent dyes

    NASA Astrophysics Data System (ADS)

    Shen, Duanwen; Liang, Kexiang; Ye, Yunpeng; Tetteh, Elizabeth; Achilefu, Samuel

    2006-02-01

    Numerous studies have shown that basic Tat peptide (48-57) internalized non-specifically in cells and localized in the nucleus. However, localization of imaging agents in cellular nucleus is not desirable because of the potential mutagenesis. When conjugated to the peptides that undergo receptor-mediated endocytosis, Tat peptide could target specific cells or pathologic tissue. We tested this hypothesis by incorporating a somatostatin receptor-avid peptide (octreotate, Oct) and two different fluorescent dyes, Cypate 2 (Cy2) and fluorescein 5'-carboxlic acid (5-FAM), into the Tat-peptide sequence. In addition to the Cy2 or 5-FAM-labeled Oct conjugated to Tat peptide (Tat) to produce Tat-Oct-Cypate2 or Tat-Oct-5-FAM, we also labeled the Tat the Tat peptide with these dyes (Tat-Cy2 and Tat-5-FAM) to serve as positive control. A somatostatin receptor-positive pancreatic tumor cell line, AR42J, was used to assess cell internalization. The results show that Tat-5-FAM and Tat-Cypate2 localized in both nucleus and cytoplasm of the cells. In contrast to Tat-Oct-Cypate2, which localized in both the cytoplasm and nucleus, Tat-Oct-5-FAM internalized in the cytoplasm but not in the nucleus of AR42J cells. The internalizations were inhibited by adding non-labeled corresponding peptides, suggesting that the endocytoses of each group of labeled and the corresponding unlabeled compounds occurred through a common pathway. Thus, fluorescent probes and endocytosis complex between octreotate and somatostatin receptors in cytoplasm could control nuclear internalization of Tat peptides.

  6. Behind the Label "Alcoholic."

    ERIC Educational Resources Information Center

    Wright, Deborah M.

    1989-01-01

    Relates individual's personal story of her childhood influenced by her parent's alcoholism, her own alcoholism as a young adult, and her experiences with counseling. Asks others not to reject her because of the label "alcoholic." (ABL)

  7. Like your labels?

    PubMed

    Field, Michele

    2010-01-01

    The descriptive “conventions” used on food labels are always evolving. Today, however, the changes are so complicated (partly driven by legislation requiring disclosures about environmental impacts, health issues, and geographical provenance) that these labels more often baffle buyers than enlighten them. In a light-handed manner, the article points to how sometimes reading label language can be like deciphering runes—and how if we are familiar with the technical terms, we can find a literal meaning, but still not see the implications. The article could be ten times longer because food labels vary according to cultures—but all food-exporting cultures now take advantage of our short attention-span when faced with these texts. The question is whether less is more—and if so, in this contest for our attention, what “contestant” is voted off. PMID:21539053

  8. Inhibition of beta-amyloid aggregation by fluorescent dye labels

    SciTech Connect

    Amaro, Mariana; Wellbrock, Thorben; Birch, David J. S.; Rolinski, Olaf J.

    2014-02-10

    The fluorescence decay of beta-amyloid's (Aβ) intrinsic fluorophore tyrosine has been used for sensing the oligomer formation of dye-labelled Aβ monomers and the results compared with previously studied oligomerization of the non-labelledpeptides. It has been demonstrated that two different sized, covalently bound probes 7-diethylaminocoumarin-3-carbonyl and Hilyte Fluor 488 (HLF), alter the rate and character of oligomerization to different extents. The ability of HLF to inhibit formation of highly ordered structures containing beta-sheets was also shown. The implications of our findings for using fluorescence methods in amyloidosis research are discussed and the advantages of this auto-fluorescence approach highlighted.

  9. Inhibition of beta-amyloid aggregation by fluorescent dye labels

    NASA Astrophysics Data System (ADS)

    Amaro, Mariana; Wellbrock, Thorben; Birch, David J. S.; Rolinski, Olaf J.

    2014-02-01

    The fluorescence decay of beta-amyloid's (Aβ) intrinsic fluorophore tyrosine has been used for sensing the oligomer formation of dye-labelled Aβ monomers and the results compared with previously studied oligomerization of the non-labelledpeptides. It has been demonstrated that two different sized, covalently bound probes 7-diethylaminocoumarin-3-carbonyl and Hilyte Fluor 488 (HLF), alter the rate and character of oligomerization to different extents. The ability of HLF to inhibit formation of highly ordered structures containing beta-sheets was also shown. The implications of our findings for using fluorescence methods in amyloidosis research are discussed and the advantages of this auto-fluorescence approach highlighted.

  10. Insulin C-peptide test

    MedlinePlus

    C-peptide ... the test depends on the reason for the C-peptide measurement. Ask your health care provider if ... C-peptide is measured to tell the difference between insulin the body produces and insulin someone injects ...

  11. Bacteriocin Inducer Peptides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Novel peptides produced by bacteriocin-producing bacteria stimulate the production of bacteriocins in vitro. The producer bacteria are cultured in the presence of a novel inducer bacteria and a peptide having a carboxy terminal sequence of VKGLT in order to achieve an increase in bacteriocin produc...

  12. Introduction to peptide synthesis.

    PubMed

    Stawikowski, Maciej; Fields, Gregg B

    2012-08-01

    A number of synthetic peptides are significant commercial or pharmaceutical products, ranging from the dipeptide sugar substitute aspartame to clinically used hormones such as oxytocin, adrenocorticotropic hormone, and calcitonin. This unit provides an overview of the field of synthetic peptides and proteins. It discusses selecting the solid support and common coupling reagents. Additional information is provided regarding common side reactions and synthesizing modified residues.

  13. Routing and Label Space Reduction in Label Switching Networks

    NASA Astrophysics Data System (ADS)

    Solano, Fernando; Caro, Luis Fernando; Stidsen, Thomas; Papadimitriou, Dimitri

    This chapter is devoted to the analysis and modeling of some problems related to the optimal usage of the label space in label switching networks. Label space problems concerning three different technologies and architectures - namely Multi-protocol Label Switching (MPLS), Ethernet VLAN-Label Switching (ELS) and All-Optical Label Switching (AOLS) - are discussed in this chapter. Each of these cases yields to different constraints of the general label space reduction problem. We propose a generic optimization model and, then, we describe some adaptations aiming at modeling each particular case. Simulation results are briefly discussed at the end of this chapter.

  14. Antimicrobial Peptides from Fish

    PubMed Central

    Masso-Silva, Jorge A.; Diamond, Gill

    2014-01-01

    Antimicrobial peptides (AMPs) are found widely distributed through Nature, and participate in the innate host defense of each species. Fish are a great source of these peptides, as they express all of the major classes of AMPs, including defensins, cathelicidins, hepcidins, histone-derived peptides, and a fish-specific class of the cecropin family, called piscidins. As with other species, the fish peptides exhibit broad-spectrum antimicrobial activity, killing both fish and human pathogens. They are also immunomodulatory, and their genes are highly responsive to microbes and innate immuno-stimulatory molecules. Recent research has demonstrated that some of the unique properties of fish peptides, including their ability to act even in very high salt concentrations, make them good potential targets for development as therapeutic antimicrobials. Further, the stimulation of their gene expression by exogenous factors could be useful in preventing pathogenic microbes in aquaculture. PMID:24594555

  15. Off-Label Drug Use

    MedlinePlus

    ... Your Local Offices Close + - Text Size Off-label Drug Use What is off-label drug use? In the United States new drugs are ... unapproved use of a drug. Is off-label drug use legal? The off-label use of FDA- ...

  16. Novel alpha-MSH peptide analogs for melanoma targeting

    NASA Astrophysics Data System (ADS)

    Flook, Adam Michael

    Skin cancer is the one of the most diagnosed cancers in the United States with increasing incidence over the past two decades. There are three major forms of skin cancer but melanoma is the deadliest. It is estimated that 76,690 new diagnoses of melanoma and 9,480 deaths will occur in 2013. Melanoma accounts for approximately 1.6% of all cancer related deaths and is the 5 th leading diagnosed cancer in the United States. The mean survival rate of patients diagnosed with metastatic melanoma is six months, with five year survival rates of less than 5%. In this project, we describe the design and characterization of novel melanoma-targeting peptide analogs for use in diagnostic imaging of both primary and metastatic melanoma lesions. Novel alpha-MSH peptide conjugates were designed to target the melanocortin-1 receptor present and over-expressed on melanoma cells. These peptides were synthesized and their in-vitro melanocortin-1 receptor binding affinities were established in murine melanoma cells. Once binding affinities were determined, the peptides were radiolabeled with 99mTc utilizing a novel direct radiolabeling technique developed in our laboratory. The peptides were purified via reverse-phase high performance liquid chromatography and in-vivo melanoma targeting and pharmacokinetic properties were determined in B16/F1 melanoma-bearing female C57BL/6 mice. Biodistribution and SPECT/CT imaging studies were performed with the promising 99m Tc-labeled peptide conjugates. All alpha-MSH peptide conjugates tested showed low nanomolar binding affinity for the melanocortin-1 receptor. All peptides were readily radiolabeld with 99mTc with greater than 95% radiochemical purity. All 99mTc-labeled peptides displayed high specific in-vivo melanoma tumor uptake while maintaining low normal organ accumulation, and were excreted through the urinary system in a timely fashion. In addition, all tested 99mTc-labeld alpha-MSH peptides demonstrated clear visualization of in

  17. A novel method to measure HLA-DM-susceptibility of peptides bound to MHC class II molecules based on peptide binding competition assay and differential IC(50) determination.

    PubMed

    Yin, Liusong; Stern, Lawrence J

    2014-04-01

    HLA-DM (DM) functions as a peptide editor that mediates the exchange of peptides loaded onto MHCII molecules by accelerating peptide dissociation and association kinetics. The relative DM-susceptibility of peptides bound to MHCII molecules correlates with antigen presentation and immunodominance hierarchy, and measurement of DM-susceptibility has been a key effort in this field. Current assays of DM-susceptibility, based on differential peptide dissociation rates measured for individually labeled peptides over a long time base, are difficult and cumbersome. Here, we present a novel method to measure DM-susceptibility based on peptide binding competition assays performed in the presence and absence of DM, reported as a delta-IC(50) (change in 50% inhibition concentration) value. We simulated binding competition reactions of peptides with various intrinsic and DM-catalyzed kinetic parameters and found that under a wide range of conditions the delta-IC(50) value is highly correlated with DM-susceptibility as measured in off-rate assay. We confirmed experimentally that DM-susceptibility measured by delta-IC(50) is comparable to that measured by traditional off-rate assay for peptides with known DM-susceptibility hierarchy. The major advantage of this method is that it allows simple, fast and high throughput measurement of DM-susceptibility for a large set of unlabeled peptides in studies of the mechanism of DM action and for identification of CD4+ T cell epitopes.

  18. Engineering cyclic peptide toxins.

    PubMed

    Clark, Richard J; Craik, David J

    2012-01-01

    Peptide-based toxins have attracted much attention in recent years for their exciting potential applications in drug design and development. This interest has arisen because toxins are highly potent and selectively target a range of physiologically important receptors. However, peptides suffer from a number of disadvantages, including poor in vivo stability and poor bioavailability. A number of naturally occurring cyclic peptides have been discovered in plants, animals, and bacteria that have exceptional stability and potentially ameliorate these disadvantages. The lessons learned from studies of the structures, stabilities, and biological activities of these cyclic peptides can be applied to the reengineering of toxins that are not naturally cyclic but are amenable to cyclization. In this chapter, we describe solid-phase chemical synthetic methods for the reengineering of peptide toxins to improve their suitability as therapeutic, diagnostic, or imaging agents. The focus is on small disulfide-rich peptides from the venoms of cone snails and scorpions, but the technology is potentially widely applicable to a number of other peptide-based toxins. PMID:22230565

  19. Evaluation of empirical rule of linearly correlated peptide selection (ERLPS) for proteotypic peptide-based quantitative proteomics.

    PubMed

    Liu, Kehui; Zhang, Jiyang; Fu, Bin; Xie, Hongwei; Wang, Yingchun; Qian, Xiaohong

    2014-07-01

    Precise protein quantification is essential in comparative proteomics. Currently, quantification bias is inevitable when using proteotypic peptide-based quantitative proteomics strategy for the differences in peptides measurability. To improve quantification accuracy, we proposed an "empirical rule for linearly correlated peptide selection (ERLPS)" in quantitative proteomics in our previous work. However, a systematic evaluation on general application of ERLPS in quantitative proteomics under diverse experimental conditions needs to be conducted. In this study, the practice workflow of ERLPS was explicitly illustrated; different experimental variables, such as, different MS systems, sample complexities, sample preparations, elution gradients, matrix effects, loading amounts, and other factors were comprehensively investigated to evaluate the applicability, reproducibility, and transferability of ERPLS. The results demonstrated that ERLPS was highly reproducible and transferable within appropriate loading amounts and linearly correlated response peptides should be selected for each specific experiment. ERLPS was used to proteome samples from yeast to mouse and human, and in quantitative methods from label-free to O18/O16-labeled and SILAC analysis, and enabled accurate measurements for all proteotypic peptide-based quantitative proteomics over a large dynamic range.

  20. Peptide receptor radionuclide therapy for advanced neuroendocrine tumors.

    PubMed

    Bodei, Lisa; Cremonesi, Marta; Kidd, Mark; Grana, Chiara M; Severi, Stefano; Modlin, Irvin M; Paganelli, Giovanni

    2014-08-01

    Peptide receptor radionuclide therapy (PRRT) consists of the systemic administration of a synthetic peptide, labeled with a suitable β-emitting radionuclide, able to irradiate tumors and their metastases via internalization through a specific receptor (usually somatostatin S2), over-expressed on the cell membrane. After almost 2 decades of experience, PRRT, with either (90)Y-octreotide or (177)Lu-octreotate, has established itself to be an efficient and effective therapeutic modality. As a treatment, it is relatively safe up to the known thresholds of absorbed and bio-effective isotope dosages and the renal and hematological toxicity profiles are acceptable if adequate protective measures are undertaken.

  1. Coumarin tags for analysis of peptides by MALDI-TOF MS and MS/MS. 2. Alexa Fluor 350 tag for increased peptide and protein Identification by LC-MALDI-TOF/TOF MS.

    PubMed

    Pashkova, Anna; Chen, Hsuan-Shen; Rejtar, Tomas; Zang, Xin; Giese, Roger; Andreev, Victor; Moskovets, Eugene; Karger, Barry L

    2005-04-01

    The goal of this study was the development of N-terminal tags to improve peptide identification using high-throughput MALDI-TOF/TOF MS. Part 1 of the study was focused on the influence of derivatization on the intensities of MALDI-TOF MS signals of peptides. In part 2, various derivatization approaches for the improvement of peptide fragmentation efficiency in MALDI-TOF/TOF MS are explored. We demonstrate that permanent cation tags, while significantly improving signal intensity in the MS mode, lead to severe suppression of MS/MS fragmentation, making these tags unsuitable for high-throughput MALDI-TOF/TOF MS analysis. In the present work, it was found that labeling with Alexa Fluor 350, a coumarin tag containing a sulfo group, along with guanidation of epsilon-amino groups of Lys, could enhance unimolecular fragmentation of peptides with the formation of a high-intensity y-ion series, while the peptide intensities in the MS mode were not severely affected. LC-MALDI-TOF/TOF MS analysis of tryptic peptides from the SCX fractions of an E. coli lysate revealed improved peptide scores, a doubling of the total number of peptides, and a 30% increase in the number of proteins identified, as a result of labeling. Furthermore, by combining the data from native and labeled samples, confidence in correct identification was increased, as many proteins were identified by different peptides in the native and labeled data sets. Additionally, derivatization was found not to impair chromatographic behavior of peptides. All these factors suggest that labeling with Alexa Fluor 350 is a promising approach to the high-throughput LC-MALDI-TOF/TOF MS analysis of proteomic samples.

  2. Genetically encoded protein photocrosslinker with a transferable mass spectrometry-identifiable label

    PubMed Central

    Yang, Yi; Song, Haiping; He, Dan; Zhang, Shuai; Dai, Shizhong; Lin, Shixian; Meng, Rong; Wang, Chu; Chen, Peng R.

    2016-01-01

    Coupling photocrosslinking reagents with mass spectrometry has become a powerful tool for studying protein–protein interactions in living systems, but it still suffers from high rates of false-positive identifications as well as the lack of information on interaction interface due to the challenges in deciphering crosslinking peptides. Here we develop a genetically encoded photo-affinity unnatural amino acid that introduces a mass spectrometry-identifiable label (MS-label) to the captured prey proteins after photocrosslinking and prey–bait separation. This strategy, termed IMAPP (In-situ cleavage and MS-label transfer After Protein Photocrosslinking), enables direct identification of photo-captured substrate peptides that are difficult to uncover by conventional genetically encoded photocrosslinkers. Taking advantage of the MS-label, the IMAPP strategy significantly enhances the confidence for identifying protein–protein interactions and enables simultaneous mapping of the binding interface under living conditions. PMID:27460181

  3. Cyanine-based probe\\tag-peptide pair fluorescence protein imaging and fluorescence protein imaging methods

    DOEpatents

    Mayer-Cumblidge, M. Uljana; Cao, Haishi

    2013-01-15

    A molecular probe comprises two arsenic atoms and at least one cyanine based moiety. A method of producing a molecular probe includes providing a molecule having a first formula, treating the molecule with HgOAc, and subsequently transmetallizing with AsCl.sub.3. The As is liganded to ethanedithiol to produce a probe having a second formula. A method of labeling a peptide includes providing a peptide comprising a tag sequence and contacting the peptide with a biarsenical molecular probe. A complex is formed comprising the tag sequence and the molecular probe. A method of studying a peptide includes providing a mixture containing a peptide comprising a peptide tag sequence, adding a biarsenical probe to the mixture, and monitoring the fluorescence of the mixture.

  4. Tumor-Penetrating Peptides

    PubMed Central

    Teesalu, Tambet; Sugahara, Kazuki N.; Ruoslahti, Erkki

    2013-01-01

    Tumor-homing peptides can be used to deliver drugs into tumors. Phage library screening in live mice has recently identified homing peptides that specifically recognize the endothelium of tumor vessels, extravasate, and penetrate deep into the extravascular tumor tissue. The prototypic peptide of this class, iRGD (CRGDKGPDC), contains the integrin-binding RGD motif. RGD mediates tumor-homing through binding to αv integrins, which are selectively expressed on various cells in tumors, including tumor endothelial cells. The tumor-penetrating properties of iRGD are mediated by a second sequence motif, R/KXXR/K. This C-end Rule (or CendR) motif is active only when the second basic residue is exposed at the C-terminus of the peptide. Proteolytic processing of iRGD in tumors activates the cryptic CendR motif, which then binds to neuropilin-1 activating an endocytic bulk transport pathway through tumor tissue. Phage screening has also yielded tumor-penetrating peptides that function like iRGD in activating the CendR pathway, but bind to a different primary receptor. Moreover, novel tumor-homing peptides can be constructed from tumor-homing motifs, CendR elements and protease cleavage sites. Pathologies other than tumors can be targeted with tissue-penetrating peptides, and the primary receptor can also be a vascular “zip code” of a normal tissue. The CendR technology provides a solution to a major problem in tumor therapy, poor penetration of drugs into tumors. The tumor-penetrating peptides are capable of taking a payload deep into tumor tissue in mice, and they also penetrate into human tumors ex vivo. Targeting with these peptides specifically increases the accumulation in tumors of a variety of drugs and contrast agents, such as doxorubicin, antibodies, and nanoparticle-based compounds. Remarkably the drug to be targeted does not have to be coupled to the peptide; the bulk transport system activated by the peptide sweeps along any compound that is present in the

  5. Site-specific protein labeling with SNAP-tags.

    PubMed

    Cole, Nelson B

    2013-09-24

    Site-specific labeling of cellular proteins with chemical probes is a powerful tool for studying protein function in living cells. A number of small peptide and protein tags have been developed that can be labeled with synthetic probes with high efficiencies and specificities and provide flexibility not available with fluorescent proteins. The SNAP-tag is a modified form of the DNA repair enzyme human O(6)-alkylguanine-DNA-alkyltransferase, and undergoes a self-labeling reaction to form a covalent bond with O(6)-benzylguanine (BG) derivatives. BG can be modified with a wide variety of fluorophores and other reporter compounds, generally without affecting the reaction with the SNAP-tag. In this unit, basic strategies for labeling SNAP-tag fusion proteins, both for live cell imaging and for in vitro analysis, are described. This includes a description of a releasable SNAP-tag probe that allows the user to chemically cleave the fluorophore from the labeled SNAP-tag fusion. In vitro labeling of purified SNAP-tag fusions is briefly described.

  6. Introduction to peptide synthesis.

    PubMed

    Fields, Gregg B

    2002-05-01

    A number of synthetic peptides are significant commercial or pharmaceutical products, ranging from the dipeptide sugar-substitute aspartame to clinically used hormones, such as oxytocin, adrenocorticotropic hormone, and calcitonin. This unit provides an overview of the field of synthetic peptides and proteins, including their purification. It discusses selecting the solid support and common coupling reagents. Additional information is provided regarding common side reactions and synthesizing modified residues.

  7. Introduction to peptide synthesis.

    PubMed

    Fields, Gregg B

    2002-02-01

    A number of synthetic peptides are significant commercial or pharmaceutical products, ranging from the dipeptide sugar-substitute aspartame to clinically used hormones, such as oxytocin, adrenocorticotropic hormone, and calcitonin. This unit provides an overview of the field of synthetic peptides and proteins, including their purification. It discusses selecting the solid support and common coupling reagents. Additional information is provided regarding common side reactions and synthesizing modified residues.

  8. Localization and characterization of neuropeptide Y-like peptides in the brain and islet organ of the anglerfish (Lophius americanus).

    PubMed

    Noe, B D; Milgram, S L; Balasubramaniam, A; Andrews, P C; Calka, J; McDonald, J K

    1989-08-01

    Results from a previous report demonstrate that more than one molecular form of neuropeptide Y-like peptide may be present in the islet organ of the anglerfish (Lophius americanus). Most of the neuropeptide Y-like immunoreactive material was anglerfish peptide YG, which is expressed in a subset of islet cells, whereas an additional neuropeptide Y-like peptide(s) was localized in islet nerves. To learn more about the neuropeptide Y-like peptides in islet nerves, we have employed immunohistochemical and biochemical methods to compare peptides found in anglerfish islets and brain. Using antisera that selectively react with either mammalian forms of neuropeptide Y or with anglerfish peptide YG, subsets of neurons were found in the brain that labelled with only one or the other of the antisera. In separate sections, other neurons that were labelled with either antiserum exhibited similar morphologies. Peptides from brains and islets were subjected to gel filtration and reverse-phase high performance liquid chromatography. Radioimmunoassays employing either the neuropeptide Y or peptide YG antisera were used to examine chromatographic eluates. Immunoreactive peptides having retention times of human neuropeptide Y and porcine neuropeptide Y were identified in extracts of both brain and islets. This indicates that peptides structurally similar to both of these peptides from the neuropeptide Y-pancreatic polypeptide family are expressed in neurons of anglerfish brain and nerve fibers of anglerfish islets. The predominant form of neuropeptide Y-like peptide in islets was anglerfish peptide YG. Neuropeptide Y-immunoreactive peptides from islet extracts that had chromatographic retention times identical to human neuropeptide Y and porcine neuropeptide Y were present in much smaller quantities.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2673525

  9. A combined tryptic peptide and winged peptide internal standard approach for the determination of α-lactalbumin in dairy products by ultra high performance liquid chromatography with tandem mass spectrometry.

    PubMed

    Lai, Shiyun; Zhang, Jingshun; Zhang, Yu; Chen, Qi; Huang, Baifen; Ren, Yiping

    2015-05-01

    A robust ultra high performance liquid chromatography with tandem mass spectrometry method at peptide level was established for measuring α-lactalbumin in various dairy products. An isotope-labeled winged peptide (VKKILDKVG*INYW*LAHKALCSEKL) with extra amino acids of the sequence of signature peptide concatenated at each end as the internal standard was spiked in samples to participate in the whole tryptic digestion process. The peptide VG*INYW*LAHK that resulted from the isotope-labeled winged peptide was used as the final isotopically labeled internal standard of the α-lactalbumin signature peptide (VGINYWLAHK) during the quantitative analysis. The contents of α-lactalbumin in samples were calculated based on the equimolar relationship between the α-lactalbumin protein and signature peptide. The optimized molar ratio of trypsin to protein (1:60) and enzymatic digestion time (5 h) could not only improve the digestion efficiency and reduce the cost, but also minimize the period of sample pretreatment. Considering the robustness of the current method using the isotopically labeled internal standard and acceptable measurement cost, its application may promote the development of nutrient investigation and quality control of α-lactalbumin in dairy products. This protein analysis method might provide a new reference strategy for food analysis and quantitative protein analysis.

  10. Effect of Fluorescently Labeling Protein Probes on Kinetics of Protein-Ligand Reactions

    PubMed Central

    Sun, Y.S.; Landry, J.P.; Fei, Y.Y.; Luo, J.T.; Wang, X.B.; Lam, K.S.

    2009-01-01

    We studied the effect of fluorescently labeling proteins on protein-ligand reactions. Un-labeled ligands (streptavidin-binding peptides and rabbit immunoglobulin G (IgG) as antigen targets) are immobilized on epoxy-functionalized glass slides. Unlabeled and Cy3-labeled protein probes from the same batch (streptavidin and goat antibodies) subsequently react with the surface-immobilized targets. By monitoring in situ the surface mass density change using an oblique-incidence reflectivity difference scanning microscope (a label-free detector), we measured kon and koff for streptavidin-peptide reactions and antibody-antigen reaction. We found that (1) equilibrium dissociation constants, defined as KD = koff/kon, for streptavidin-peptide reactions increases by a factor of 3 ~ 4 when the solution-phase streptavidin is labeled with Cy3 dye; and (2) KD for reactions of solution-phase goat anti-rabbit antibodies with rabbit IgG targets also change significantly when the goat antibodies are labeled with Cy3 dye. PMID:18991423

  11. Label-Free Quantitative Proteomics in Yeast.

    PubMed

    Léger, Thibaut; Garcia, Camille; Videlier, Mathieu; Camadro, Jean-Michel

    2016-01-01

    Label-free bottom-up shotgun MS-based proteomics is an extremely powerful and simple tool to provide high quality quantitative analyses of the yeast proteome with only microgram amounts of total protein. Although the experimental design of this approach is rather straightforward and does not require the modification of growth conditions, proteins or peptides, several factors must be taken into account to benefit fully from the power of this method. Key factors include the choice of an appropriate method for the preparation of protein extracts, careful evaluation of the instrument design and available analytical capabilities, the choice of the quantification method (intensity-based vs. spectral count), and the proper manipulation of the selected quantification algorithm. The elaboration of this robust workflow for data acquisition, processing, and analysis provides unprecedented insight into the dynamics of the yeast proteome. PMID:26483028

  12. Electroactive Silica Nanoparticles for Biological Labeling

    SciTech Connect

    Wang, Jun; Liu, Guodong; Lin, Yuehe

    2006-08-29

    A novel electrochemical immuno-biosensor based on poly(guanine)-functionalized silica nanoparticle labels and mediator-generated catalytic reaction was described. The functionalized silica NPs conjugates were characterized by atomic force microscopy, X-ray photoelectron spectroscopy, and electrochemistry. This immunobiosensor is very sensitive and the limit of detection was found to be down to 0.2 ng/ml (4 pM), which was attributed to signal amplification by poly[G] functionalized silica NPs and guanine catalytic oxidation. Attractive feature of this approach is feasible to develop a cheap, sensitive and portable device for multiplexed diagnoses of different proteins. This method is simple, selective and reproducible for trace protein analysis and can be extended to study protein/protein, peptide/protein, and DNA/ protein interactions.

  13. Label-Free Quantitative Proteomics in Yeast.

    PubMed

    Léger, Thibaut; Garcia, Camille; Videlier, Mathieu; Camadro, Jean-Michel

    2016-01-01

    Label-free bottom-up shotgun MS-based proteomics is an extremely powerful and simple tool to provide high quality quantitative analyses of the yeast proteome with only microgram amounts of total protein. Although the experimental design of this approach is rather straightforward and does not require the modification of growth conditions, proteins or peptides, several factors must be taken into account to benefit fully from the power of this method. Key factors include the choice of an appropriate method for the preparation of protein extracts, careful evaluation of the instrument design and available analytical capabilities, the choice of the quantification method (intensity-based vs. spectral count), and the proper manipulation of the selected quantification algorithm. The elaboration of this robust workflow for data acquisition, processing, and analysis provides unprecedented insight into the dynamics of the yeast proteome.

  14. Labeling lake water with tritium

    USGS Publications Warehouse

    Frederick, B.J.

    1963-01-01

    A method of packaging tritiated water in a manner that facilitates safe handling in environmental labeling operations, and procedures followed in labeling a large body of water with a small volume of tritiated water are described. ?? 1963.

  15. 99m tc labeled liposomes

    SciTech Connect

    Phillips, W.T.; Klipper, R.W.; Timmons, J.H.; Rudolph, A.S.

    1992-10-27

    This patent describes a method of preparing stable gamma-emitting radionuclide-labeled alkyleneamine oxime, the incubating being for a period of time sufficient to form labeled liposome-encapsulated protein.

  16. Decode the Sodium Label Lingo

    MedlinePlus

    ... For Preschooler For Gradeschooler For Teen Decode the Sodium Label Lingo Published January 24, 2013 Print Email Reading food labels can help you slash sodium. Here's how to decipher them. "Sodium free" or " ...

  17. Differences in the production of spliced antigenic peptides by the standard proteasome and the immunoproteasome.

    PubMed

    Dalet, Alexandre; Stroobant, Vincent; Vigneron, Nathalie; Van den Eynde, Benoît J

    2011-01-01

    Peptide splicing allows the production of antigenic peptides composed of two fragments initially non-contiguous in the parental protein. The proposed mechanism of splicing is a transpeptidation occurring within the proteasome. Three spliced peptides, derived from FGF-5, melanoma protein gp100 and nuclear protein SP110, have been described. Here, we compared the production of these spliced peptides by the standard proteasome and the immunoproteasome. Differential isotope labelling was used to quantify (by mass spectrometry) the fragments contained in digests obtained with precursor peptides and purified proteasomes. The results show that both the standard and the immunoproteasomes can produce spliced peptides although they differ in their efficiency of production of each peptide. The FGF-5 and gp100 peptides are more efficiently produced by the standard proteasome, whereas the SP110 peptide is more efficiently produced by the immunoproteasome. This seems to result from differences in the production of the two splicing partners, which depends on a balance between cleavages liberating or destroying those fragments. By showing that splicing depends on the efficiency of production of the splicing partners, these results also support the transpeptidation model of peptide splicing. Furthermore, given the presence of immunoproteasomes in dendritic cells and cells exposed to IFN-γ, the findings may be relevant for vaccine design.

  18. Improved identification of wheat gluten proteins through alkylation of cysteine residues and peptide-based mass spectrometry

    PubMed Central

    Rombouts, Ine; Lagrain, Bert; Brunnbauer, Markus; Delcour, Jan A.; Koehler, Peter

    2013-01-01

    The concentration and composition of wheat gluten proteins and the presence, concentration and location of cysteine residues therein are important for wheat flour quality. However, it is difficult to identify gluten proteins, as they are an extremely polymorphic mixture of prolamins. We here present methods for cysteine labeling of wheat prolamins with 4-vinylpyridine (4-VP) and iodoacetamide (IDAM) which, as compared to label-free analysis, substantially improve identification of cysteine-containing peptides in enzymic prolamin digests by electrospray ionization - tandem mass spectrometry. Both chymotrypsin and thermolysin yielded cysteine-containing peptides from different gluten proteins, but more proteins could be identified after chymotryptic digestion. In addition, to the best of our knowledge, we were the first to label prolamins with isotope coded affinity tags (ICAT), which are commonly used for quantitative proteomics. However, more peptides were detected after labeling gluten proteins with 4-VP and IDAM than with ICAT. PMID:23880742

  19. Improved identification of wheat gluten proteins through alkylation of cysteine residues and peptide-based mass spectrometry.

    PubMed

    Rombouts, Ine; Lagrain, Bert; Brunnbauer, Markus; Delcour, Jan A; Koehler, Peter

    2013-01-01

    The concentration and composition of wheat gluten proteins and the presence, concentration and location of cysteine residues therein are important for wheat flour quality. However, it is difficult to identify gluten proteins, as they are an extremely polymorphic mixture of prolamins. We here present methods for cysteine labeling of wheat prolamins with 4-vinylpyridine (4-VP) and iodoacetamide (IDAM) which, as compared to label-free analysis, substantially improve identification of cysteine-containing peptides in enzymic prolamin digests by electrospray ionization--tandem mass spectrometry. Both chymotrypsin and thermolysin yielded cysteine-containing peptides from different gluten proteins, but more proteins could be identified after chymotryptic digestion. In addition, to the best of our knowledge, we were the first to label prolamins with isotope coded affinity tags (ICAT), which are commonly used for quantitative proteomics. However, more peptides were detected after labeling gluten proteins with 4-VP and IDAM than with ICAT.

  20. Targeting pre-miRNA by Peptide Nucleic Acids

    PubMed Central

    Avitabile, Concetta; Saviano, Michele; D'Andrea, Luca; Bianchi, Nicoletta; Fabbri, Enrica; Brognara, Eleonora; Gambari, Roberto; Romanelli, Alessandra

    2012-01-01

    PNAs conjugated to carrier peptides have been employed for the targeting of miRNA precursor, with the aim to develop molecules able to interfere in the pre-miRNA processing. The capability of the molecules to bind pre-miRNA has been tested in vitro by fluorescence assayes on Thiazole Orange labeled molecules and in vivo, in K562 cells, evaluating the amount of miRNA produced after treatment of cells with two amounts of PNAs. PMID:22699795

  1. A protein/peptide assay using peptide-resin adduct: application to the calmodulin/RS20 complex.

    PubMed

    Guimard, L; Afshar, M; Haiech, J; Calas, B

    1994-08-15

    To obtain equilibrium and kinetic constants of a protein/peptide complex, we have developed a rapid procedure which uses peptides specifically linked to a resin. With this peptide-resin adduct, bound and free 125I-labeled protein could be easily separated by simple centrifugation. The feasibility of the method was demonstrated with the calmodulin/RS20 complex, where RS20 is the putative calmodulin binding peptide of the smooth muscle myosin light chain kinase (smMLCK). In addition to the wild-type calmodulin (SYNCAM) expressed in Escherichia coli, we also examined calmodulin mutants with charge reversals called SYNCAM12A (DEE 118-120-->KKK) and SYNCAM18A (EEE 82-84-->KKK and DEE 118-120-->KKK). The kinetic analysis of the interaction between SYNCAM and RS20 associated with titration experiments allowed us to measure dissociation constants (KD) in the range of 10(-9) M, in good agreement with previously published data. Moreover, the binding assays showed that SYN-CAM18A did not interact with RS20, whereas SYN-CAM12A did with a KD around 10(-8) M. The lack of binding of SYNCAM18A to RS20 provides an explanation for the lack of smMLCK activation by SYNCAM18A. Altogether, these results demonstrate that peptide-resin can be used as a tool for separating bound from free protein, thus enabling a rapid and reliable quantification of the protein/peptide interaction.

  2. Reactive sulfhydryl groups of sarcoplasmic reticulum ATPase. II. Site of labeling with iodoacetamide and its fluorescent derivative.

    PubMed

    Yamashita, T; Kawakita, M

    1987-02-01

    Iodoacetamide (IAA) and its fluorescent derivative, 5-(2-iodoacetamidoethyl) amino-naphthalene-1-sulfonate (IAEDANS) specifically bind to a site on the C-terminal half of sarcoplasmic reticulum (SR) Ca2+,Mg2+-ATPase. The location of this specific binding site was identified. SR membranes were treated with 150 microM [14C]IAA at pH 7.0 and 30 degrees C. One mole of IAA per mole of ATPase was bound in 6 h without affecting the Ca2+-transport activity. [14C]IAA-labeled SR membranes were cleaved with BrCN, and 14C-labeled peptide fragments were separated by Sephadex LH-60 chromatography and then digested further with trypsin. A radioactive peptide (Ala-Cys 674-Cys-Phe-Ala-Arg) was purified by Sephadex LH-20 chromatography and C18 reversed phase HPLC (Cys denotes the [14C]IAA-binding site). IAEDANS-labeling was carried out by reacting SR membranes with 50 microM IAEDANS for 5 h, at pH 7.0 and 30 degrees C. A fluorescent peptide was successfully purified by the same procedures as for the IAA-labeled peptide, and the amino acid sequence analysis of this peptide revealed that the IAEDANS labeling site was identical with the IAA binding site.

  3. Protected amine labels: a versatile molecular scaffold for multiplexed nominal mass and sub-Da isotopologue quantitative proteomic reagents.

    PubMed

    Ficarro, Scott B; Biagi, Jessica M; Wang, Jinhua; Scotcher, Jenna; Koleva, Rositsa I; Card, Joseph D; Adelmant, Guillaume; He, Huan; Askenazi, Manor; Marshall, Alan G; Young, Nicolas L; Gray, Nathanael S; Marto, Jarrod A

    2014-04-01

    We assemble a versatile molecular scaffold from simple building blocks to create binary and multiplexed stable isotope reagents for quantitative mass spectrometry. Termed Protected Amine Labels (PAL), these reagents offer multiple analytical figures of merit including, (1) robust targeting of peptide N-termini and lysyl side chains, (2) optimal mass spectrometry ionization efficiency through regeneration of primary amines on labeled peptides, (3) an amino acid-based mass tag that incorporates heavy isotopes of carbon, nitrogen, and oxygen to ensure matched physicochemical and MS/MS fragmentation behavior among labeled peptides, and (4) a molecularly efficient architecture, in which the majority of hetero-atom centers can be used to synthesize a variety of nominal mass and sub-Da isotopologue stable isotope reagents. We demonstrate the performance of these reagents in well-established strategies whereby up to four channels of peptide isotopomers, each separated by 4 Da, are quantified in MS-level scans with accuracies comparable to current commercial reagents. In addition, we utilize the PAL scaffold to create isotopologue reagents in which labeled peptide analogs differ in mass based on the binding energy in carbon and nitrogen nuclei, thereby allowing quantification based on MS or MS/MS spectra. We demonstrate accurate quantification for reagents that support 6-plex labeling and propose extension of this scheme to 9-channels based on a similar PAL scaffold. Finally, we provide exemplar data that extend the application of isotopologe-based quantification reagents to medium resolution, quadrupole time-of-flight mass spectrometers.

  4. Gd(3+) Spin Labels Report the Conformation and Solvent Accessibility of Solution and Vesicle-Bound Melittin.

    PubMed

    Manukovsky, Nurit; Frydman, Veronica; Goldfarb, Daniella

    2015-10-29

    Although Gd(3+)-based spin labels have been shown to be an alternative to nitroxides for double electron-electron resonance (DEER) distance measurements at high fields, their ability to provide solvent accessibility information, as nitroxides do, has not been explored. In addition, the effect of the label type on the measured distance distribution has not been sufficiently characterized. In this work, we extended the applicability of Gd(3+) spin labels to solvent accessibility measurements on a peptide in model membranes, namely, large unilamellar vesicles (LUVs) using W-band (2)H Mims electron-nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) techniques and Gd(3+)-ADO3A-labeled melittin. In addition, we carried out Gd(3+)-Gd(3+) DEER distance measurements to probe the peptide conformation in solution and when bound to LUVs. A comparison with earlier results reported for the same system with nitroxide labels shows that, although in both cases the peptide binds parallel to the membrane surface, the Gd(3+)-ADO3A label tends to protrude from the membrane into the solvent, whereas the nitroxide does the opposite. This can be explained on the basis of the hydrophilicity of the Gd(3+)-ADO3A labels in contrast with the hydrophobicity of nitroxides. The distance distributions obtained from different labels are accordingly different, with the Gd(3+)-ADO3A yielding consistently broader distributions. These discrepancies are most pronounced when the peptide termini are labeled, which implies that such labeling positions may be inadvisible. PMID:26001213

  5. Antimicrobial Peptides from Plants.

    PubMed

    Tam, James P; Wang, Shujing; Wong, Ka H; Tan, Wei Liang

    2015-01-01

    Plant antimicrobial peptides (AMPs) have evolved differently from AMPs from other life forms. They are generally rich in cysteine residues which form multiple disulfides. In turn, the disulfides cross-braced plant AMPs as cystine-rich peptides to confer them with extraordinary high chemical, thermal and proteolytic stability. The cystine-rich or commonly known as cysteine-rich peptides (CRPs) of plant AMPs are classified into families based on their sequence similarity, cysteine motifs that determine their distinctive disulfide bond patterns and tertiary structure fold. Cystine-rich plant AMP families include thionins, defensins, hevein-like peptides, knottin-type peptides (linear and cyclic), lipid transfer proteins, α-hairpinin and snakins family. In addition, there are AMPs which are rich in other amino acids. The ability of plant AMPs to organize into specific families with conserved structural folds that enable sequence variation of non-Cys residues encased in the same scaffold within a particular family to play multiple functions. Furthermore, the ability of plant AMPs to tolerate hypervariable sequences using a conserved scaffold provides diversity to recognize different targets by varying the sequence of the non-cysteine residues. These properties bode well for developing plant AMPs as potential therapeutics and for protection of crops through transgenic methods. This review provides an overview of the major families of plant AMPs, including their structures, functions, and putative mechanisms. PMID:26580629

  6. Electron transfer in peptides.

    PubMed

    Shah, Afzal; Adhikari, Bimalendu; Martic, Sanela; Munir, Azeema; Shahzad, Suniya; Ahmad, Khurshid; Kraatz, Heinz-Bernhard

    2015-02-21

    In this review, we discuss the factors that influence electron transfer in peptides. We summarize experimental results from solution and surface studies and highlight the ongoing debate on the mechanistic aspects of this fundamental reaction. Here, we provide a balanced approach that remains unbiased and does not favor one mechanistic view over another. Support for a putative hopping mechanism in which an electron transfers in a stepwise manner is contrasted with experimental results that support electron tunneling or even some form of ballistic transfer or a pathway transfer for an electron between donor and acceptor sites. In some cases, experimental evidence suggests that a change in the electron transfer mechanism occurs as a result of donor-acceptor separation. However, this common understanding of the switch between tunneling and hopping as a function of chain length is not sufficient for explaining electron transfer in peptides. Apart from chain length, several other factors such as the extent of the secondary structure, backbone conformation, dipole orientation, the presence of special amino acids, hydrogen bonding, and the dynamic properties of a peptide also influence the rate and mode of electron transfer in peptides. Electron transfer plays a key role in physical, chemical and biological systems, so its control is a fundamental task in bioelectrochemical systems, the design of peptide based sensors and molecular junctions. Therefore, this topic is at the heart of a number of biological and technological processes and thus remains of vital interest.

  7. Antimicrobial Peptides from Plants

    PubMed Central

    Tam, James P.; Wang, Shujing; Wong, Ka H.; Tan, Wei Liang

    2015-01-01

    Plant antimicrobial peptides (AMPs) have evolved differently from AMPs from other life forms. They are generally rich in cysteine residues which form multiple disulfides. In turn, the disulfides cross-braced plant AMPs as cystine-rich peptides to confer them with extraordinary high chemical, thermal and proteolytic stability. The cystine-rich or commonly known as cysteine-rich peptides (CRPs) of plant AMPs are classified into families based on their sequence similarity, cysteine motifs that determine their distinctive disulfide bond patterns and tertiary structure fold. Cystine-rich plant AMP families include thionins, defensins, hevein-like peptides, knottin-type peptides (linear and cyclic), lipid transfer proteins, α-hairpinin and snakins family. In addition, there are AMPs which are rich in other amino acids. The ability of plant AMPs to organize into specific families with conserved structural folds that enable sequence variation of non-Cys residues encased in the same scaffold within a particular family to play multiple functions. Furthermore, the ability of plant AMPs to tolerate hypervariable sequences using a conserved scaffold provides diversity to recognize different targets by varying the sequence of the non-cysteine residues. These properties bode well for developing plant AMPs as potential therapeutics and for protection of crops through transgenic methods. This review provides an overview of the major families of plant AMPs, including their structures, functions, and putative mechanisms. PMID:26580629

  8. Phosphorylation-dependent changes in the spatial relationship between Ca-ATPase polypeptide chains in sarcoplasmic reticulum membranes.

    PubMed

    Bigelow, D J; Squier, T C; Inesi, G

    1992-04-01

    In order to investigate possible structural changes associated with the coupling mechanisms of the Ca-ATPase in sarcoplasmic reticulum membranes, we have utilized fluorescence resonance energy transfer between spectroscopic probes covalently bound to different domains of the ATPase. Using time-correlated single photon counting, we have directly measured the energy transfer efficiency between 5-[2-[(iodoacetyl)amino]ethyl]aminonaphthalene-1-sulfonic acid (IAEDANS), that is specifically bound to the B trypic fragment at cysteines 670 and 674 and acceptors covalently bound either near the nucleotide binding site, i.e. fluorescein 5-isothiocyanate at lysine 515, also on the B fragment, or maleimide-directed probes specifically located on the A1, tryptic fragment, i.e. 4-dimethylaminoazobenzene-4'-maleimide (DABmal) or fluorescein-5-maleimide (Fmal), probably at cysteines 344 and 364. All of these donor-acceptor pairs exhibit energy transfer both within and between Ca-ATPase molecules allowing us to investigate spatial relationships between the A1 and B domains and between different ATPase polypeptide chains. Differentiation between the intra- and intermolecular components of energy transfer was accomplished in two ways: 1) by comparing the transfer efficiencies in native membranes before and after detergent solubilization and 2) by reconstituting ATPase chains that have already been labeled with either the donor or acceptor chromophores. Using this approach, we find no significant change in the intramolecular transfer efficiency between any of these donor-acceptor pairs either upon binding of calcium to the high affinity sites or upon stabilization of the phosphoenzyme intermediate, indicating that there are no large structural changes within the B tryptic fragment or, alternatively, between the A1 and B fragments. With respect to intermolecular energy transfer, we observe no effect of calcium binding on the unliganded enzyme with either donor-acceptor pair. However

  9. A Dual-Purpose Linker for Alpha Helix Stabilization and Imaging Agent Conjugation to Glucagon-Like Peptide-1 Receptor Ligands

    PubMed Central

    Zhang, Liang; Navaratna, Tejas; Liao, Jianshan; Thurber, Greg M.

    2016-01-01

    Peptides display many characteristics of efficient imaging agents such as rapid targeting, fast background clearance, and low non-specific cellular uptake. However, poor stability, low affinity, and loss of binding after labeling often preclude their use in vivo. Using the glucagon-like peptide-1 receptor (GLP-1R) ligands exendin and GLP-1 as a model system, we designed a novel alpha helix stabilizing linker to simultaneously address these limitations. The stabilized and labeled peptides showed an increase in helicity, improved protease resistance, negligible loss or an improvement in binding affinity, and excellent in vivo targeting. The ease of incorporating azidohomoalanine in peptides and efficient reaction with the dialkyne linker enables this technique to potentially be used as a general method for labeling alpha helices. This strategy should be useful for imaging beta cells in diabetes research and in developing and testing other peptide targeting agents. PMID:25594741

  10. Novel Antimicrobial Peptides with High Anticancer Activity and Selectivity

    PubMed Central

    Chen, Kuan-Hao; Yu, Hui-Yuan; Chih, Ya-Han; Cheng, Hsi-Tsung; Chou, Yu-Ting; Cheng, Jya-Wei

    2015-01-01

    We describe a strategy to boost anticancer activity and reduce normal cell toxicity of short antimicrobial peptides by adding positive charge amino acids and non-nature bulky amino acid β-naphthylalanine residues to their termini. Among the designed peptides, K4R2-Nal2-S1 displayed better salt resistance and less toxicity to hRBCs and human fibroblast than Nal2-S1 and K6-Nal2-S1. Fluorescence microscopic studies indicated that the FITC-labeled K4R2-Nal2-S1 preferentially binds cancer cells and causes apoptotic cell death. Moreover, a significant inhibition in human lung tumor growth was observed in the xenograft mice treated with K4R2-Nal2-S1. Our strategy provides new opportunities in the development of highly effective and selective antimicrobial and anticancer peptide-based therapeutics. PMID:25970292

  11. BIOSYNTHESIS OF MYCOBACILLIN, A NEW ANTIFUNGAL PEPTIDE I.

    PubMed Central

    Banerjee, Arun B.; Bose, S. K.

    1964-01-01

    Banerjee, Arun B. (University of Calcutta, Calcutta, India), and S. K. Bose. Biosynthesis of mycobacillin, a new antifungal peptide. I. Role of nucleic acid. J. Bacteriol. 87:1397–1401. 1964.—The biosynthesis of mycobacillin, a cyclic polypeptide antifungal antibiotic, was studied in relation to the effect of chloramphenicol, 6-azathymine, and 5-bromouracil on the process. It was found that chloramphenicol inhibits both mycobacillin synthesis and growth, whereas nucleic acid base analogues inhibit only growth and nucleic acid synthesis but not mycobacillin formation. A change in the concentration of labeled aspartic acid in the general metabolic pool led to a corresponding change in the specific activity of aspartic acid isolated from different peptide fragments of the mycobacillin molecule, suggesting that mycobacillin synthesis occurs by way of linear addition of amino acid to the peptide chain. PMID:14188719

  12. Development and characterization of novel 8-plex DiLeu isobaric labels for quantitative proteomics and peptidomics

    PubMed Central

    Frost, Dustin C.; Greer, Tyler; Xiang, Feng; Liang, Zhidan; Li, Lingjun

    2015-01-01

    Rationale Relative quantification of proteins via their enzymatically digested peptide products determines disease biomarker candidate lists in discovery studies. Isobaric label-based strategies using TMT and iTRAQ allow for up to 10 samples to be multiplexed in one experiment, but their expense limits their use. The demand for cost-effective tagging reagents capable of multiplexing many samples led us to develop an 8-plex version of our isobaric labeling reagent, DiLeu. Methods The original 4-plex DiLeu reagent was extended to an 8-plex set by coupling isotopic variants of dimethylated leucine to an alanine balance group designed to offset the increasing mass of the label’s reporter group. Tryptic peptides from a single protein digest, a protein mixture digest, and Saccharomyces cerevisiae lysate digest were labeled with 8-plex DiLeu and analyzed via nanoLC-MS2 on a Q-Exactive Orbitrap mass spectrometer. Characteristics of 8-plex DiLeu-labeled peptides, including quantitative accuracy and fragmentation, were examined. Results An 8-plex set of DiLeu reagents with 1 Da-spaced reporters was synthesized at a yield of 36%. The average cost to label eight 100 μg peptide samples was calculated to be approximately $15. Normalized collision energy tests on the Q-Exactive revealed that a higher-energy collisional dissociation value of 27 generated the optimum number of high-quality spectral matches. Relative quantification of DiLeu-labeled peptides yielded normalized median ratios accurate to within 12% of their expected values. Conclusions Cost-effective 8-plex DiLeu reagents can be synthesized and applied to relative peptide and protein quantification. These labels increase the multiplexing capacity of our previous 4-plex implementation without requiring high-resolution instrumentation to resolve reporter ion signals. PMID:25981542

  13. Characterization and sequencing of an active-site cysteine-containing peptide from the xylanase of a thermotolerant Streptomyces.

    PubMed

    Keskar, S S; Rao, M B; Deshpande, V V

    1992-02-01

    The kinetics of chemical modification of the xylanase from a thermotolerant Streptomyces T7 indicated the involvement of 1 mol of cysteine residue/mol of enzyme [Keskar, Srinivasan & Deshpande (1989) Biochem. J. 261, 49-55]. The chromophoric reagent N-(2,4-dinitroanilino)maleimide (DAM) reacts covalently with thiol groups of xylanase with complete inactivation. Protection against inactivation was provided by the substrate (xylan). The purified xylanase that had been modified with DAM was digested with pepsin and the peptides were purified by gel filtration followed by peptide mapping. The active-site peptide was distinguished from the other thiol-containing peptides by comparison of the peptides generated by labelling the enzyme in the presence and in the absence of the substrate. The peptide mapping of the modified enzyme in the absence of xylan showed three yellow peptides, whereas in the presence of xylan only two yellow peptides were detected. The active-site peptide protected by the substrate failed to form the complex with DAM. The modified active-site peptide was isolated and sequenced. Gas-phase sequencing provided the following sequence: Ser-Val-Ile-Met-Xaa-Ile-Asp-His-Ile-Arg-Phe. This is the first report on the isolation and sequencing of the active-site peptide from a xylanase. The comparison of reactive cysteine-containing peptide sequence with the catalytic regions of other glucanases revealed the presence of a conserved aspartic acid residue.

  14. Signal peptide of cellulase.

    PubMed

    Yan, Shaomin; Wu, Guang

    2014-06-01

    Cellulase is an enzyme playing a crucial role in biotechnology industries ranging from textile to biofuel because of tremendous amount of cellulose produced in plant. In order to improve cellulase productivity, huge resource has been spent in search for good cellulases from microorganism in remote areas and in creation of ideal cellulase by engineering. However, not much attention is given to the secretion of cellulases from cell into extracellular space, where a cellulase plays its enzymatic role. In this minireview, the signal peptides, which lead secreted proteins to specific secretion systems and scatter in literature, are reviewed. The patterns of signal peptides are checked against 4,101 cellulases documented in UniProtKB, the largest protein database in the world, to determine how these cellulases are secreted. Simultaneous review on both literature and cellulases from the database not only provides updated knowledge on signal peptides but also indicates the gap in our research.

  15. Synthetic antibiofilm peptides.

    PubMed

    de la Fuente-Núñez, César; Cardoso, Marlon Henrique; de Souza Cândido, Elizabete; Franco, Octavio Luiz; Hancock, Robert E W

    2016-05-01

    Bacteria predominantly exist as multicellular aggregates known as biofilms that are associated with at least two thirds of all infections and exhibit increased adaptive resistance to conventional antibiotic therapies. Therefore, biofilms are major contributors to the global health problem of antibiotic resistance, and novel approaches to counter them are urgently needed. Small molecules of the innate immune system called host defense peptides (HDPs) have emerged as promising templates for the design of potent, broad-spectrum antibiofilm agents. Here, we review recent developments in the new field of synthetic antibiofilm peptides, including mechanistic insights, synergistic interactions with available antibiotics, and their potential as novel antimicrobials against persistent infections caused by biofilms. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert. PMID:26724202

  16. Learning with imperfectly labeled patterns

    NASA Technical Reports Server (NTRS)

    Chittineni, C. B.

    1979-01-01

    The problem of learning in pattern recognition using imperfectly labeled patterns is considered. The performance of the Bayes and nearest neighbor classifiers with imperfect labels is discussed using a probabilistic model for the mislabeling of the training patterns. Schemes for training the classifier using both parametric and non parametric techniques are presented. Methods for the correction of imperfect labels were developed. To gain an understanding of the learning process, expressions are derived for success probability as a function of training time for a one dimensional increment error correction classifier with imperfect labels. Feature selection with imperfectly labeled patterns is described.

  17. Peptide reporters of kinase activity in whole cell lysates

    PubMed Central

    Wu, Ding; Sylvester, Juliesta E.; Parker, Laurie L.; Zhou, Guangchang; Kron, Stephen J.

    2010-01-01

    Kinase assays are used to screen for small-molecule inhibitors that may show promise as targeted pharmaceutical therapies. Using cell lysates instead of purified kinases provides a more accurate estimate of inhibitor sensitivity and selectivity in a biological setting. This review summarizes the range of homogeneous (solution-phase) and heterogeneous (solid-supported) formats available for using peptide substrates to monitor kinase activities in cell lysates. With a focus on heterogeneous kinase assays, the peptide substrate Abltide is used as a model to optimize presentation geometries and the modular arrangement of short sequences for kinase recognition. We present results from peptides immobilized on two- and three-dimensional surfaces such as hydrogels on 96-well plates and glass slides, and fluorescent Luminex beads. We discuss methods to increase assay sensitivity using chemifluorescent ELISAs, antibody-based recognition, and label-free mass spectrometry. Monitoring the activity of specific kinases in cell lysates presents challenges that can be overcome by manipulating peptide substrates to optimize assay conditions. In particular, signal-to-background ratios were improved by 1) adding long branched hydrophilic linkers between the substrate and the surface, 2) changing the orientation of peptides relative to the surface, and 3) including peptide ligands in cis or in trans to recruit kinases to the surface. By improving the accessibility of immobilized peptide substrates to kinases in solution, the apparent rate of phosphorylation increased and assays were more sensitive to changes in endogenous kinase activities. These strategies can be generalized to improve the reactivity of most peptide substrates used in heterogeneous kinase assays with cell lysates. PMID:20593469

  18. Peptide receptor radionuclide therapy with somatostatin analogues in neuroendocrine tumors.

    PubMed

    Giovacchini, Giampiero; Nicolas, Guillaume; Forrer, Flavio

    2012-06-01

    Neuroendocrine tumors (NETs) are rare tumors with variable malignant behavior. The majority of NETs express increased levels of somatostatin (SST) receptors, particularly SST2 receptors. Radiolabeled peptides specific for the SST2 receptors may be used for diagnosis of NETs and for peptide receptor radionuclide therapy (PRRT). [(111)In-DTPA(0)]-octreotide has been the first peptide used for PRRT. This radiolabeled peptide, emitting Auger electrons, often induced symptomatic relief, but objective morphological responses were rarely documented. After the introduction of the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) other peptides, primarily [DOTA(0),Tyr(3)]octreotate (DOTATATE) and [DOTA(0),Tyr(3)]octreotide (DOTATOC) were labeled with (90)Y or (177)Lu and used for therapy applications. The rate of objective response obtained with these radiolabeled peptides ranges between 6% and 46%, owing to differences in inclusion criteria adopted in different studies, length and type of therapy, and criteria of evaluation of the response. The present data in the literature do not allow defining the most suitable peptide and radionuclide for the treatment of NETs. Instead emerging evidence indicates that a combination of nuclides with different physical characteristics might be more effective than the use of a single nuclide. Kidney and bone marrow toxicity are the limiting factors for PRRT. Mild toxicity is often encountered while severe toxicity is rarer. Toxicity could be reduced and therapeutic efficacy enhanced by patient-specific dosimetry. Future directions include different issues of PRRT, such as defining the most suitable treatment scheme, evaluation of new peptides with different affinity profiles to other SST receptor subtypes, and reduction of toxicity. PMID:22292758

  19. Biomimetic peptide nanosensors.

    PubMed

    Cui, Yue; Kim, Sang N; Naik, Rajesh R; McAlpine, Michael C

    2012-05-15

    The development of a miniaturized sensing platform tailored for sensitive and selective detection of a variety of biochemical analytes could offer transformative fundamental and technological opportunities. Due to their high surface-to-volume ratios, nanoscale materials are extremely sensitive sensors. Likewise, peptides represent robust substrates for selective recognition due to the potential for broad chemical diversity within their relatively compact size. Here we explore the possibilities of linking peptides to nanosensors for the selective detection of biochemical targets. Such systems raise a number of interesting fundamental challenges: What are the peptide sequences, and how can rational design be used to derive selective binders? What nanomaterials should be used, and what are some strategies for assembling hybrid nanosensors? What role does molecular modeling play in elucidating response mechanisms? What is the resulting performance of these sensors, in terms of sensitivity, selectivity, and response time? What are some potential applications? This Account will highlight our early attempts to address these research challenges. Specifically, we use natural peptide sequences or sequences identified from phage display as capture elements. The sensors are based on a variety of nanomaterials including nanowires, graphene, and carbon nanotubes. We couple peptides to the nanomaterial surfaces via traditional surface functionalization methods or self-assembly. Molecular modeling provides detailed insights into the hybrid nanostructure, as well as the sensor detection mechanisms. The peptide nanosensors can distinguish chemically camouflaged mixtures of vapors and detect chemical warfare agents with sensitivities as low as parts-per-billion levels. Finally, we anticipate future uses of this technology in biomedicine: for example, devices based on these sensors could detect disease from the molecular components in human breath. Overall, these results provide a

  20. Preparation of Radiopharmaceuticals Labeled with Metal Radionuclides

    SciTech Connect

    Welch, M.J.

    2012-02-16

    The overall goal of this project was to develop methods for the production of metal-based radionuclides, to develop metal-based radiopharmaceuticals and in a limited number of cases, to translate these agents to the clinical situation. Initial work concentrated on the application of the radionuclides of Cu, Cu-60, Cu-61 and Cu-64, as well as application of Ga-68 radiopharmaceuticals. Initially Cu-64 was produced at the Missouri University Research Reactor and experiments carried out at Washington University. A limited number of studies were carried out utilizing Cu-62, a generator produced radionuclide produced by Mallinckrodt Inc. (now Covidien). In these studies, copper-62-labeled pyruvaldehyde Bis(N{sup 4}-methylthiosemicarbazonato)-copper(II) was studied as an agent for cerebral myocardial perfusion. A remote system for the production of this radiopharmaceutical was developed and a limited number of patient studies carried out with this agent. Various other copper radiopharmaceuticals were investigated, these included copper labeled blood imaging agents as well as Cu-64 labeled antibodies. Cu-64 labeled antibodies targeting colon cancer were translated to the human situation. Cu-64 was also used to label peptides (Cu-64 octriatide) and this is one of the first applications of a peptide radiolabeled with a positron emitting metal radionuclide. Investigations were then pursued on the preparation of the copper radionuclides on a small biomedical cyclotron. A system for the production of high specific activity Cu-64 was developed and initially the Cu-64 was utilized to study the hypoxic imaging agent Cu-64 ATSM. Utilizing the same target system, other positron emitting metal radionuclides were produced, these were Y-86 and Ga-66. Radiopharmaceuticals were labeled utilizing both of these radionuclides. Many studies were carried out in animal models on the uptake of Cu-ATSM in hypoxic tissue. The hypothesis is that Cu-ATSM retention in vivo is dependent upon the

  1. Invertebrate FMRFamide related peptides.

    PubMed

    Krajniak, Kevin G

    2013-06-01

    In 1977 the neuropeptide FMRFamide was isolated from the clam, Macrocallista nimbosa. Since then several hundred FMRFamide-related peptides (FaRPs) have been isolated from invertebrate animals. Precursors to the FaRPs likely arose in the cnidarians. With the transition to a bilateral body plan FaRPs became a fixture in the invertebrate phyla. They have come to play a critical role as neurotransmitters, neuromodulators, and neurohormones. FaRPs regulate a variety of body functions including, feeding, digestion, circulation, reproduction, movement. The evolution of the molecular form and function of these omnipresent peptides will be considered.

  2. Dicyclopropylmethyl peptide backbone protectant.

    PubMed

    Carpino, Louis A; Nasr, Khaled; Abdel-Maksoud, Adel Ali; El-Faham, Ayman; Ionescu, Dumitru; Henklein, Peter; Wenschuh, Holger; Beyermann, Michael; Krause, Eberhard; Bienert, Michael

    2009-08-20

    The N-dicyclopropylmethyl (Dcpm) residue, introduced into amino acids via reaction of dicyclopropylmethanimine hydrochloride with an amino acid ester followed by sodium cyanoborohydride or triacetoxyborohydride reduction, can be used as an amide bond protectant for peptide synthesis. Examples which demonstrate the amelioration of aggregation effects include syntheses of the alanine decapeptide and the prion peptide (106-126). Avoidance of cyclization to the aminosuccinimide followed substitution of Fmoc-(Dcpm)Gly-OH for Fmoc-Gly-OH in the assembly of sequences containing the sensitive Asp-Gly unit.

  3. Technetium-99m labelling of glycosylated somatostatin-14.

    PubMed

    Du, J; Hiltunen, J; Marquez, M; Nilsson, S; Holmberg, A R

    2001-08-01

    This study presents a technetium-99m labelling method based on organometallic chemistry. It describes the simple mixing of a 99mTc(I)-carbonyl compound [99mTc(OH2)3(CO)3]+ with a histidine-tagged somatostatin-dextran (SMS-Dx-His) conjugate. Somatostatin and histidine was coupled to periodate activated dextran. The linkage was stabilised by reductive amination. The conjugate was then radiolabelled with 99mTc by using the 99mTc(CO)3 core. The labelling efficiency was 65-80% and the radiochemical purity > 95%. In the in vitro cysteine challenge, the result showed that 25% of the radiolabel was released after 1 h incubation at 37 degrees C (cysteine-conjugate at 1000:1 molar ratio). The radiolabelled SMS-Dx-His showed similar HPLC profile as the unlabelled conjugate. This labelling method, employing non reducing conditions, is useful for the labelling of peptides containing disulphide bonds. It should be possible to be used also for labelling with rhenium-188 for therapeutic applications.

  4. Efficient Covalent Bond Formation in Gas-Phase Peptide-Peptide Ion Complexes with the Photoleucine Stapler

    NASA Astrophysics Data System (ADS)

    Shaffer, Christopher J.; Andrikopoulos, Prokopis C.; Řezáč, Jan; Rulíšek, Lubomír; Tureček, František

    2016-04-01

    Noncovalent complexes of hydrophobic peptides GLLLG and GLLLK with photoleucine (L*) tagged peptides G(L* n L m )K (n = 1,3, m = 2,0) were generated as singly charged ions in the gas phase and probed by photodissociation at 355 nm. Carbene intermediates produced by photodissociative loss of N2 from the L* diazirine rings underwent insertion into X-H bonds of the target peptide moiety, forming covalent adducts with yields reaching 30%. Gas-phase sequencing of the covalent adducts revealed preferred bond formation at the C-terminal residue of the target peptide. Site-selective carbene insertion was achieved by placing the L* residue in different positions along the photopeptide chain, and the residues in the target peptide undergoing carbene insertion were identified by gas-phase ion sequencing that was aided by specific 13C labeling. Density functional theory calculations indicated that noncovalent binding to GL*L*L*K resulted in substantial changes of the (GLLLK + H)+ ground state conformation. The peptide moieties in [GL*L*LK + GLLLK + H]+ ion complexes were held together by hydrogen bonds, whereas dispersion interactions of the nonpolar groups were only secondary in ground-state 0 K structures. Born-Oppenheimer molecular dynamics for 100 ps trajectories of several different conformers at the 310 K laboratory temperature showed that noncovalent complexes developed multiple, residue-specific contacts between the diazirine carbons and GLLLK residues. The calculations pointed to the substantial fluidity of the nonpolar side chains in the complexes. Diazirine photochemistry in combination with Born-Oppenheimer molecular dynamics is a promising tool for investigations of peptide-peptide ion interactions in the gas phase.

  5. Identification of high-affinity VEGFR3-binding peptides through a phage-displayed random peptide library

    PubMed Central

    Wu, Yan; Li, Cai-Yun

    2015-01-01

    Objective Vascular endothelial growth factor (VEGF) interaction with its receptor, VEGFR-3/Flt-4, regulates lymphangiogenesis. VEGFR-3/Flt-4 expression in cancer cells has been correlated with clinical stage, lymph node metastasis, and lymphatic invasion. The objective of this study is to identify a VEGFR-3/Flt-4-interacting peptide that could be used to inhibit VEGFR-3 for ovarian cancer therapy. Methods The extracellular fragment of recombinant human VEGFR-3/Flt-4 (rhVEGFR-3/Flt-4) fused with coat protein pIII was screened against a phage-displayed random peptide library. Using affinity enrichment and enzyme-linked immunosorbent assay (ELISA) screening, positive clones of phages were amplified. Three phage clones were selected after four rounds of biopanning, and the specific binding of the peptides to rhVEGFR-3 was detected by ELISA and compared with that of VEGF-D. Immunohistochemistry and immunofluorescence analyses of ovarian cancer tissue sections was undertaken to demonstrate the specificity of the peptides. Results After four rounds of biopanning, ELISA confirmed the specificity of the enriched bound phage clones for rhVEGFR-3. Sequencing and translation identified three different peptides. Non-competitive ELISA revealed that peptides I, II, and III had binding affinities for VEGFR-3 with Kaff (affinity constant) of 16.4±8.6 µg/mL (n=3), 9.2±2.1 µg/mL (n=3), and 174.8±31.1 µg/mL (n=3), respectively. In ovarian carcinoma tissue sections, peptide III (WHWLPNLRHYAS), which had the greatest binding affinity, also co-localized with VEGFR-3 in endothelial cells lining lymphatic vessels; its labeling of ovarian tumors in vivo was also confirmed. Conclusion These finding showed that peptide III has high specificity and activity and, therefore, may represent a potential therapeutic approach to target VEGF-VEGFR-3 signaling for the treatment or diagnosis of ovarian cancer. PMID:26197772

  6. Identification of novel peptides for horse meat speciation in highly processed foodstuffs.

    PubMed

    Claydon, Amy J; Grundy, Helen H; Charlton, Adrian J; Romero, M Rosario

    2015-01-01

    There is a need for robust analytical methods to support enforcement of food labelling legislation. Proteomics is emerging as a complementary methodology to existing tools such as DNA and antibody-based techniques. Here we describe the development of a proteomics strategy for the determination of meat species in highly processed foods. A database of specific peptides for nine relevant animal species was used to enable semi-targeted species determination. This principle was tested for horse meat speciation, and a range of horse-specific peptides were identified as heat stable marker peptides for the detection of low levels of horse meat in mixtures with other species.

  7. Identification of novel peptides for horse meat speciation in highly processed foodstuffs.

    PubMed

    Claydon, Amy J; Grundy, Helen H; Charlton, Adrian J; Romero, M Rosario

    2015-01-01

    There is a need for robust analytical methods to support enforcement of food labelling legislation. Proteomics is emerging as a complementary methodology to existing tools such as DNA and antibody-based techniques. Here we describe the development of a proteomics strategy for the determination of meat species in highly processed foods. A database of specific peptides for nine relevant animal species was used to enable semi-targeted species determination. This principle was tested for horse meat speciation, and a range of horse-specific peptides were identified as heat stable marker peptides for the detection of low levels of horse meat in mixtures with other species. PMID:26258799

  8. Antihypertensive peptides from food proteins.

    PubMed

    Aluko, Rotimi E

    2015-01-01

    Bioactive peptides are encrypted within the primary structure of food proteins where they remain inactive until released by enzymatic hydrolysis. Once released from the parent protein, certain peptides have the ability to modulate the renin-angiotensin system (RAS) because they decrease activities of renin or angiotensin-converting enzyme (ACE), the two main enzymes that regulate mammalian blood pressure. These antihypertensive peptides can also enhance the endothelial nitric oxide synthase (eNOS) pathway to increase nitric oxide (NO) levels within vascular walls and promote vasodilation. The peptides can block the interactions between angiotensin II (vasoconstrictor) and angiotensin receptors, which can contribute to reduced blood pressure. This review focuses on the methods that are involved in antihypertensive peptide production from food sources, including fractionation protocols that are used to enrich bioactive peptide content and enhance peptide activity. It also discusses mechanisms that are believed to be involved in the antihypertensive activity of these peptides.

  9. Autoradiographic localization of a gluten peptide during organ culture of human duodenal mucosa

    SciTech Connect

    Fluge, G.; Aksnes, L.

    1983-01-01

    An 125I-labeled subfraction of Frazer's fraction III (molecular weight, 8,000) was added to the culture medium during organ culture of duodenal biopsies from two patients with celiac disease in exacerbation. The isotope-labeled gluten peptide was localized by autoradiography after 6, 12, and 24 h of culture. At 6 h, labeling was located mainly in the basal layers of the biopsies. The tissue was well preserved. After 12 h in culture, the labeling had spread to the lamina propria and the crypts. A few grains were located over enterocytes and desquamated cells. Moderate histological signs of toxicity were observed. After 24 h, there was marked toxic deterioration, comparable to that seen after culture with alpha-gliadin. Labeling had spread throughout the entire section. There seemed to be no specificity of the binding, for the entire section was affected. Culture with the identical gluten fraction, in the radionegative state, produced histological deterioration comparable to that seen after exposure to the isotope-labeled peptide. Gluten peptides are presented to the target cells in a unique way during organ culture, different from in vivo conditions. This may influence the results when the organ culture method is used to investigate the pathogenesis of celiac disease.

  10. Effect of Ca2+-Mg2+ exchange on the flexibility and/or conformation of the small domain in monomeric actin.

    PubMed

    Nyitrai, M; Hild, G; Lakos, Z; Somogyi, B

    1998-05-01

    A fluorescence resonance energy transfer (FRET) parameter, f' (defined as the average transfer efficiency, (E), normalized by the actual fluorescence intensity of the donor in the presence of acceptor, F(DA)), was previously shown to be capable of monitoring both changes in local flexibility of the protein matrix and major conformational transitions. The temperature profile of this parameter was used to detect the change of the protein flexibility in the small domain of the actin monomer (G-actin) upon the replacement of Ca2+ by Mg2+. The Cys-374 residue of the actin monomer was labeled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) to introduce a fluorescence donor and the Lys-61 residue with fluorescein-5-isothiocyanate (FITC) to serve as an acceptor. The f' increases with increasing temperature over the whole temperature range for Mg-G-actin. This parameter increases similarly in the case of Ca-G-actin up to 26 degrees C, whereas an opposite tendency appears above this temperature. These data indicate that there is a conformational change in Ca-G-actin above 26 degrees C that was not detected in the case of Mg-G-actin. In the temperature range between 6 degrees C and 26 degrees C the slope of the temperature profile of f' is the same for Ca-G-actin and Mg-G-actin, suggesting that the flexibility of the protein matrix between the two labels is identical in the two forms of actin.

  11. Evaluation of a ⁶⁴Cu‑labeled 1,4,7‑triazacyclononane, 1‑glutaric acid‑4,7 acetic acid (NODAGA)‑galactose‑bombesin analogue as a PET imaging probe in a gastrin‑releasing peptide receptor‑expressing prostate cancer xenograft model.

    PubMed

    Kim, Min Hwan; Park, Ji Ae; Woo, Sang-Keun; Lee, Kyo Chul; An, Gwang Il; Kim, Byoung Soo; Kim, Kwang Il; Lee, Tae Sup; Kim, Chan Wha; Kim, Kyeong Min; Kang, Joo Hyun; Lee, Yong Jin

    2015-03-01

    Gastrin‑releasing peptide receptor (GRPR) is overexpressed by a variety of human tumors and in particular, identified to be upregulated in prostate cancers. The current study aimed to develop clinically translatable BBN analogue‑based radioligands for positron emission tomography (PET) of GRPR‑positive tumors. We developed radiolabeled BBN analogues and modified radiolabeled galacto‑BBN analogues and then investigated their tumor‑targeting efficacy in vivo. The chelator 1,4,7‑triazacyclononane, 1‑glutaric acid‑4,7 acetic acid (NODAGA) was used to radiolabel the peptides with 64Cu. The peptides were evaluated by measuring cell‑based receptor‑binding affinities. Biodistribution experiments and small animal imaging using PET were performed in nude mice bearing subcutaneous PC3 human prostate cancer xenografts. The conjugates were radiolabeled with yields >99%. The stability assay showed that [64Cu]NODAGA‑BBN and [64Cu]NODAGA‑galacto‑BBN remained stable in both human and mouse serum for 1 h at 37˚C. PET images of PC3 tumor‑bearing nude mice were acquired at 1, 3, 24, 48 and 72 h after injection. [64Cu]NODAGA‑galacto‑BBN showed retention in tumors for 72 h, low liver uptake, and rapid renal clearance. PET imaging results were also confirmed by biodistrubution 1 and 3 h after injection. [64Cu]NODAGA‑BBN and [64Cu]NODAGA‑galacto‑BBN are promising new PET probes for GRPR‑positive prostate cancer.

  12. Site-specific labeling of proteins via sortase: protocols for the molecular biologist.

    PubMed

    Popp, Maximilian Wei-Lin

    2015-01-01

    Creation of site-specifically labeled protein bioconjugates is an important tool for the molecular biologist and cell biologist. Chemical labeling methods, while versatile with respect to the types of moieties that can be attached, suffer from lack of specificity, often targeting multiple positions within a protein. Here we describe protocols for the chemoenzymatic labeling of proteins at the C-terminus using the bacterial transpeptidase, sortase A. We detail a protocol for the purification of an improved pentamutant variant of the Staphylococcus aureus enzyme (SrtA 5(o)) that exhibits vastly improved kinetics relative to the wild-type enzyme. Importantly, a protocol for the construction of peptide probes compatible with sortase labeling using techniques that can be adapted to any cellular/molecular biology lab with no existing infrastructure for synthetic chemistry is described. Finally, we provide an example of how to optimize the labeling reaction using the improved SrtA 5(o) variant. PMID:25560076

  13. The interplay of T1- and T2-relaxation on T1-weighted MRI of hMSCs induced by Gd-DOTA-peptides.

    PubMed

    Cao, Limin; Li, Binbin; Yi, Peiwei; Zhang, Hailu; Dai, Jianwu; Tan, Bo; Deng, Zongwu

    2014-04-01

    Three Gd-DOTA-peptide complexes with different peptide sequence are synthesized and used as T1 contrast agent to label human mesenchymal stem cells (hMSCs) for magnetic resonance imaging study. The peptides include a universal cell penetrating peptide TAT, a linear MSC-specific peptide EM7, and a cyclic MSC-specific peptide CC9. A significant difference in labeling efficacy is observed between the Gd-DOTA-peptides as well as a control Dotarem. All Gd-DOTA-peptides as well as Dotarem induce significant increase in T1 relaxation rate which is in favor of T1-weighted MR imaging. Gd-DOTA-CC9 yields the maximum labeling efficacy but poor T1 contrast enhancement. Gd-DOTA-EM7 yields the minimum labeling efficacy but better T1 contrast enhancement. Gd-DOTA-TAT yields a similar labeling efficacy as Gd-DOTA-CC9 and similar T1 contrast enhancement as Gd-DOTA-EM7. The underlying mechanism that governs T1 contrast enhancement effect is discussed. Our results suggest that T1 contrast enhancement induced by Gd-DOTA-peptides depends not only on the introduced cellular Gd content, but more importantly on the effect that Gd-DOTA-peptides exert on the T1-relaxation and T2-relaxation processes/rates. Both T1 and particularly T2 relaxation rate have to be taken into account to interpret T1 contrast enhancement. In addition, the interpretation has to be based on cellular instead of aqueous longitudinal and transverse relaxivities of Gd-DOTA-peptides.

  14. Identification of Asp isomerization in proteins by ¹⁸O labeling and tandem mass spectrometry.

    PubMed

    Zhang, Jennifer; Katta, Viswanatham

    2012-01-01

    Isomerization of aspartic acid (Asp) to isoaspartic acid (isoAsp) via succinimide intermediate is a common route of degradation for proteins that can affect their structural integrity. As Asp/isoAsp is isobaric in mass, it is difficult to identify the site of modification by LC-MS/MS peptide mapping. Here, we describe an approach to label the Asp residue involved in isomerization at the protein level by hydrolyzing the succinimide intermediate in H₂¹⁸O. Tryptic digestion of this labeled protein will result in peptides containing the site of isomerization being 2 Da heavier than the ¹⁶O-containing counterparts, due to ¹⁸O incorporation during the hydrolysis process. Comparison of tandem mass spectra of isomerized peptides with and without ¹⁸O incorporation allows easy identification of the Asp residue involved. This method proved to be especially useful in identifying the sites when isomerization occurs in Asp-Asp motifs.

  15. Effect of fluorescently labeling protein probes on kinetics of protein-ligand reactions.

    PubMed

    Sun, Y S; Landry, J P; Fei, Y Y; Zhu, X D; Luo, J T; Wang, X B; Lam, K S

    2008-12-01

    We studied the effect of fluorescently labeling proteins on protein-ligand reactions. Unlabeled ligands (streptavidin-binding peptides and rabbit immunoglobulin G (IgG) as antigen targets) are immobilized on epoxy-functionalized glass slides. Unlabeled and Cy3-labeled protein probes from the same batch (streptavidin and goat antibodies) subsequently react with the surface-immobilized targets. By monitoring in situ the surface mass density change using an oblique-incidence reflectivity difference scanning microscope (a label-free detector), we measured k(on) and k(off) for streptavidin-peptide reactions and antibody-antigen reaction. We found that (1) equilibrium dissociation constants, defined as K(D) = k(off)/k(on), for streptavidin-peptide reactions increases by a factor of 3-4 when the solution-phase streptavidin is labeled with Cy3 dye and (2) K(D) for reactions of solution-phase goat anti-rabbit antibodies with rabbit IgG targets also change significantly when the goat antibodies are labeled with Cy3 dye. PMID:18991423

  16. Brain Peptides and Psychopharmacology

    ERIC Educational Resources Information Center

    Arehart-Treichel, Joan

    1976-01-01

    Proteins isolated from the brain and used as drugs can improve and apparently even transfer mental states and behavior. Much of the pioneering work and recent research with humans and animals is reviewed and crucial questions that are being posed about the psychologically active peptides are related. (BT)

  17. Peptide and peptide library cyclization via bromomethylbenzene derivatives.

    PubMed

    Hacker, David E; Almohaini, Mohammed; Anbazhagan, Aruna; Ma, Zhong; Hartman, Matthew C T

    2015-01-01

    Cyclization confers several advantages to peptides, cumulatively serving to make them more drug-like. In this protocol, cyclic peptides are generated via bis-alkylation of cysteine-containing peptides using α,α'-dibromo-m-xylene. The reactions are robust and high yielding. Multiple reaction platforms for the application of this versatile strategy are described herein: the cyclization of solid-phase-synthesized peptides, both in solution and on resin, as well as the cyclization of in vitro translated mRNA-peptide fusion libraries on oligo(dT) resin.

  18. Peptides and Ageing.

    PubMed

    Khavinson, Vladimir Kh

    2002-01-01

    A technology has been developed for manufacturing of biologically active complex peptide preparations from extracts of different tissues. In particular, the pineal preparation (Epithalamin) augments the in vitro outgrowth of explants from the pineal gland but not from other tissues, the latter being stimulated by peptide preparations from respective tissues. Epithalamin increases melatonin production by the pineal gland of rats, improves immunological parameters in rats and mice, produces anticarcinogenic effects in different experimental models, stimulates antioxidant defenses, and restores the reproductive function in old rats. These effects are combined in the ability of Epithalamin to increase the lifespan in rats, mice, and fruit flies. Many of these effects are reproduced in clinical trials, which have demonstrated the geroprotector activity of Epithalamin in humans. Among the effects of the thymic preparation Thymalin, those related to its ability to stimulate immunity are the most prominent. This ability is associated with anticarcinogenic and geroprotector activities. Clinical trials of the peptide preparations obtained from other organs including the prostate, the cerebral cortex, and the eye retina, have demonstrated beneficial effects reflected by the improvement of the conditions of respective organs. Based on the data about the amino acid compositions of the peptide preparations, novel principles of the design of biologically active short peptides possessing tissue-specific activities has been developed. Dipeptides specific for the thymus and tetrapeptides specific for the heart, liver, brain cortex, and pineal glands stimulate the in vitro outgrowth of explants of respective organs. Interestingly, for eye retina and the pineal gland, a common tetrapeptide Ala-Glu-Asp-Gly (Epitalon) has been designed, probably reflecting the common embryonal origin of these two organs. Epitalon reproduces the effects of Epithalamin including those related to its

  19. Multiplex localization of sequential peptide epitopes by use of a planar microbead chip.

    PubMed

    Schmidt, Carsten; Rödiger, Stefan; Gruner, Melanie; Moncsek, Anja; Stohwasser, Ralf; Hanack, Katja; Schierack, Peter; Schröder, Christian

    2016-02-18

    Epitope mapping is crucial for the characterization of protein-specific antibodies. Commonly, small overlapping peptides are chemically synthesized and immobilized to determine the specific peptide sequence. In this study, we report the use of a fast and inexpensive planar microbead chip for epitope mapping. We developed a generic strategy for expressing recombinant peptide libraries instead of using expensive synthetic peptide libraries. A biotin moiety was introduced in vivo at a defined peptide position using biotin ligase. Peptides in crude Escherichia coli lysate were coupled onto streptavidin-coated microbeads by incubation, thereby avoiding tedious purification procedures. For read-out we used a multiplex planar microbead chip with size- and fluorescence-encoded microbead populations. For epitope mapping, up to 18 populations of peptide-loaded microbeads (at least 20 microbeads per peptide) displaying the primary sequence of a protein were analyzed simultaneously. If an epitope was recognized by an antibody, a secondary fluorescence-labeled antibody generated a signal that was quantified, and the mean value of all microbeads in the population was calculated. We mapped the epitopes for rabbit anti-PA28γ (proteasome activator 28γ) polyclonal serum, for a murine monoclonal antibody against PA28γ, and for a murine monoclonal antibody against the hamster polyoma virus major capsid protein VP1 as models. In each case, the identification of one distinct peptide sequence out of up to 18 sequences was possible. Using this approach, an epitope can be mapped multiparametrically within three weeks.

  20. Peptide vectors for gene delivery: from single peptides to multifunctional peptide nanocarriers.

    PubMed

    Raad, Markus de; Teunissen, Erik A; Mastrobattista, Enrico

    2014-07-01

    The therapeutic use of nucleic acids relies on the availability of sophisticated delivery systems for targeted and intracellular delivery of these molecules. Such a gene delivery should possess essential characteristics to overcome several extracellular and intracellular barriers. Peptides offer an attractive platform for nonviral gene delivery, as several functional peptide classes exist capable of overcoming these barriers. However, none of these functional peptide classes contain all the essential characteristics required to overcome all of the barriers associated with successful gene delivery. Combining functional peptides into multifunctional peptide vectors will be pivotal for improving peptide-based gene delivery systems. By using combinatorial strategies and high-throughput screening, the identification of multifunctional peptide vectors will accelerate the optimization of peptide-based gene delivery systems.

  1. Antagonistic peptide technology for functional dissection of CLE peptides revisited.

    PubMed

    Czyzewicz, Nathan; Wildhagen, Mari; Cattaneo, Pietro; Stahl, Yvonne; Pinto, Karine Gustavo; Aalen, Reidunn B; Butenko, Melinka A; Simon, Rüdiger; Hardtke, Christian S; De Smet, Ive

    2015-08-01

    In the Arabidopsis thaliana genome, over 1000 putative genes encoding small, presumably secreted, signalling peptides can be recognized. However, a major obstacle in identifying the function of genes encoding small signalling peptides is the limited number of available loss-of-function mutants. To overcome this, a promising new tool, antagonistic peptide technology, was recently developed. Here, this antagonistic peptide technology was tested on selected CLE peptides and the related IDA peptide and its usefulness in the context of studies of peptide function discussed. Based on the analyses, it was concluded that the antagonistic peptide approach is not the ultimate means to overcome redundancy or lack of loss-of-function lines. However, information collected using antagonistic peptide approaches (in the broad sense) can be very useful, but these approaches do not work in all cases and require a deep insight on the interaction between the ligand and its receptor to be successful. This, as well as peptide ligand structure considerations, should be taken into account before ordering a wide range of synthetic peptide variants and/or generating transgenic plants.

  2. Site-specific conformational determination in thermal unfolding studies of helical peptides using vibrational circular dichroism with isotopic substitution

    PubMed Central

    Silva, R. A. G. D.; Kubelka, Jan; Bour, Petr; Decatur, Sean M.; Keiderling, Timothy A.

    2000-01-01

    Understanding the detailed mechanism of protein folding requires dynamic, site-specific stereochemical information. The short time response of vibrational spectroscopies allows evaluation of the distribution of populations in rapid equilibrium as the peptide unfolds. Spectral shifts associated with isotopic labels along with local stereochemical sensitivity of vibrational circular dichroism (VCD) allow determination of the segment sequence of unfolding. For a series of alanine-rich peptides that form α-helices in aqueous solution, we used isotopic labeling and VCD to demonstrate that the α-helix noncooperatively unwinds from the ends with increasing temperature. For these blocked peptides, the C-terminal is frayed at 5°C. Ab initio level theoretical simulations of the IR and VCD band shapes are used to analyze the spectra and to confirm the conformation of the labeled components. The VCD signals associated with the labeled residues are amplified by coupling to the nonlabeled parts of the molecule. Thus small labeled segments are detectable and stereochemically defined in moderately large peptides in this report of site-specific peptide VCD conformational analysis. PMID:10880566

  3. Fluorescently labeled adrenomedullin allows real-time monitoring of adrenomedullin receptor trafficking in living cells.

    PubMed

    Schönauer, Ria; Kaiser, Anette; Holze, Cathleen; Babilon, Stefanie; Köbberling, Johannes; Riedl, Bernd; Beck-Sickinger, Annette G

    2015-12-01

    The human adrenomedullin (ADM) is a 52 amino acid peptide hormone belonging to the calcitonin family of peptides, which plays a major role in the development and regulation of cardiovascular and lymphatic systems. For potential use in clinical applications, we aimed to investigate the fate of the peptide ligand after binding and activation of the adrenomedullin receptor (AM1), a heterodimer consisting of the calcitonin receptor-like receptor (CLR), a G protein-coupled receptor, associated with the receptor activity-modifying protein 2 (RAMP2). Full length and N-terminally shortened ADM peptides were synthesized using Fmoc/tBu solid phase peptide synthesis and site-specifically labeled with the fluorophore carboxytetramethylrhodamine (Tam) either by amide bond formation or copper(I)-catalyzed azide alkyne cycloaddition. For the first time, Tam-labeled ligands allowed the observation of co-internalization of the whole ligand-receptor complex in living cells co-transfected with fluorescent fusion proteins of CLR and RAMP2. Application of a fluorescent probe to track lysosomal compartments revealed that ADM together with the CLR/RAMP2-complex is routed to the degradative pathway. Moreover, we found that the N-terminus of ADM is not a crucial component of the peptide sequence in terms of AM1 internalization behavior. PMID:26767744

  4. Ultrasensitive detection of closely related angiotensin I peptides using capillary electrophoresis with near-infrared laser-induced fluorescence detection.

    PubMed

    Baars, M J; Patonay, G

    1999-02-01

    A novel near-infrared (NIR) fluorescent dye (NN382, LICOR, Inc.) was evaluated as an ultrasensitive peptide-labeling reagent for use with capillary electrophoresis (CE). Six angiotensin I (Ang-I) variants were selected as model peptides for the derivatization and separation studies. The closely related decapeptides were labeled with the NIR dye, separated using CE, and detected by NIR laser-induced fluorescence. Derivatization of the peptides was achieved under aqueous conditions using 2.5-500 pmol of Ang-I in a 50-microL sample (5 x 10(-8)-1 x 10(-5)M), and between 1.3 and 254 amol of the labeled peptides were injected on column. The fluorescence response was linear over a 200-fold range (correlation r > or = 0.9986). The limit of detection (SNR = 3, signal/RMS noise) ranged from 100 to 300 zmol, for the six Ang-I variants. Four of six peptides were resolved from each other and excess dye using capillary zone electrophoresis with a simple 50 mM phosphate run buffer, pH 7.2. Two pairs of coeluting peptides were successfully resolved using micellar electrokinetic chromatography with a nonionic surfactant, Triton X-100. The NIR amine-labeling reagent NN382 is a viable alternative to using visible fluorophores for CE methods requiring high sensitivity. PMID:9989384

  5. CONSTANd : A Normalization Method for Isobaric Labeled Spectra by Constrained Optimization.

    PubMed

    Maes, Evelyne; Hadiwikarta, Wahyu Wijaya; Mertens, Inge; Baggerman, Geert; Hooyberghs, Jef; Valkenborg, Dirk

    2016-08-01

    In quantitative proteomics applications, the use of isobaric labels is a very popular concept as they allow for multiplexing, such that peptides from multiple biological samples are quantified simultaneously in one mass spectrometry experiment. Although this multiplexing allows that peptide intensities are affected by the same amount of instrument variability, systematic effects during sample preparation can also introduce a bias in the quantitation measurements. Therefore, normalization methods are required to remove this systematic error. At present, a few dedicated normalization methods for isobaric labeled data are at hand. Most of these normalization methods include a framework for statistical data analysis and rely on ANOVA or linear mixed models. However, for swift quality control of the samples or data visualization a simple normalization technique is sufficient. To this aim, we present a new and easy-to-use data-driven normalization method, named CONSTANd. The CONSTANd method employs constrained optimization and prior information about the labeling strategy to normalize the peptide intensities. Further, it allows maintaining the connection to any biological effect while reducing the systematic and technical errors. As a result, peptides can not only be compared directly within a multiplexed experiment, but are also comparable between other isobaric labeled datasets from multiple experimental designs that are normalized by the CONSTANd method, without the need to include a reference sample in every experimental setup. The latter property is especially useful when more than six, eight or ten (TMT/iTRAQ) biological samples are required to detect differential peptides with sufficient statistical power and to optimally make use of the multiplexing capacity of isobaric labels. PMID:27302888

  6. Biochemical functionalization of peptide nanotubes with phage displayed peptides

    NASA Astrophysics Data System (ADS)

    Swaminathan, Swathi; Cui, Yue

    2016-09-01

    The development of a general approach for the biochemical functionalization of peptide nanotubes (PNTs) could open up existing opportunities in both fundamental studies as well as a variety of applications. PNTs are spontaneously assembled organic nanostructures made from peptides. Phage display has emerged as a powerful approach for identifying selective peptide binding motifs. Here, we demonstrate for the first time the biochemical functionalization of PNTs via peptides identified from a phage display peptide library. The phage-displayed peptides are shown to recognize PNTs. These advances further allow for the development of bifunctional peptides for the capture of bacteria and the self-assembly of silver particles onto PNTs. We anticipate that these results could provide significant opportunities for using PNTs in both fundamental studies and practical applications, including sensors and biosensors nanoelectronics, energy storage devices, drug delivery, and tissue engineering.

  7. Biochemical functionalization of peptide nanotubes with phage displayed peptides.

    PubMed

    Swaminathan, Swathi; Cui, Yue

    2016-09-01

    The development of a general approach for the biochemical functionalization of peptide nanotubes (PNTs) could open up existing opportunities in both fundamental studies as well as a variety of applications. PNTs are spontaneously assembled organic nanostructures made from peptides. Phage display has emerged as a powerful approach for identifying selective peptide binding motifs. Here, we demonstrate for the first time the biochemical functionalization of PNTs via peptides identified from a phage display peptide library. The phage-displayed peptides are shown to recognize PNTs. These advances further allow for the development of bifunctional peptides for the capture of bacteria and the self-assembly of silver particles onto PNTs. We anticipate that these results could provide significant opportunities for using PNTs in both fundamental studies and practical applications, including sensors and biosensors nanoelectronics, energy storage devices, drug delivery, and tissue engineering. PMID:27479451

  8. Label and Label-Free Detection Techniques for Protein Microarrays

    PubMed Central

    Syahir, Amir; Usui, Kenji; Tomizaki, Kin-ya; Kajikawa, Kotaro; Mihara, Hisakazu

    2015-01-01

    Protein microarray technology has gone through numerous innovative developments in recent decades. In this review, we focus on the development of protein detection methods embedded in the technology. Early microarrays utilized useful chromophores and versatile biochemical techniques dominated by high-throughput illumination. Recently, the realization of label-free techniques has been greatly advanced by the combination of knowledge in material sciences, computational design and nanofabrication. These rapidly advancing techniques aim to provide data without the intervention of label molecules. Here, we present a brief overview of this remarkable innovation from the perspectives of label and label-free techniques in transducing nano-biological events.

  9. Antimicrobial peptides: premises and promises.

    PubMed

    Reddy, K V R; Yedery, R D; Aranha, C

    2004-12-01

    Antimicrobial peptides (AMPs) are an important component of the natural defences of most living organisms against invading pathogens. These are relatively small (< 10kDa), cationic and amphipathic peptides of variable length, sequence and structure. During the past two decades several AMPs have been isolated from a wide variety of animals, both vertebrates and invertebrates, and plants as well as from bacteria and fungi. Most of these peptides are obtained from different sources like macrophages, neutrophils, epithelial cells, haemocytes, fat body, reproductive tract, etc. These peptides exhibit broad-spectrum activity against a wide range of microorganisms including Gram-positive and Gram-negative bacteria, protozoa, yeast, fungi and viruses. A few peptides have also been found to be cytotoxic to sperm and tumour cells. AMPs are classified based on the three dimensional structural studies carried out with the help of NMR. The peptides are broadly classified into five major groups namely (a) peptides that form alpha-helical structures, (b) peptides rich in cysteine residues, (c) peptides that form beta-sheet, (d) peptides rich in regular amino acids namely histatin, arginine and proline and (e) peptides composed of rare and modified amino acids. Most of these peptides are believed to act by disrupting the plasma membrane leading to the lysis of the cell. AMPs have been found to be excellent candidates for developing novel antimicrobial agents and a few of these peptides show antimicrobial activity against pathogens causing sexually transmitted infection (STI), including HIV/HSV. Peptides, namely magainin and nisin have been shown to demonstrate contraceptive properties in vitro and in vivo. A few peptides have already entered clinical trials for the treatment of impetigo, diabetic foot ulcers and gastric helicobacter infections. In this review, we discuss the source, structures and mode of action with special reference to therapeutic considerations of various AMPs

  10. A Peptide Filtering Relation Quantifies MHC Class I Peptide Optimization

    PubMed Central

    Goldstein, Leonard D.; Howarth, Mark; Cardelli, Luca; Emmott, Stephen; Elliott, Tim; Werner, Joern M.

    2011-01-01

    Major Histocompatibility Complex (MHC) class I molecules enable cytotoxic T lymphocytes to destroy virus-infected or cancerous cells, thereby preventing disease progression. MHC class I molecules provide a snapshot of the contents of a cell by binding to protein fragments arising from intracellular protein turnover and presenting these fragments at the cell surface. Competing fragments (peptides) are selected for cell-surface presentation on the basis of their ability to form a stable complex with MHC class I, by a process known as peptide optimization. A better understanding of the optimization process is important for our understanding of immunodominance, the predominance of some T lymphocyte specificities over others, which can determine the efficacy of an immune response, the danger of immune evasion, and the success of vaccination strategies. In this paper we present a dynamical systems model of peptide optimization by MHC class I. We incorporate the chaperone molecule tapasin, which has been shown to enhance peptide optimization to different extents for different MHC class I alleles. Using a combination of published and novel experimental data to parameterize the model, we arrive at a relation of peptide filtering, which quantifies peptide optimization as a function of peptide supply and peptide unbinding rates. From this relation, we find that tapasin enhances peptide unbinding to improve peptide optimization without significantly delaying the transit of MHC to the cell surface, and differences in peptide optimization across MHC class I alleles can be explained by allele-specific differences in peptide binding. Importantly, our filtering relation may be used to dynamically predict the cell surface abundance of any number of competing peptides by MHC class I alleles, providing a quantitative basis to investigate viral infection or disease at the cellular level. We exemplify this by simulating optimization of the distribution of peptides derived from Human

  11. Antimicrobial Peptides as Infection Imaging Agents: Better Than Radiolabeled Antibiotics

    PubMed Central

    Akhtar, Muammad Saeed; Imran, Muhammad Babar; Nadeem, Muhammad Afzal; Shahid, Abubaker

    2012-01-01

    Nuclear medicine imaging techniques offer whole body imaging for localization of number and site of infective foci inspite of limitation of spatial resolution. The innate human immune system contains a large member of important elements including antimicrobial peptides to combat any form of infection. However, development of antibiotics against bacteria progressed rapidly and gained popularity over antimicrobial peptides but even powerful antimicrobials failed to reduce morbidity and mortality due to emergence of mutant strains of bacteria resulting in antimicrobial resistance. Differentiation between infection and inflammation using radiolabeled compounds with nuclear medicine techniques has always been a dilemma which is still to be resolved. Starting from nonspecific tracers to specific radiolabeled tracers, the question is still unanswered. Specific radiolabeled tracers included antibiotics and antimicrobial peptides which bind directly to the bacteria for efficient localization with advanced nuclear medicine equipments. However, there are merits and demerits attributed to each. In the current paper, radiolabeled antibiotics and radiolabeled peptides for infection localization have been discussed starting with the background of primitive nonspecific tracers. Radiolabeled antimicrobial peptides have certain merits compared with labeled antibiotics which make them superior agents for localization of infective focus. PMID:22675369

  12. Principles of protein labeling techniques.

    PubMed

    Obermaier, Christian; Griebel, Anja; Westermeier, Reiner

    2015-01-01

    Protein labeling methods prior to separation and analysis have become indispensable approaches for proteomic profiling. Basically, three different types of tags are employed: stable isotopes, mass tags, and fluorophores. While proteins labeled with stable isotopes and mass tags are measured and differentiated by mass spectrometry, fluorescent labels are detected with fluorescence imagers. The major purposes for protein labeling are monitoring of biological processes, reliable quantification of compounds and specific detection of protein modifications and isoforms in multiplexed samples, enhancement of detection sensitivity, and simplification of detection workflows. Proteins can be labeled during cell growth by incorporation of amino acids containing different isotopes, or in biological fluids, cells or tissue samples by attaching specific groups to the ε-amino group of lysine, the N-terminus, or the cysteine residues. The principles and the modifications of the different labeling approaches on the protein level are described; benefits and shortcomings of the methods are discussed.

  13. Applications of Convertible Isonitriles in the Ligation and Macrocyclization of Multicomponent Reaction-Derived Peptides and Depsipeptides.

    PubMed

    Wessjohann, Ludger A; Morejón, Micjel C; Ojeda, Gerardo M; Rhoden, Cristiano R B; Rivera, Daniel G

    2016-08-01

    Peptide ligation and macrocyclization are among the most relevant approaches in the field of peptide chemistry. Whereas a variety of strategies relying on coupling reagents and native chemical ligation are available, there is a continuous need for efficient peptide ligation and cyclization methods. Herein we report on the utilization of convertible isonitriles as effective synthetic tools for the ligation and macrocyclization of peptides arising from isocyanide-based multicomponent reactions. The strategy relies on the use of convertible isonitriles-derived from Fukuyama amines-and peptide carboxylic acids in Ugi and Passerini reactions to afford N-alkylated peptides and depsipeptides, respectively, followed by conversion of the C-terminal amide onto either N-peptidoacyl indoles or pyrroles. Such activated peptides proved efficient in the ligation to peptidic, lipidic and fluorescently labeled amines and in macrocyclization protocols. As a result, a wide set of N-substituted peptides (with methyl, glycosyl and amino acids as N-substituents), cyclic N-methylated peptides and a depsipeptide were produced in good yields using conditions that involve either classical heating or microwave irradiation. This report improves the repertoire of peptide covalent modification methods by exploiting the synthetic potential of multicomponent reactions and convertible isonitriles. PMID:27390908

  14. A new method to specifically label thiophosphorylatable proteins with extrinsic probes. Labeling of serine-19 of the regulatory light chain of smooth muscle myosin.

    PubMed

    Facemyer, K C; Cremo, C R

    1992-01-01

    We present a new method to specifically and stably label proteins by attaching extrinsic probes to amino acids that are thiophosphorylated by protein kinases and ATP gamma S. The method was demonstrated for labeling of a thiophosphorylatable serine of the isolated regulatory light chain of smooth muscle myosin. We stoichiometrically blocked the single thiol (Cys-108) either by forming a reversible intermolecular disulfide bond or by reacting with iodoacetic acid. The protein was stoichiometrically thiophosphorylated at Ser-19 by myosin light chain kinase and ATP gamma S. The nucleophilic sulfur of the protein phosphorothioate was coupled at pH 7.9 and 25 degrees C to the fluorescent haloacetate [3H]-5-[[2-[(iodoacetyl)-amino]ethyl]amino]naphthalene-1- sulfonic acid ([3H]IAEDANS) by displacement of the iodide. Typical labeling efficiencies were 70-100%. The labeling was specific for the thiophosphorylated Ser-19, as determined from the sequences of two labeled peptides isolated from a tryptic digest of the labeled protein. [3H]IAEDANS attached to the thiophosphorylated Ser-19 was stable at pH 3-10 at 25 degrees C, and to boiling in high concentrations of reductant. The labeled light chains were efficiently exchanged for unlabeled regulatory light chains of the whole myosin molecule. The resulting labeled myosin had normal ATPase activities in the absence of actin, indicating that the modification of Ser-19 and the exchange of the labeled light chain into myosin did not significantly disrupt the protein. The labeled myosin partially retained the elevated actin-activated Mg(2+)-ATPase activity which is characteristic of thiophosphorylated myosin. This indicates that labeling of the thiophosphate group with [3H]IAEDANS did not completely disrupt the functional properties of the thiophosphorylated protein in the presence of actin.

  15. Antibody Production with Synthetic Peptides.

    PubMed

    Lee, Bao-Shiang; Huang, Jin-Sheng; Jayathilaka, Lasanthi P; Lee, Jenny; Gupta, Shalini

    2016-01-01

    Peptides (usually 10-20 amino acid residues in length) can be used as effectively as proteins in raising antibodies producing both polyclonal and monoclonal antibodies routinely with titers higher than 20,000. Peptide antigens do not function as immunogens unless they are conjugated to proteins. Production of high quality antipeptide antibodies is dependent upon peptide sequence selection, the success of peptide synthesis, peptide-carrier protein conjugation, the humoral immune response in the host animal, the adjuvant used, the peptide dose administered, the injection method, and the purification of the antibody. Peptide sequence selection is probably the most critical step in the production of antipeptide antibodies. Although the process for designing peptide antigens is not exact, several guidelines and computational B-cell epitope prediction methods can help maximize the likelihood of producing antipeptide antibodies that recognize the protein. Antibodies raised by peptides have become essential tools in life science research. Virtually all phospho-specific antibodies are now produced using phosphopeptides as antigens. Typically, 5-20 mg of peptide is enough for antipeptide antibody production. It takes 3 months to produce a polyclonal antipeptide antibody in rabbits that yields ~100 mL of serum which corresponds to ~8-10 mg of the specific antibody after affinity purification using a peptide column. PMID:27515072

  16. Appliance energy labeling takes effect

    SciTech Connect

    Not Available

    1980-06-01

    Consumers buying household appliances will be helped by energy-efficiency labels and minimum efficiency standards required for refrigerators and refrigerator/freezers, freezers, dishwashers, water heaters, clothes washers, room air conditioners, and furnaces. The ENERGYGUIDE labels must be displayed in the store and in catalogs. Two voluntary efficiency programs were combined in the Energy Policy and Conservation Act (EPCA) requiring labels by 1980. Shoppers may compare the efficiencies of appliances and compute the actual cost differential over the lifetime of the equipment. Manufacturers have responded with more-efficient models, but the impact of efficient appliances on energy consumption will be small. A sample label with the required information is illustrated. (DCK)

  17. Toward milk speciation through the monitoring of casein proteotypic peptides.

    PubMed

    Cuollo, Marina; Caira, Simonetta; Fierro, Olga; Pinto, Gabriella; Picariello, Gianluca; Addeo, Francesco

    2010-06-15

    The possibility of detecting extraneous milk in singles species cheese-milk has been explored. A mass spectrometry (MS)-based procedure has been developed to detect 'signature peptides', corresponding to the predefined subset of 'proteotypic peptides', as matchless analytical surrogates of the parent caseins. Tryptic digests of skimmed milk samples from four species were analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. Amongst the candidate signature peptides that are able to differentiate milks from the four species, the alpha(s1)-casein (CN) f8-22 peptide was selected as a convenient marker for bovine, ovine and water buffalo milk while the f4-22 peptide was selected as a marker for the two caprine alpha(s1)-CN A and B variants, which differ by a Pro(16) (B)->Leu(16) (A) substitution. MALDI analysis of the digest allowed the detection of alpha(s1)-CN f8-22 and caprine alpha(s1)-CN f4-22. The accurate evaluation of caprine milk in a quaternary mixture required the development of a liquid chromatography/electrospray ionization (LC/ESI)-MS procedure. Five synthetic signature peptide analogues, which differed from their natural counterparts by a single amino acid substitution, were used as internal standards to quantify the alpha(s1)-CN, which was chosen as a reference milk protein, from the different species. The limits of detection were 0.5% (1% for caprine) for either the MALDI or the LC/ESI-MS method. The isotopic-label-free quantification of isoform- or variant-specific signature peptides has disclosed a convenient approach for targeting proteins in complex mixtures.

  18. The structure, dynamics and orientation of antimicrobial peptides in membranes by multidimensional solid-state NMR spectroscopy.

    PubMed

    Bechinger, B

    1999-12-15

    Linear peptide antibiotics have been isolated from amphibians, insects and humans and used as templates to design cheaper and more potent analogues for medical applications. Peptides such as cecropins or magainins are < or = 40 amino acids in length. Many of them have been prepared by solid-phase peptide synthesis with isotopic labels incorporated at selected sites. Structural analysis by solid-state NMR spectroscopy and other biophysical techniques indicates that these peptide antibiotics strongly interact with lipid membranes. In bilayer environments they exhibit amphipathic alpha-helical conformations and alignments of the helix axis parallel to the membrane surface. This contrasts the transmembrane orientations observed for alamethicin or gramicidin A. Models that have been proposed to explain the antibiotic and pore-forming activities of membrane-associated peptides, as well as other experimental results, include transmembrane helical bundles, wormholes, carpets, detergent-like effects or the in-plane diffusion of peptide-induced bilayer instabilities.

  19. Cyanine-based probe\\tag-peptide pair for fluorescence protein imaging and fluorescence protein imaging methods

    DOEpatents

    Mayer-Cumblidge, M. Uljana; Cao, Haishi

    2010-08-17

    A molecular probe comprises two arsenic atoms and at least one cyanine based moiety. A method of producing a molecular probe includes providing a molecule having a first formula, treating the molecule with HgOAc, and subsequently transmetallizing with AsCl.sub.3. The As is liganded to ethanedithiol to produce a probe having a second formula. A method of labeling a peptide includes providing a peptide comprising a tag sequence and contacting the peptide with a biarsenical molecular probe. A complex is formed comprising the tag sequence and the molecular probe. A method of studying a peptide includes providing a mixture containing a peptide comprising a peptide tag sequence, adding a biarsenical probe to the mixture, and monitoring the fluorescence of the mixture.

  20. D-(Ala1)-peptide T-amide is transported from blood to brain by a saturable system

    SciTech Connect

    Barrera, C.M.; Kastin, A.J.; Banks, W.A.

    1987-12-01

    It is becoming increasingly evident that peptides can cross the blood-brain barrier. The entry into the central nervous system of a commercially available analog of Peptide T, an octapeptide derived from the human immunodeficiency virus envelope glycoprotein 120, was studied in several experiments. It was found that /sup 125/I-Peptide T analog given intravenously in the periphery entered the brain in an intact form, as confirmed by HPLC, to a greater extent than did the labeled albumin control. This entry occurred despite the very low lipid solubility, measured by the octanol/buffer partition coefficient, for the iodinated analog. The rate of entry was decreased by unlabeled Peptide T analog, but not by iodo-tyrosine. Saturable transport out of the brain was not observed after intraventricular administration. Thus, results with /sup 125/I-Peptide T analog indicate that saturable systems can transport peptides from the blood into the central nervous system.

  1. Labeled Cocaine Analogs

    SciTech Connect

    Goodman, Mark M.; Shi, Bing Zhi; Keil, Robert N.

    1999-03-30

    Novel methods for positron emission tomography or single photon emission spectroscopy using tracer compounds having the structure: ##STR1## where X in .beta. configuration is phenyl, naphthyl; 2,3 or 4-iodophenyl; 2,3 or 4-(trimethylsilyl)phenyl; 3,4,5 or 6-iodonaphthyl; 3,4,5 or 6-(trimethylsilyl)naphthyl; 2,3 or 4-(trialkylstannyl)phenyl; or 3,4,5 or 6-(trialkylstannyl)napthyl Y in .beta. configuration is 2-fluoroethoxy, 3-fluoropropoxy, 4-fluorobutoxy, 2-fluorocyclopropoxy, 2 or 3-fluorocyclobutoxy, R,S 1'-fluoroisopropoxy, R 1'-fluoroisopropoxy, S 1'-fluoroisopropoxy, 1',3'-difluoroisopropoxy, R,S 1'-fluoroisobutoxy, R 1'-fluoroisobutoxy, S 1'-fluoroisobutoxy, R,S 4'-fluoroisobutoxy, R 4'-fluoroisobutoxy, S 4'-fluoroisobutoxy, or 1',1'-di(fluoromethyl)isobutoxy, The compounds bind dopamine transporter protein and can be labeled with .sup.18 F or .sup.123 I for imaging.

  2. 78 FR 66826 - Prior Label Approval System: Generic Label Approval

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-07

    ... the Agency (76 FR 75809). FSIS also proposed to combine the regulations that provide for the approval... preamble (76 FR 75814), FSIS wrote: . . . statements on labels that are defined in FSIS's regulations or... ``Product Labeling: Definition of the Term ``Natural'' and related materials (71 FR 70503, Dec. 5, 2006)...

  3. Laser labeling, a safe technology to label produce

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Labeling of the produce has gained marked attention in recent years. Laser labeling technology involves the etching of required information on the surface using a low energy CO2 laser beam. The etching forms alphanumerical characters by pinhole dot matrix depressions. These openings can lead to wat...

  4. Laser labeling, a safe technology to label produce

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Laser labeling of fruits and vegetables is an alternative means to label produce. Low energy CO2 laser beams etch the surface showing the contrasting underlying layer. These etched surfaces can promote water loss and potentially allow for entry of decay organisms. The long-term effects of laser labe...

  5. 76 FR 75809 - Prior Label Approval System: Generic Label Approval

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-05

    ... poultry products will take effect January 1, 2012 (75 FR 82148, Dec. 29, 2010). These mandatory features... limited types of labels (e.g., labels for raw, single ingredient meat and poultry products) (48 FR 11410... Agency. On March 25, 1992, FSIS published an Advance Notice of Proposed Rulemaking (ANPRM) (57 FR...

  6. One-pot peptide and protein conjugation: a combination of enzymatic transamidation and click chemistry.

    PubMed

    Rachel, N M; Pelletier, J N

    2016-02-11

    Enzymatic transamidation and copper-catalyzed azide-alkyne cycloaddition (CuAAC) were combined to yield covalently conjugated peptides and proteins. The addition of glutathione preserved enzymatic activity in the presence of copper. Tuning the reaction kinetics was key to success, providing up to 95% conversion. This one-pot reaction allowed for targeted fluorescent protein labeling. PMID:26741126

  7. Peptide mass fingerprinting.

    PubMed

    Thiede, Bernd; Höhenwarter, Wolfgang; Krah, Alexander; Mattow, Jens; Schmid, Monika; Schmidt, Frank; Jungblut, Peter R

    2005-03-01

    Peptide mass fingerprinting by MALDI-MS and sequencing by tandem mass spectrometry have evolved into the major methods for identification of proteins following separation by two-dimensional gel electrophoresis, SDS-PAGE or liquid chromatography. One main technological goal of proteome analyses beside high sensitivity and automation was the comprehensive analysis of proteins. Therefore, the protein species level with the essential information on co- and post-translational modifications must be achieved. The power of peptide mass fingerprinting for protein identification was described here, as exemplified by the identification of protein species with high molecular masses (spectrin alpha and beta), low molecular masses (elongation factor EF-TU fragments), splice variants (alpha A crystallin), aggregates with disulfide bridges (alkylhydroperoxide reductase), and phosphorylated proteins (heat shock protein 27). Helpful tools for these analyses were the use of the minimal protein identifier concept and the software program MS-Screener to remove mass peaks assignable to contaminants and neighbor spots.

  8. Effects of peptide YY on gallbladder motility

    SciTech Connect

    Conter, R.L.; Roslyn, J.J.; Taylor, I.L.

    1987-06-01

    The effects of peptide YY (PYY) on cholecystokinin-stimulated gallbladder contraction were investigated in the prairie dog model. Twelve animals underwent laparotomy with catheter placement into the gallbladder and common bile duct (vent). The gallbladder was continuously perfused with (/sup 14/C)polyethylene glycol-labeled lactated Ringer at 0.03 ml/min, and vent effluent was collected at 2.5-min intervals. All animals received 20 min of intravenous infusion of cholecystokinin octapeptide (CCK-OP), 2.5 ng x kg/sup -1/ x min/sup -1/, immediately followed by 60-min infusions of either lactated Ringer (LR) or synthetic PYY, 10 or 50 ng x kg/sup -1/ x min/sup -1/. When LR was infused after CCK-OP, gallbladder filling increased by 15.4 +/- 10.5% with minimal changes in gallbladder pressure. Infusion of PYY/sub 10/ resulted in a significant increase in gallbladder volume and filling with a significant decrease in intragallbladder pressure. Similar findings were noted with PYY/sub 50/. These data indicate that synthetic PYY significantly augments gallbladder filling after CCK-OP-stimulated gallbladder contraction. These finding, coupled with the observation that PYY inhibits pancreatic secretion, suggest that this peptide may be the anti-CCK hormone and may have an important role in regulating biliary activity postprandially.

  9. Synthesis of tritium labeled Ac-(Nle/sup 4/, D-Phe/sup 7/)-. cap alpha. -MSH/sub 4-11/-NH/sub 2/: a superpotent melanotropin with prolonged biological activity

    SciTech Connect

    Wilkes, B.D.; Hruby, V.J.; Yamamura, H.I.; Akiyama, K.; Castrucci, A.M. de; Hadley, M.E.; Andrews, J.R.; Wan, Y.P.

    1984-03-05

    Ac-(Nle/sup 4/, D-Phe/sup 7/)-..cap alpha..-MSH/sub 4-11/-NH/sub 2/ an octapeptide, is a melanotropin analogue (Ac-Nle-Glu-His-D-Phe-Arg-Trp-Gly-Lys-NH/sub 2/), which is a superpotent agonist of frog and lizard skin melanocytes and mouse S 91 (Cloudman) melanoma cells. This melanotropin possesses ultraprolonged activity on melanocytes, both in vitro and in vivo, and the peptide is resistant to inactivation by serum enzymes. The tritium-labeled congener was prepared by direct incorporation of (/sup 3/H)-labeled norleucine into the peptide. The melanotropic activity of the labeled peptide is identical to the unlabeled analogue. This labeled peptide should be useful for studies on the localization and characterization of melanotropin receptors.

  10. 18O-Labeled Proteome Reference as Global Internal Standards for Targeted Quantification by Selected Reaction Monitoring-Mass Spectrometry

    SciTech Connect

    Kim, Jong Seo; Fillmore, Thomas L.; Liu, Tao; Robinson, Errol W.; Hossain, Mahmud; Champion, Boyd L.; Moore, Ronald J.; Camp, David G.; Smith, Richard D.; Qian, Weijun

    2011-10-11

    Selected reaction monitoring-mass spectrometry (SRM-MS) is an emerging technology for high throughput targeted protein quantification and verification in biological and biomarker discovery studies; however, the cost associated with the use of stable isotope labeled synthetic peptides as internal standards is prohibitive for quantitatively screening large numbers of candidate proteins as often required in the pre-verification phase of biomarker discovery. Herein we present the proof-of-concept experiments of using an 18O-labeled 'universal' reference as comprehensive internal standards for quantitative SRM-MS analysis. With an 18O-labeled whole proteome sample as reference, every peptide of interest will have its own corresponding heavy isotope labeled internal standard, thus providing an ideal approach for quantitative screening of a large number of candidates using SRM-MS. Our results showed that the 18O incorporation efficiency using a recently improved protocol was >99.5% for most peptides investigated, a level comparable to 13C/15N labeled synthetic peptides in terms of heavy isotope incorporation. The accuracy, reproducibility, and linear dynamic range of quantification were further assessed based on known ratios of standard proteins spiked into mouse plasma with an 18O-labeled mouse plasma reference. A dynamic range of four orders of magnitude in relative concentration was obtained with high reproducibility (i.e., coefficient of variance <10%) based on the 16O/18O peak area ratios. Absolute and relative quantification of C-reactive protein and prostate-specific antigen were demonstrated by coupling an 18O-labeled reference with standard additions of protein standards. Collectively, our results demonstrated that the use of 18O-labeled reference provides a convenient and effective strategy for quantitative SRM screening of large number of candidate proteins.

  11. Identification of essential lysines involved in substrate binding of vacuolar H+-pyrophosphatase.

    PubMed

    Lee, Chien-Hsien; Pan, Yih-Jiuan; Huang, Yun-Tzu; Liu, Tseng-Huang; Hsu, Shen-Hsing; Lee, Ching-Hung; Chen, Yen-Wei; Lin, Shih-Ming; Huang, Lin-Kun; Pan, Rong-Long

    2011-04-01

    H+-translocating pyrophosphatase (H+-PPase; EC 3.6.1.1) drives proton transport against an electrochemical potential gradient by hydrolyzing pyrophosphate (PPi) and is found in various endomembranes of higher plants, bacteria, and some protists. H+-PPase contains seven highly conserved lysines. We examined the functional roles of these lysines, which are, for the most part, found in the cytosolic regions of mung bean H+-PPase by site-directed mutagenesis. Construction of mutants that each had a cytosolic and highly conserved lysine substituted with an alanine resulted in dramatic drops in the PPi hydrolytic activity. The effects caused by ions on the activities of WT and mutant H+-PPases suggest that Lys-730 may be in close proximity to the Mg2+-binding site, and the great resistance of the K694A and K695A mutants to fluoride inhibition suggests that these lysines are present in the active site. The modifier fluorescein 5'-isothiocyanate (FITC) labeled a lysine at the H+-PPase active site but did not inhibit the hydrolytic activities of K250A, K250N, K250T, and K250S, which suggested that Lys-250 is essential for substrate binding and may be involved in proton translocation. Analysis of tryptic digests indicated that Lys-711 and Lys-717 help maintain the conformation of the active site. Proteolytic evidence also demonstrated that Lys-250 is the primary target of trypsin and confirmed its crucial role in H+-PPase hydrolysis.

  12. Expression of messenger RNAs for peptides and tyrosine hydroxylase in primary sensory neurons that innervate arterial baroreceptors and chemoreceptors.

    PubMed

    Czyzyk-Krzeska, M F; Bayliss, D A; Lawson, E E; Millhorn, D E

    1991-08-01

    Retrograde fiber tracing and in situ hybridization were used to determine expression of mRNAs for preprotachykinin A (ppTA), calcitonin gene related peptide (CGRP), preproenkephalin A (ENK), neuropeptide tyrosine (NPY) and somatostatin (SOM) as well as tyrosine hydroxylase (TH) in the petrosal ganglia primary sensory neurons which innervate carotid sinus baroreceptors and carotid body chemoreceptors. Perfusion of the carotid sinus with the retrogradely transported dye (Fluoro-Gold) labeled primary sensory neurons in petrosal ganglion. Numerous somata in the petrosal ganglion labeled with dye contained mRNAs for all the above peptides, except SOM. Moreover, TH mRNA was found in a substantial number of retrogradely labeled cells in the petrosal ganglion. This study provides information concerning which of the numerous peptides identified in sensory neurons of petrosal ganglion may be involved in modulation of the arterial baroreceptor and chemoreceptor reflexes. PMID:1681484

  13. Nutrition Marketing on Food Labels

    ERIC Educational Resources Information Center

    Colby, Sarah E.; Johnson, LuAnn; Scheett, Angela; Hoverson, Bonita

    2010-01-01

    Objective: This research sought to determine how often nutrition marketing is used on labels of foods that are high in saturated fat, sodium, and/or sugar. Design and Setting: All items packaged with food labels (N = 56,900) in all 6 grocery stores in Grand Forks, ND were surveyed. Main Outcome Measure(s): Marketing strategy, nutrient label…

  14. Meat and Poultry Labeling Terms

    MedlinePlus

    ... Food Standards and Labels: The Facts Labeling and Marketing Information [ Top of Page ] OVEN PREPARED: Product is fully cooked and ready to eat. [ Top of Page ] YOUNG TURKEY: Turkeys of either sex that are less than 8 months of age according to present regulations. [ Top of Page ] Last ...

  15. A New Strategy of Using O18-Labeled Iodoacetic Acid for Mass Spectrometry-Based Protein Quantitation

    NASA Astrophysics Data System (ADS)

    Wang, Shunhai; Kaltashov, Igor A.

    2012-07-01

    A new O18 labeling protocol is designed to assist quantitation of cysteine-containing proteins using LC/MS. Unlike other O18 labeling strategies, the labeling is carried out at the intact protein level (prior to its digestion) during reduction/alkylation of cysteine side chains using O18-labeled iodoacetic acid (IAA). The latter can be easily prepared by exchanging carboxylic oxygen atoms of commercially available IAA in O18-enriched water at low pH. Since incorporation of the O18 label in the protein occurs at the whole protein, rather than peptide level, the quantitation results are not peptide-dependent. The excellent stability of the label in mild pH conditions provides flexibility and robustness needed of sample processing steps following the labeling. In contrast to generally costly isotope labeling reagents, this approach uses only two relatively inexpensive commercially available reagents (IAA and H2O18). The feasibility of the new method is demonstrated using an 80 kDa human serum transferrin (hTf) as a model, where linear quantitation is achieved across a dynamic range spanning three orders of magnitude. The new approach can be used in quantitative proteomics applications and is particularly suitable for a variety of tasks in the biopharmaceutical sector, ranging from pharmacokinetic studies to quality control of protein therapeutics.

  16. Characterization of mammalian glucose transport proteins using photoaffinity labeling techniques

    SciTech Connect

    Wadzinski, B.E.

    1989-01-01

    A carrier-free radioiodinated phenylazide derivative of forskolin, 3-iodo-4-azidophenethylamido-7-O-succinyl-deacetyl-forskolin (({sup 125}I)IAPS-forskolin), has been shown to be a highly selective photoaffinity probe for the human erythrocyte glucose transported and the glucose transport proteins found in several mammalian tissues and cultured cells where the glucose transport protein is present at a low concentration. The photoincorporation of ({sup 125}I)IAPS-forskolin into these glucose transporters was blocked by D- (but not L-) glucose, cytochalasin B, and forskolin. In addition to labeling the mammalian glucose transport proteins, ({sup 125}I)IAPS-forskolin also labeled the L-arabinose transporter from E. coli. In muscle and adipose tissues, glucose transport is markedly increased in response to insulin. ({sup 125}I)IAPS-forskolin was shown to selectivity tag the glucose transporter in membranes derived from these cells. In addition, the covalent derivatization of the transport protein in subcellular fractions of the adipocyte has provided a means to study the hormonal regulation of glucose transport. ({sup 125}I)IAPS-forskolin has also been used to label the purified human erythrocyte glucose transporter. The site of insertion has therefore been localized by analysis of the radiolabeled peptides which were produced following chemical and proteolytic digestion of the labeled transport protein.

  17. Sandmeyer reaction repurposed for the site-selective, non-oxidizing radioiodination of fully-deprotected peptides: studies on the endogenous opioid peptide α-neoendorphin.

    PubMed

    Pickett, Julie E; Nagakura, Kunihiko; Pasternak, Anna R; Grinnell, Steven G; Majumdar, Susruta; Lewis, Jason S; Pasternak, Gavril W

    2013-08-01

    Standard radioiodination methods lack site-selectivity and either mask charges (Bolton-Hunter) or involve oxidative reaction conditions (chloramine-T). Opioid peptides are very sensitive to certain structural modifications, making these labeling methods untenable. In our model opioid peptide, α-neoendorphin, we replaced a tyrosyl hydroxyl with an iodine, and in cell lines stably expressing mu, delta, or kappa opioid receptors, we saw no negative effects on binding. We then optimized a repurposed Sandmeyer reaction using copper(I) catalysts with non-redoxing/non-nucleophilic ligands, bringing the radiochemical yield up to around 30%, and site-selectively incorporated radioactive iodine into this position under non-oxidizing reaction conditions, which should be broadly compatible with most peptides. The (125)I- and (131)I-labeled versions of the compound bound with high affinity to opioid receptors in mouse brain homogenates, thus demonstrating the general utility of the labeling strategy and of the peptide for exploring opioid binding sites. PMID:23796454

  18. Specific glucagon-related peptides isolated from anglerfish islets are metabolic cleavage products of (pre)proglucagon-II.

    PubMed

    Noe, B D; Andrews, P C

    1986-01-01

    Sequence analyses of cDNAs prepared from anglerfish islet mRNA have demonstrated the presence of mRNAs coding for two different preproglucagons, aPPG-I and aPPG-II. Each of these precursors was predicted to contain 29 residue and 34 residue glucagon-related peptides as potential cleavage products. Recently, several glucagon-related peptides found in extracts of anglerfish islets have been isolated and characterized. In order to determine whether any of these peptides could be identified as metabolic cleavage products in anglerfish islets, differentially radiolabeled Mr 2,500-8,000 peptides from islet extracts were subjected to reverse phase HPLC under varying conditions. The potential cleavage products aPPG-II[52-80] and aPPG-II[89-122] could be readily identified among the extract peptides. Both peptides became labeled appropriately (as predicted from their sequences) with 13 different amino acids and demonstrated glucagon-like immunoreactivity in a radioimmunoassay. Conversely, a third peptide (aPPG-II[89-119]) could be found among the labeled products in small amounts only. These results demonstrate that glucagon-II[52-80] and aGLP-II[89-112] are primary cleavage products of aPPG-II and suggest that aGLP-IIc[89-119] may be a peptide generated more slowly by post-translational modification of aGLP-II. PMID:3526301

  19. The importance of peptide detectability for protein identification, quantification, and experiment design in MS/MS proteomics

    PubMed Central

    Li, Yong Fuga; Arnold, Randy J.; Tang, Haixu

    2010-01-01

    Peptide detectability is defined as the probability that a peptide is identified in an LC-MS/MS experiment and has been useful in providing solutions to protein inference and label-free quantification. Previously, predictors for peptide detectability trained on standard or complex samples were proposed. Although the models trained on complex samples may benefit from the large training data sets, it is unclear to what extent they are affected by the unequal abundances of identified proteins. To address this challenge and improve detectability prediction, we present a new algorithm for the iterative learning of peptide detectability from complex mixtures. We provide evidence that the new method approximates detectability with useful accuracy and, based on its design, can be used to interpret the outcome of other learning strategies. We studied the properties of peptides from the bacterium Deinococcus radiodurans and found that at standard quantities, its tryptic peptides can be roughly classified as either detectable or undetectable, with a relatively small fraction having medium detectability. We extend the concept of detectability from peptides to proteins and apply the model to predict the behavior of a replicate LC-MS/MS experiment from a single analysis. Finally, our study summarizes a theoretical framework for peptide/protein identification and label-free quantification. PMID:21067214

  20. Synthesis and antiviral activity of PB1 component of the influenza A RNA polymerase peptide fragments.

    PubMed

    Matusevich, O V; Egorov, V V; Gluzdikov, I A; Titov, M I; Zarubaev, V V; Shtro, A A; Slita, A V; Dukov, M I; Shurygina, A-P S; Smirnova, T D; Kudryavtsev, I V; Vasin, A V; Kiselev, O I

    2015-01-01

    This study is devoted to the antiviral activity of peptide fragments from the PB1 protein - a component of the influenza A RNA polymerase. The antiviral activity of the peptides synthesized was studied in MDCK cell cultures against the pandemic influenza strain A/California/07/2009 (H1N1) pdm09. We found that peptide fragments 6-13, 6-14, 26-30, 395-400, and 531-540 of the PB1 protein were capable of suppressing viral replication in cell culture. Terminal modifications i.e. N-acetylation and C-amidation increased the antiviral properties of the peptides significantly. Peptide PB1 (6-14) with both termini modified showed maximum antiviral activity, its inhibitory activity manifesting itself during the early stages of viral replication. It was also shown that the fluorescent-labeled analog of this peptide was able to penetrate into the cell. The broad range of virus-inhibiting activity of PB1 (6-14) peptide was confirmed using a panel of influenza A viruses of H1, H3 and H5 subtypes including those resistant to oseltamivir, the leading drug in anti-influenza therapy. Thus, short peptide fragments of the PB1 protein could serve as leads for future development of influenza prevention and/or treatment agents.

  1. CTHRSSVVC Peptide as a Possible Early Molecular Imaging Target for Atherosclerosis.

    PubMed

    Silva, Rosemeire A; Giordano, Ricardo J; Gutierrez, Paulo S; Rocha, Viviane Z; Rudnicki, Martina; Kee, Patrick; Abdalla, Dulcinéia S P; Puech-Leão, Pedro; Caramelli, Bruno; Arap, Wadih; Pasqualini, Renata; Meneghetti, José C; Marques, Fabio L N; Khoobchandani, Menka; Katti, Kattesh V; Lugão, Ademar B; Kalil, Jorge

    2016-01-01

    The purpose of our work was to select phages displaying peptides capable of binding to vascular markers present in human atheroma, and validate their capacity to target the vascular markers in vitro and in low-density lipoprotein receptor knockout (LDLr(-/-)) mouse model of atherosclerosis. By peptide fingerprinting on human atherosclerotic tissues, we selected and isolated four different peptides sequences, which bind to atherosclerotic lesions and share significant similarity to known human proteins with prominent roles in atherosclerosis. The CTHRSSVVC-phage peptide displayed the strongest reactivity with human carotid atherosclerotic lesions (p < 0.05), when compared to tissues from normal carotid arteries. This peptide sequence shares similarity to a sequence present in the fifth scavenger receptor cysteine-rich (SRCR) domain of CD163, which appeared to bind to CD163, and subsequently, was internalized by macrophages. Moreover, the CTHRSSVVC-phage targets atherosclerotic lesions of a low-density lipoprotein receptor knockout (LDLr(-/-)) mouse model of atherosclerosis in vivo to High-Fat diet group versus Control group. Tetraazacyclododecane-1,4,7,10-tetraacetic acid-CTHRSSVVC peptide (DOTA-CTHRSSVVC) was synthesized and labeled with (111)InCl₃ in >95% yield as determined by high performance liquid chromatography (HPLC), to validate the binding of the peptide in atherosclerotic plaque specimens. The results supported our hypothesis that CTHRSSVVC peptide has a remarkable sequence for the development of theranostics approaches in the treatment of atherosclerosis and other diseases. PMID:27563889

  2. Selective Deletion of the Internal Lysine Residue from the Peptide Sequence by Collisional Activation

    NASA Astrophysics Data System (ADS)

    Banerjee, Shibdas; Mazumdar, Shyamalava

    2012-11-01

    The gas-phase peptide ion fragmentation chemistry is always the center of attraction in proteomics to analyze the amino acid sequence of peptides and proteins. In this work, we describe the formation of an anomalous fragment ion, which corresponds to the selective deletion of the internal lysine residue from a series of lysine containing peptides upon collisional activation in the ion trap. We detected several water-loss fragment ions and the maximum number of water molecules lost from a particular fragment ion was equal to the number of lysine residues in that fragment. As a consequence of this water-loss phenomenon, internal lysine residues were found to be deleted from the peptide ion. The N,N-dimethylation of all the amine functional groups of the peptide stopped the internal lysine deletion reaction, but selective N-terminal α-amino acetylation had no effect on this process indicating involvement of the side chains of the lysine residues. The detailed mechanism of the lysine deletion was investigated by multistage CID of the modified and unmodified peptides, by isotope labeling and by energy resolved CID studies. The results suggest that the lysine deletion might occur through a unimolecular multistep mechanism involving a seven-membered cyclic imine intermediate formed by the loss of water from a lysine residue in the protonated peptide. This intermediate subsequently undergoes degradation reaction to deplete the interior imine ring from the peptide backbone leading to the deletion of an internal lysine residue.

  3. CTHRSSVVC Peptide as a Possible Early Molecular Imaging Target for Atherosclerosis

    PubMed Central

    Silva, Rosemeire A.; Giordano, Ricardo J.; Gutierrez, Paulo S.; Rocha, Viviane Z.; Rudnicki, Martina; Kee, Patrick; Abdalla, Dulcinéia S. P.; Puech-Leão, Pedro; Caramelli, Bruno; Arap, Wadih; Pasqualini, Renata; Meneghetti, José C.; Marques, Fabio L. N.; Khoobchandani, Menka; Katti, Kattesh V.; Lugão, Ademar B.; Kalil, Jorge

    2016-01-01

    The purpose of our work was to select phages displaying peptides capable of binding to vascular markers present in human atheroma, and validate their capacity to target the vascular markers in vitro and in low-density lipoprotein receptor knockout (LDLr−/−) mouse model of atherosclerosis. By peptide fingerprinting on human atherosclerotic tissues, we selected and isolated four different peptides sequences, which bind to atherosclerotic lesions and share significant similarity to known human proteins with prominent roles in atherosclerosis. The CTHRSSVVC-phage peptide displayed the strongest reactivity with human carotid atherosclerotic lesions (p < 0.05), when compared to tissues from normal carotid arteries. This peptide sequence shares similarity to a sequence present in the fifth scavenger receptor cysteine-rich (SRCR) domain of CD163, which appeared to bind to CD163, and subsequently, was internalized by macrophages. Moreover, the CTHRSSVVC-phage targets atherosclerotic lesions of a low-density lipoprotein receptor knockout (LDLr−/−) mouse model of atherosclerosis in vivo to High-Fat diet group versus Control group. Tetraazacyclododecane-1,4,7,10-tetraacetic acid-CTHRSSVVC peptide (DOTA-CTHRSSVVC) was synthesized and labeled with 111InCl3 in >95% yield as determined by high performance liquid chromatography (HPLC), to validate the binding of the peptide in atherosclerotic plaque specimens. The results supported our hypothesis that CTHRSSVVC peptide has a remarkable sequence for the development of theranostics approaches in the treatment of atherosclerosis and other diseases. PMID:27563889

  4. 21 CFR 201.72 - Potassium labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 4 2014-04-01 2014-04-01 false Potassium labeling. 201.72 Section 201.72 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.72 Potassium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the potassium...

  5. 21 CFR 201.72 - Potassium labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 4 2012-04-01 2012-04-01 false Potassium labeling. 201.72 Section 201.72 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.72 Potassium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the potassium...

  6. 21 CFR 201.72 - Potassium labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 4 2013-04-01 2013-04-01 false Potassium labeling. 201.72 Section 201.72 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.72 Potassium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the potassium...

  7. 21 CFR 610.60 - Container label.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Container label. 610.60 Section 610.60 Food and... GENERAL BIOLOGICAL PRODUCTS STANDARDS Labeling Standards § 610.60 Container label. (a) Full label. The following items shall appear on the label affixed to each container of a product capable of bearing a...

  8. 16 CFR 460.12 - Labels.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 16 Commercial Practices 1 2012-01-01 2012-01-01 false Labels. 460.12 Section 460.12 Commercial Practices FEDERAL TRADE COMMISSION TRADE REGULATION RULES LABELING AND ADVERTISING OF HOME INSULATION § 460.12 Labels. If you are a manufacturer, you must label all packages of your insulation. The labels...

  9. 16 CFR 460.12 - Labels.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 16 Commercial Practices 1 2014-01-01 2014-01-01 false Labels. 460.12 Section 460.12 Commercial Practices FEDERAL TRADE COMMISSION TRADE REGULATION RULES LABELING AND ADVERTISING OF HOME INSULATION § 460.12 Labels. If you are a manufacturer, you must label all packages of your insulation. The labels...

  10. 16 CFR 460.12 - Labels.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 16 Commercial Practices 1 2011-01-01 2011-01-01 false Labels. 460.12 Section 460.12 Commercial Practices FEDERAL TRADE COMMISSION TRADE REGULATION RULES LABELING AND ADVERTISING OF HOME INSULATION § 460.12 Labels. If you are a manufacturer, you must label all packages of your insulation. The labels...

  11. 16 CFR 460.12 - Labels.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 16 Commercial Practices 1 2010-01-01 2010-01-01 false Labels. 460.12 Section 460.12 Commercial Practices FEDERAL TRADE COMMISSION TRADE REGULATION RULES LABELING AND ADVERTISING OF HOME INSULATION § 460.12 Labels. If you are a manufacturer, you must label all packages of your insulation. The labels...

  12. 16 CFR 460.12 - Labels.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 16 Commercial Practices 1 2013-01-01 2013-01-01 false Labels. 460.12 Section 460.12 Commercial Practices FEDERAL TRADE COMMISSION TRADE REGULATION RULES LABELING AND ADVERTISING OF HOME INSULATION § 460.12 Labels. If you are a manufacturer, you must label all packages of your insulation. The labels...

  13. 21 CFR 201.71 - Magnesium labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 4 2012-04-01 2012-04-01 false Magnesium labeling. 201.71 Section 201.71 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.71 Magnesium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the magnesium...

  14. 21 CFR 201.71 - Magnesium labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 4 2013-04-01 2013-04-01 false Magnesium labeling. 201.71 Section 201.71 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.71 Magnesium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the magnesium...

  15. 21 CFR 201.71 - Magnesium labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 4 2014-04-01 2014-04-01 false Magnesium labeling. 201.71 Section 201.71 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.71 Magnesium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the magnesium...

  16. 9 CFR 317.4 - Labeling approval.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... labeling of such final labeling has been submitted for approval to the Food Labeling Division, Regulatory... Secretary upon request. (b) The Food Labeling Division shall permit submission for approval of only sketch... Food Labeling Division, Regulatory Programs, Food Safety and Inspection Service, U.S. Department...

  17. 21 CFR 201.72 - Potassium labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 4 2010-04-01 2010-04-01 false Potassium labeling. 201.72 Section 201.72 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.72 Potassium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the potassium...

  18. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... contains sodium bicarbonate, sodium phosphate, or sodium biphosphate as an active ingredient for oral... 21 Food and Drugs 4 2011-04-01 2011-04-01 false Sodium labeling. 201.64 Section 201.64 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.64 Sodium labeling. (a) The labeling...

  19. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... contains sodium bicarbonate, sodium phosphate, or sodium biphosphate as an active ingredient for oral... 21 Food and Drugs 4 2012-04-01 2012-04-01 false Sodium labeling. 201.64 Section 201.64 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.64 Sodium labeling. (a) The labeling...

  20. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... contains sodium bicarbonate, sodium phosphate, or sodium biphosphate as an active ingredient for oral... 21 Food and Drugs 4 2013-04-01 2013-04-01 false Sodium labeling. 201.64 Section 201.64 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.64 Sodium labeling. (a) The labeling...

  1. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... contains sodium bicarbonate, sodium phosphate, or sodium biphosphate as an active ingredient for oral... 21 Food and Drugs 4 2014-04-01 2014-04-01 false Sodium labeling. 201.64 Section 201.64 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.64 Sodium labeling. (a) The labeling...

  2. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... contains sodium bicarbonate, sodium phosphate, or sodium biphosphate as an active ingredient for oral... 21 Food and Drugs 4 2010-04-01 2010-04-01 false Sodium labeling. 201.64 Section 201.64 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.64 Sodium labeling. (a) The labeling...

  3. Rapid Covalent Fluorescence Labeling of Membrane Proteins on Live Cells via Coiled-Coil Templated Acyl Transfer.

    PubMed

    Reinhardt, Ulrike; Lotze, Jonathan; Mörl, Karin; Beck-Sickinger, Annette G; Seitz, Oliver

    2015-10-21

    Fluorescently labeled proteins enable the microscopic imaging of protein localization and function in live cells. In labeling reactions targeted against specific tag sequences, the size of the fluorophore-tag is of major concern. The tag should be small to prevent interference with protein function. Furthermore, rapid and covalent labeling methods are desired to enable the analysis of fast biological processes. Herein, we describe the development of a method in which the formation of a parallel coiled coil triggers the transfer of a fluorescence dye from a thioester-linked coil peptide conjugate onto a cysteine-modified coil peptide. This labeling method requires only small tag sequences (max 23 aa) and occurs with high tag specificity. We show that size matching of the coil peptides and a suitable thioester reactivity allow the acyl transfer reaction to proceed within minutes (rather than hours). We demonstrate the versatility of this method by applying it to the labeling of different G-protein coupled membrane receptors including the human neuropeptide Y receptors 1, 2, 4, 5, the neuropeptide FF receptors 1 and 2, and the dopamine receptor 1. The labeled receptors are fully functional and able to bind the respective ligand with high affinity. Activity is not impaired as demonstrated by activation, internalization, and recycling experiments. PMID:26367072

  4. Distribution of tempo-dichlorotriazine spin label on immunoglobulin molecule. Interpretation of ESR spectra

    SciTech Connect

    Nezlin, R.

    1986-03-05

    Spin label TEMPO-dichlorotriazine (DT) has been used previously for determination of the rotational relaxation times of immunoglobulin (Ig) molecules and evaluation of their flexibility. Well defined outer wide extrema as well as sharp inner extrema are characteristic for ESR spectra of spin labeled Ig molecules. Such patterns of the spectrum can be accounted for either by the existence of the spin label in two states, one corresponding to its rapid and another to its restricted rotation or by varying environments of the spin label located in different areas of the Ig molecule. To choose between these possibilities, the distribution of /sup 14/C-TEMPO-DT on human IgG1(k) was studied. The same amount of the label per mg of protein was found in H and L chains as well as in the Fab fragment, and a smaller amount in the pFc'. The label was detected in most of the L chain tryptic peptides. Thus, the spin label is distributed nearly uniformly on IgG molecule, which is due to the regular distribution of amino acid residues reacted with the spin label. ESR spectra can be interpreted as a sum of individual spectra.

  5. Self-aggregation properties of spin-labeled zervamicin IIA as studied by PELDOR spectroscopy.

    PubMed

    Milov, A D; Tsvetkov, Yu D; Gorbunova, E Yu; Mustaeva, L G; Ovchinnikova, T V; Raap, J

    2002-09-01

    In this article, the pulsed double electron-electron resonance in electron spin-echo (PELDOR) technique is applied to study the self-aggregation of spin-labeled zervamicin IIA, a hexadecapeptide antibiotic of fungal origin, which is known to form ion channels in a phospholipid double layer. Measurements of the ion channel forming properties and the antibiotic activity of the analog indicate that replacement of the C-terminal phenylalaninol by the amino-2,2,6,6-tetramethylpiperidinyloxy (TEMPO) residue does not influence the biophysical and biological properties. The dipole-dipole interaction between the spin labels of the fully biologically active peptide analog was studied in frozen (77 K) glassy solutions in different ratios of toluene-methanol. The spin-labeled zervamicin IIA molecules were shown to form aggregates. An average distance between the spin labels in the aggregates was estimated to be in the range of 25-35 A (depending on the solvent composition), indicating that the amphiphilic helical peptide molecules are oriented in an antiparallel fashion. Increasing of methanol content in the solution results in a loosening of the aggregate structure. It was shown that the fraction of aggregated zervamicin IIA molecules is less than 44-67% depending on the solvent composition. The general usefulness of the method to obtain structural long-range information in a range of several tens of angstroms is demonstrated by comparison with the peptide cluster of trichogin GA IV. PMID:12124850

  6. Macrocyclization of Unprotected Peptide Isocyanates.

    PubMed

    Vinogradov, Alexander A; Choo, Zi-Ning; Totaro, Kyle A; Pentelute, Bradley L

    2016-03-18

    A chemistry for the facile two-component macrocyclization of unprotected peptide isocyanates is described. Starting from peptides containing two glutamic acid γ-hydrazide residues, isocyanates can be readily accessed and cyclized with hydrazides of dicarboxylic acids. The choice of a nucleophilic linker allows for the facile modulation of biochemical properties of a macrocyclic peptide. Four cyclic NYAD-1 analogues were synthesized using the described method and displayed a range of biological activities. PMID:26948900

  7. Macrocyclization of Unprotected Peptide Isocyanates.

    PubMed

    Vinogradov, Alexander A; Choo, Zi-Ning; Totaro, Kyle A; Pentelute, Bradley L

    2016-03-18

    A chemistry for the facile two-component macrocyclization of unprotected peptide isocyanates is described. Starting from peptides containing two glutamic acid γ-hydrazide residues, isocyanates can be readily accessed and cyclized with hydrazides of dicarboxylic acids. The choice of a nucleophilic linker allows for the facile modulation of biochemical properties of a macrocyclic peptide. Four cyclic NYAD-1 analogues were synthesized using the described method and displayed a range of biological activities.

  8. Peptide Aptamers: Development and Applications

    PubMed Central

    Reverdatto, Sergey; Burz, David S.; Shekhtman, Alexander

    2015-01-01

    Peptide aptamers are small combinatorial proteins that are selected to bind to specific sites on their target molecules. Peptide aptamers consist of short, 5-20 amino acid residues long sequences, typically embedded as a loop within a stable protein scaffold. Various peptide aptamer scaffolds and in vitro and in vivo selection techniques are reviewed with emphasis on specific biomedical, bioimaging, and bioanalytical applications. PMID:25866267

  9. Labeled Cocaine Analogs

    SciTech Connect

    Goodman, Mark M.; Shi, Bing Zhi; Keil, Robert N.

    1999-01-26

    Novel compounds having the structure: ##STR1## where X in .beta. configuration is phenyl, naphthyl; 2,3 or 4-iodophenyl; 2,3 or 4-(trimethylsilyl)phenyl; 3,4,5 or 6-iodonaphthyl; 3,4,5 or 6-(trimethylsilyl)naphthyl; 2,3 or 4-(trialkylstannyl)phenyl; or 3,4,5 or 6-(trialkylstannyl)naphthyl Y in .beta. configuration is Y.sub.1 or Y.sub.2, where Y.sub.1 is 2-fluoroethoxy, 3-fluoropropoxy, 4-fluorobutoxy, 2-fluorocyclopropoxy, 2 or 3-fluorocyclobutoxy, R,S 1'-fluoroisopropoxy, R 1'-fluoroisopropoxy, S 1'-fluoroisopropoxy, 1',3'-difluoroisopropoxy, R,S 1'-fluoroisobutoxy, R 1'-fluoroisobutoxy, S 1'-fluoroisobutoxy, R,S 4'-fluoroisobutoxy, R 4'-fluoroisobutoxy, S 4'-fluoroisobutoxy, or 1',1'-di(fluoromethyl)isobutoxy, and Y.sub.2 is 2-methanesulfonyloxy ethoxy, 3-methanesulfonyloxy propoxy, 4-methanesulfonyloxy butoxy, 2-methanesulfonyloxy cyclopropoxy, 2 or 3-methanesulfonyloxy cyclobutoxy, 1'methanesulfonyloxy isopropoxy, 1'-fluoro, 3'-methanesulfonyloxy isopropoxy, 1'-methanesulfonyloxy, 3'-fluoro isopropoxy, 1'-methanesulfonyloxy isobutoxy, or 4'-methanesulfonyloxy isobutoxy bind dopamine transporter protein and can be labeled with .sup.18 F or .sup.123 I for imaging.

  10. Synthesis Of Labeled Metabolites

    DOEpatents

    Martinez, Rodolfo A.; Silks, III, Louis A.; Unkefer, Clifford J.; Atcher, Robert

    2004-03-23

    The present invention is directed to labeled compounds, for example, isotopically enriched mustard gas metabolites including: [1,1',2,2'-.sup.13 C.sub.4 ]ethane, 1,1'-sulfonylbis[2-(methylthio); [1,1',2,2'-.sup.13 C.sub.4 ]ethane, 1-[[2-(methylsulfinyl)ethyl]sulfonyl]-2-(methylthio); [1,1',2,2'-.sup.13 C.sub.4 ]ethane, 1,1'-sulfonylbis[2-(methylsulfinyl)]; and, 2,2'-sulfinylbis([1,2-.sup.13 C.sub.2 ]ethanol of the general formula ##STR1## where Q.sup.1 is selected from the group consisting of sulfide (--S--), sulfone (--S(O)--), sulfoxide (--S(O.sub.2)--) and oxide (--O--), at least one C* is .sup.13 C, X is selected from the group consisting of hydrogen and deuterium, and Z is selected from the group consisting of hydroxide (--OH), and --Q.sup.2 --R where Q.sup.2 is selected from the group consisting of sulfide (--S--), sulfone(--S(O)--), sulfoxide (--S(O.sub.2)--) and oxide (--O--), and R is selected from the group consisting of hydrogen, a C.sub.1 to C.sub.4 lower alkyl, and amino acid moieties, with the proviso that when Z is a hydroxide and Q.sup.1 is a sulfide, then at least one X is deuterium.

  11. Comparison of alternative nucleophiles for Sortase A-mediated bioconjugation and application in neuronal cell labelling.

    PubMed

    Baer, Samuel; Nigro, Julie; Madej, Mariusz P; Nisbet, Rebecca M; Suryadinata, Randy; Coia, Gregory; Hong, Lisa P T; Adams, Timothy E; Williams, Charlotte C; Nuttall, Stewart D

    2014-05-01

    The Sortase A (SrtA) enzyme from Staphylococcus aureus catalyses covalent attachment of protein substrates to pentaglycine cross-bridges in the Gram positive bacterial cell wall. In vitro SrtA-mediated protein ligation is now an important protein engineering tool for conjugation of substrates containing the LPXTGX peptide recognition sequence to oligo-glycine nucleophiles. In order to explore the use of alternative nucleophiles in this system, five different rhodamine-labelled compounds, with N-terminal nucleophilic amino acids, triglycine, glycine, and lysine, or N-terminal non-amino acid nucleophiles ethylenediamine and cadaverine, were synthesized. These compounds were tested for their relative abilities to function as nucleophiles in SrtA-mediated bioconjugation reactions. N-Terminal triglycine, glycine and ethylenediamine were all efficient in labelling a range of LPETGG containing recombinant antibody and scaffold proteins and peptides, while reduced activity was observed for the other nucleophiles across the range of proteins and peptides studied. Expansion of the range of available nucleophiles which can be utilised in SrtA-mediated bioconjugation expands the range of potential applications for this technology. As a demonstration of the utility of this system, SrtA coupling was used to conjugate the triglycine rhodamine-labelled nucleophile to the C-terminus of an Im7 scaffold protein displaying Aβ, a neurologically important peptide implicated in Alzheimer's disease. Purified, labelled protein showed Aβ-specific targeting to mammalian neuronal cells. Demonstration of targeting neuronal cells with a chimeric protein illustrates the power of this system, and suggests that SrtA-mediated direct cell-surface labelling and visualisation is an achievable goal. PMID:24643508

  12. Affinity labeling and characterization of the active site histidine of glucosephosphate isomerase

    SciTech Connect

    Gibson, D.R.; Gracy, R.W.; Hartman, F.C.

    1980-10-10

    N-bromoacetylethanolamine phosphate was found to act as a specific affinity label for the active center of glucosephosphate isomerase. The inactivation process followed pseudo-first order kinetics, was irreversible, and exhibited rate saturation kinetics with minimal half-lives of inactivation of 4.5 and 6.3 min for the enzyme isolated from human placenta and rabbit muscle, respectively. The pH dependence of the inactivation process closely paralleled the pH dependence of the overall catalytic process with pK/sub a/ values at pH 6.4 and 9.0. The stoichiometry of labeling of either enzyme, as determined with N-bromo(/sup 14/C/sub 2/)acetylethanolamine phosphate, was 1 eq of the affinity label/subunit of enzyme. After acid hydrolysis and amino acid analysis of the radioactive affinity-labeled human enzyme, only radioactive 3-carboxymethyl histidine was found. In the case of the rabbit enzyme, the only radioactive derivative obtained was 1-carboxymethyl histidine. Active site tryptic peptides were isolated by solvent extraction, thin layer peptide fingerprinting, and ion exchange chromatography before and after removal of the phosphate from the active site peptide. Amino acid analysis of the labeled peptides from the two species were very similar. Using high sensitivity methods for sequence analysis, the primary structure of the active site was established as Val-Leu-His-Ala-Glu-Asn-Val-Asp (Gly,Thr,Ser) Glu-Ile (Thr-Gly-His-Lys-Glx)-Tyr-Phe. Apparent sequence homology between the catalytic center of glucosephosphate isomerase and triosephosphate isomerase suggest that the two enzymes may have evolved from a common ancestral gene.

  13. Improving Peptide Applications Using Nanotechnology.

    PubMed

    Narayanaswamy, Radhika; Wang, Tao; Torchilin, Vladimir P

    2016-01-01

    Peptides are being successfully used in various fields including therapy and drug delivery. With advancement in nanotechnology and targeted delivery carrier systems, suitable modification of peptides has enabled achievement of many desirable goals over-riding some of the major disadvantages associated with the delivery of peptides in vivo. Conjugation or physical encapsulation of peptides to various nanocarriers, such as liposomes, micelles and solid-lipid nanoparticles, has improved their in vivo performance multi-fold. The amenability of peptides to modification in chemistry and functionalization with suitable nanocarriers are very relevant aspects in their use and have led to the use of 'smart' nanoparticles with suitable linker chemistries that favor peptide targeting or release at the desired sites, minimizing off-target effects. This review focuses on how nanotechnology has been used to improve the number of peptide applications. The paper also focuses on the chemistry behind peptide conjugation to nanocarriers, the commonly employed linker chemistries and the several improvements that have already been achieved in the areas of peptide use with the help of nanotechnology.

  14. Improving Peptide Applications Using Nanotechnology.

    PubMed

    Narayanaswamy, Radhika; Wang, Tao; Torchilin, Vladimir P

    2016-01-01

    Peptides are being successfully used in various fields including therapy and drug delivery. With advancement in nanotechnology and targeted delivery carrier systems, suitable modification of peptides has enabled achievement of many desirable goals over-riding some of the major disadvantages associated with the delivery of peptides in vivo. Conjugation or physical encapsulation of peptides to various nanocarriers, such as liposomes, micelles and solid-lipid nanoparticles, has improved their in vivo performance multi-fold. The amenability of peptides to modification in chemistry and functionalization with suitable nanocarriers are very relevant aspects in their use and have led to the use of 'smart' nanoparticles with suitable linker chemistries that favor peptide targeting or release at the desired sites, minimizing off-target effects. This review focuses on how nanotechnology has been used to improve the number of peptide applications. The paper also focuses on the chemistry behind peptide conjugation to nanocarriers, the commonly employed linker chemistries and the several improvements that have already been achieved in the areas of peptide use with the help of nanotechnology. PMID:26279082

  15. Peptides that influence membrane topology

    NASA Astrophysics Data System (ADS)

    Wong, Gerard C. L.

    2014-03-01

    We examine the mechanism of a range of polypeptides that influence membrane topology, including antimicrobial peptides, cell penetrating peptides, viral fusion peptides, and apoptosis proteins, and show how a combination of geometry, coordination chemistry, and soft matter physics can be used to approach a unified understanding. We will also show how such peptides can impact biomedical problems such as auto-immune diseases (psoriasis, lupus), infectious diseases (viral and bacterial infections), and mitochondrial pathologies (under-regulated apoptosis leads to neurodegenerative diseases whereas over-regulated apoptosis leads to cancer.)

  16. Fragmentation pathways of protonated peptides.

    PubMed

    Paizs, Béla; Suhai, Sándor

    2005-01-01

    The fragmentation pathways of protonated peptides are reviewed in the present paper paying special attention to classification of the known fragmentation channels into a simple hierarchy defined according to the chemistry involved. It is shown that the 'mobile proton' model of peptide fragmentation can be used to understand the MS/MS spectra of protonated peptides only in a qualitative manner rationalizing differences observed for low-energy collision induced dissociation of peptide ions having or lacking a mobile proton. To overcome this limitation, a deeper understanding of the dissociation chemistry of protonated peptides is needed. To this end use of the 'pathways in competition' (PIC) model that involves a detailed energetic and kinetic characterization of the major peptide fragmentation pathways (PFPs) is proposed. The known PFPs are described in detail including all the pre-dissociation, dissociation, and post-dissociation events. It is our hope that studies to further extend PIC will lead to semi-quantative understanding of the MS/MS spectra of protonated peptides which could be used to develop refined bioinformatics algorithms for MS/MS based proteomics. Experimental and computational data on the fragmentation of protonated peptides are reevaluated from the point of view of the PIC model considering the mechanism, energetics, and kinetics of the major PFPs. Evidence proving semi-quantitative predictability of some of the ion intensity relationships (IIRs) of the MS/MS spectra of protonated peptides is presented. PMID:15389847

  17. Biodiscovery of aluminum binding peptides

    NASA Astrophysics Data System (ADS)

    Adams, Bryn L.; Sarkes, Deborah A.; Finch, Amethist S.; Hurley, Margaret M.; Stratis-Cullum, Dimitra

    2013-05-01

    Cell surface peptide display systems are large and diverse libraries of peptides (7-15 amino acids) which are presented by a display scaffold hosted by a phage (virus), bacteria, or yeast cell. This allows the selfsustaining peptide libraries to be rapidly screened for high affinity binders to a given target of interest, and those binders quickly identified. Peptide display systems have traditionally been utilized in conjunction with organic-based targets, such as protein toxins or carbon nanotubes. However, this technology has been expanded for use with inorganic targets, such as metals, for biofabrication, hybrid material assembly and corrosion prevention. While most current peptide display systems employ viruses to host the display scaffold, we have recently shown that a bacterial host, Escherichia coli, displaying peptides in the ubiquitous, membrane protein scaffold eCPX can also provide specific peptide binders to an organic target. We have, for the first time, extended the use of this bacterial peptide display system for the biodiscovery of aluminum binding 15mer peptides. We will present the process of biopanning with macroscopic inorganic targets, binder enrichment, and binder isolation and discovery.

  18. Characterization of the secondary structure of calmodulin in complex with a calmodulin-binding domain peptide

    SciTech Connect

    Roth, S.M.; Schneider, D.M.; Strobel, L.A.; Wand, A.J. Univ. of Illinois, Urbana ); Van Berkum, M.F.A.; Means, A.R. )

    1992-02-11

    The interaction between calcium-saturated chicken calmodulin and a peptide corresponding to the calmodulin-binding domain of the chicken smooth muscle myosin light chain kinase has been studied by multinuclear and multidimensional nuclear magnetic resonance methods. Extensive {sup 1}H and {sup 15}N resonance assignments of calmodulin in the complex have been obtained from the analysis of two- and three-dimensional nuclear magnetic resonance spectra. The assignment of calmodulin in the complex was facilitated by the use of selective labeling of the protein with {alpha}-{sup 15}N-labeled valine, alanine, lysine, leucine, and glycine. These provided reference points during the main-chain-directed analysis of three-dimensional spectra of complexes prepared with uniformly {sup 15}N-labeled calmodulin. The pattern of nuclear Overhauser effects (NOE) seen among main-chain amide NH, C{sub {alpha}}H, and C{sub {beta}}H hydrogens indicates that the secondary structure of the globular domains of calmodulin in the complex closely corresponds to that observed in the calcium-saturated state of the protein in the absence of bound peptide. However, the backbone conformation of residues 76-84 adopts an extended chain conformation upon binding of the peptide in contrast to its helical conformation in the absence of peptide. A sufficient number of NOEs between the globular domains of calmodulin and the bound peptide have been found to indicate that the N- and C-terminal regions of the peptide interact with the C- and N-terminal domains of calmodulin, respectively. The significance of these results are discussed in terms of recently proposed models for the structure of calmodulin-peptide complexes.

  19. Peptides and Food Intake

    PubMed Central

    Sobrino Crespo, Carmen; Perianes Cachero, Aránzazu; Puebla Jiménez, Lilian; Barrios, Vicente; Arilla Ferreiro, Eduardo

    2014-01-01

    The mechanisms for controlling food intake involve mainly an interplay between gut, brain, and adipose tissue (AT), among the major organs. Parasympathetic, sympathetic, and other systems are required for communication between the brain satiety center, gut, and AT. These neuronal circuits include a variety of peptides and hormones, being ghrelin the only orexigenic molecule known, whereas the plethora of other factors are inhibitors of appetite, suggesting its physiological relevance in the regulation of food intake and energy homeostasis. Nutrients generated by food digestion have been proposed to activate G-protein-coupled receptors on the luminal side of enteroendocrine cells, e.g., the L-cells. This stimulates the release of gut hormones into the circulation such as glucagon-like peptide-1 (GLP-1), oxyntomodulin, pancreatic polypeptides, peptide tyrosine tyrosine, and cholecystokinin, which inhibit appetite. Ghrelin is a peptide secreted from the stomach and, in contrast to other gut hormones, plasma levels decrease after a meal and potently stimulate food intake. Other circulating factors such as insulin and leptin relay information regarding long-term energy stores. Both hormones circulate at proportional levels to body fat content, enter the CNS proportionally to their plasma levels, and reduce food intake. Circulating hormones can influence the activity of the arcuate nucleus (ARC) neurons of the hypothalamus, after passing across the median eminence. Circulating factors such as gut hormones may also influence the nucleus of the tractus solitarius (NTS) through the adjacent circumventricular organ. On the other hand, gastrointestinal vagal afferents converge in the NTS of the brainstem. Neural projections from the NTS, in turn, carry signals to the hypothalamus. The ARC acts as an integrative center, with two major subpopulations of neurons influencing appetite, one of them coexpressing neuropeptide Y and agouti-related protein (AgRP) that increases food

  20. Peptides and food intake.

    PubMed

    Sobrino Crespo, Carmen; Perianes Cachero, Aránzazu; Puebla Jiménez, Lilian; Barrios, Vicente; Arilla Ferreiro, Eduardo

    2014-01-01

    The mechanisms for controlling food intake involve mainly an interplay between gut, brain, and adipose tissue (AT), among the major organs. Parasympathetic, sympathetic, and other systems are required for communication between the brain satiety center, gut, and AT. These neuronal circuits include a variety of peptides and hormones, being ghrelin the only orexigenic molecule known, whereas the plethora of other factors are inhibitors of appetite, suggesting its physiological relevance in the regulation of food intake and energy homeostasis. Nutrients generated by food digestion have been proposed to activate G-protein-coupled receptors on the luminal side of enteroendocrine cells, e.g., the L-cells. This stimulates the release of gut hormones into the circulation such as glucagon-like peptide-1 (GLP-1), oxyntomodulin, pancreatic polypeptides, peptide tyrosine tyrosine, and cholecystokinin, which inhibit appetite. Ghrelin is a peptide secreted from the stomach and, in contrast to other gut hormones, plasma levels decrease after a meal and potently stimulate food intake. Other circulating factors such as insulin and leptin relay information regarding long-term energy stores. Both hormones circulate at proportional levels to body fat content, enter the CNS proportionally to their plasma levels, and reduce food intake. Circulating hormones can influence the activity of the arcuate nucleus (ARC) neurons of the hypothalamus, after passing across the median eminence. Circulating factors such as gut hormones may also influence the nucleus of the tractus solitarius (NTS) through the adjacent circumventricular organ. On the other hand, gastrointestinal vagal afferents converge in the NTS of the brainstem. Neural projections from the NTS, in turn, carry signals to the hypothalamus. The ARC acts as an integrative center, with two major subpopulations of neurons influencing appetite, one of them coexpressing neuropeptide Y and agouti-related protein (AgRP) that increases food

  1. Time-Resolved Fluorescence Anisotropy Study of the Interaction Between DNA and a Peptide Truncated from the p53 Protein Core Domain.

    PubMed

    Liu, Chengxuan; Liang, Gaiting; Liu, Zhen; Zu, Lily

    2014-03-01

    Time-resolved fluorescence anisotropy spectroscopy was applied to study the interaction between a peptide truncated from the binding site of tumor suppressor p53 protein and the DNAs covalently labeled with 6-carboxyfluorescein (FAM) dye. Fluorescence intensity quenching and changes of anisotropy decay lifetime were monitored when FAM labeled DNA formed complex with the peptide. The results demonstrated that the sequence of DNA could not define the binding specificity between the peptide and DNA. But the anisotropy decay of FAM can be used to examine the binding affinity of the peptide to DNA. The fluorescent dynamics of FAM can also be used to represent the rigidity of the complex formed between the peptide and DNA.

  2. Precision of heavy-light peptide ratios measured by maldi-tof mass spectrometry.

    PubMed

    Anderson, N Leigh; Razavi, Morteza; Pearson, Terry W; Kruppa, Gary; Paape, Rainer; Suckau, Detlef

    2012-03-01

    We have investigated the precision of peptide quantitation by MALDI-TOF mass spectrometry (MS) using six pairs of proteotypic peptides (light) and same-sequence stable isotope labeled synthetic internal standards (heavy). These were combined in two types of dilution curves spanning 100-fold and 2000-fold ratios. Coefficients of variation (CV; standard deviation divided by mean value) were examined across replicate MALDI spots using a reflector acquisition method requiring 100 000 counts for the most intense peak in each summed spectrum. The CV of light/heavy peptide centroid peak area ratios determined on four replicate spots per sample, averaged across 11 points of a 100-fold dilution curve and over all six peptides, was 2.2% (ranging from 1.5 to 3.7% among peptides) at 55 fmol total (light + heavy) of each peptide applied per spot, and 2.5% at 11 fmol applied. The average CV of measurements at near-equivalence (light = heavy, the center of the dilution curve) for the six peptides was 1.0%, about 17-fold lower CV than that observed when five peptides were ratioed to a sixth peptide (i.e., a different-sequence internal standard). Response curves across the 100-fold range were not completely linear but could be closely modeled by a power law fit giving R(2) values >0.998 for all peptides. The MALDI-TOF MS method was used to determine the endogenous level of a proteotypic peptide (EDQYHYLLDR) of human protein C inhibitor (PCI) in a plasma digest after enrichment by capture on a high affinity antipeptide antibody, a technique called stable isotope standards and capture by anti-peptide antibodies (SISCAPA). The level of PCI was determined to be 770 ng/mL with a replicate measurement CV of 1.5% and a >14 000-fold target enrichment via SISCAPA-MALDI-TOF. These results indicate that MALDI-TOF technology can provide precise quantitation of high-to-medium abundance peptide biomarkers over a 100-fold dynamic range when ratioed to same-sequence labeled internal standards

  3. 21 CFR 1302.04 - Location and size of symbol on label and labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 9 2012-04-01 2012-04-01 false Location and size of symbol on label and labeling. 1302.04 Section 1302.04 Food and Drugs DRUG ENFORCEMENT ADMINISTRATION, DEPARTMENT OF JUSTICE LABELING... and labeling. The symbol shall be prominently located on the label or the labeling of the...

  4. 21 CFR 1302.04 - Location and size of symbol on label and labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 9 2013-04-01 2013-04-01 false Location and size of symbol on label and labeling. 1302.04 Section 1302.04 Food and Drugs DRUG ENFORCEMENT ADMINISTRATION, DEPARTMENT OF JUSTICE LABELING... and labeling. The symbol shall be prominently located on the label or the labeling of the...

  5. Using fluorine nuclear magnetic resonance to probe the interaction of membrane-active peptides with the lipid bilayer.

    PubMed

    Buer, Benjamin C; Chugh, Jeetender; Al-Hashimi, Hashim M; Marsh, E Neil G

    2010-07-13

    A variety of biologically active peptides exert their function through direct interactions with the lipid membrane of the cell. These surface interactions are generally transient and highly dynamic, making them hard to study. Here we have examined the feasibility of using solution phase (19)F nuclear magnetic resonance (NMR) to study peptide-membrane interactions. Using the antimicrobial peptide MSI-78 as a model system, we demonstrate that peptide binding to either small unilamellar vesicles (SUVs) or bicelles can readily be detected by simple one-dimensional (19)F NMR experiments with peptides labeled with l-4,4,4-trifluoroethylglycine. The (19)F chemical shift associated with the peptide-membrane complex is sensitive both to the position of the trifluoromethyl reporter group (whether in the hydrophobic face or positively charged face of the amphipathic peptide) and to the curvature of the lipid bilayer (whether the peptide is bound to SUVs or bicelles). (19)F spin echo experiments using the Carr-Purcell-Meiboom-Gill pulse sequence were used to measure the transverse relaxation (T(2)) of the nucleus and thereby examine the local mobility of the MSI-78 analogues bound to bicelles. The fluorine probe positioned in the hydrophobic face of the peptide relaxes at a rate that correlates with the tumbling of the bicelle, suggesting that it is relatively immobile, whereas the probe at the positively charged face relaxes more slowly, indicating this position is much more dynamic. These results are in accord with structural models of MSI-78 bound to lipids and point to the feasibility of using fluorine-labeled peptides to monitor peptide-membrane interactions in living cells.

  6. Deglycosylation of chondroitin sulfate proteoglycan and derived peptides

    SciTech Connect

    Campbell, S.C.; Krueger, R.C.; Schwartz, N.B. )

    1990-01-30

    In order to define the domain structure of proteoglycans as well as identify primary amino acid sequences specific for attachment of the various carbohydrate substituents, reliable techniques for deglycosylating proteoglycans are required. In this study, deglycosylation of cartilage chondroitin sulfate proteoglycan (CSPG) with minimal core protein cleavage was accomplished by digestion with chondroitinase ABC and keratanase, followed by treatment with anhydrous HF in pyridine. Nearly complete deglycosylation of secreted proteoglycan was verified within 45 min of HF treatment by loss of incorporated ({sup 3}H)glucosamine label from the proteoglycan as a function of time of treatment, as well as by direct analysis of carbohydrate content and xylosyltransferase acceptor activity of unlabeled core protein preparations. The deglycosylated CSPG preparations were homogeneous and of high molecular weight. Comparison of the intact deglycosylated core protein preparations with newly synthesized unprocessed precursors suggested that extensive proteolytic cleavage of the core protein did not occur during normal intracellular processing. Furthermore, peptide patterns generated after clostripain digestion of core protein precursor and of deglycosylated secreted proteoglycan were comparable. With the use of the clostripain digestion procedure, peptides were produced from unlabeled proteoglycan, and two predominant peptides from the most highly glycosylated regions were isolated, characterized, and deglycosylated. These peptides were found to follow similar kinetics of deglycosylation and to acquire xylose activity comparable to the intact core protein.

  7. Amino Acid and Peptide Requirement of Fusiformis necrophorus

    PubMed Central

    Wahren, Ann; Holme, Tord

    1973-01-01

    Uptake of individual amino acids and peptides by Fusiformis necrophorus was studied in growing cultures and resting cell suspensions. The cells were able to incorporate 16 of 17 14C-labeled amino acids into cell protein, the exception being proline. Proline could neither be formed by the cells from any of the other tested amino acids nor be synthesized from glucose or serine when these were used as energy sources. The addition of di- and tripeptides, the octapeptides vasopressin and oxytocin, and the poly (24) peptide ACTH did not stimulate cell growth, but a marked stimulatory effect was noted after the addition of poly-l-proline (mean molecular weight 2,000). It is concluded that cells of F. necrophorus (i) possess transport systems for most amino acids but not for proline, (ii) are dependent on exogenous proline in the form of proline-containing peptides for growth, and (iii) may be cultivated in a defined amino acid medium provided the proline requirement is met by the addition of a proline-containing peptide. PMID:4745417

  8. Vasoactive intestinal peptide and electrical activity influence neuronal survival

    SciTech Connect

    Brenneman, D.E.; Eiden, L.E.

    1986-02-01

    Blockage of electrical activity in dissociated spinal cord cultures results in a significant loss of neurons during a critical period in development. Decreases in neuronal cell numbers and SVI-labeled tetanus toxin fixation produced by electrical blockage with tetrodotoxin (TTX) were prevented by addition of vasoactive intestinal peptide (VIP) to the nutrient medium. The most effective concentration of VIP was 0.1 nM. At higher concentrations, the survival-enhancing effect of VIP on TTX-treated cultures was attenuated. Addition of the peptide alone had no significant effect on neuronal cell counts or tetanus toxin fixation. With the same experimental conditions, two closely related peptides, PHI-27 (peptide, histidyl-isoleucine amide) and secretin, were found not to increase the number of neurons in TTX-treated cultures. Interference with VIP action by VIP antiserum resulted in neuronal losses that were not significantly different from those observed after TTX treatment. These data indicate that under conditions of electrical blockade a neurotrophic action of VIP on neuronal survival can be demonstrated.

  9. C-Peptide Prevents Hippocampal Apoptosis in Type 1 Diabetes

    PubMed Central

    Li, Zhen-guo; Zhang, Weixian

    2002-01-01

    To explore mechanisms underlying central nervous system (CNS) complications in diabetes, we examined hippocampal neuronal apoptosis and loss, and the effect of C-peptide replacement in type 1 diabetic BB/W rats. Apoptosis was demonstrated after 8 months of diabetes, by DNA fragmentation, increased number of apoptotic cells, and an elevated ratio of Bax/Bcl-xL, accompanied by reduced neuronal density in the hippocampus. No apoptotic activity was detected and neuronal density was unchanged in 2-month diabetic hippocampus, whereas insulin-like growth factor (IGF) activities were impaired. In type 1 diabetic BB/W rats replaced with C-peptide, no TdT-mediated dUTP nick-end labeling (TUNEL)- positive cells were shown and DNA laddering was not evident in hippocampus at either 2 or 8 months. C-peptide administration prevented the preceding perturbation of IGF expression and reduced the elevated ratio of Bax/Bcl-xL. Our data suggest that type 1 diabetes causes a duration-dependent programmed cell death of the hippocampus, which is partially prevented by C-peptide. PMID:12546277

  10. Identification and Tumour-Binding Properties of a Peptide with High Affinity to the Disialoganglioside GD2

    PubMed Central

    Müller, Jan; Reichel, Robin; Vogt, Sebastian; Müller, Stefan P.; Sauerwein, Wolfgang; Brandau, Wolfgang; Eggert, Angelika

    2016-01-01

    Neuroectodermal tumours are characterized by aberrant processing of disialogangliosides concomitant with high expression of GD2 or GD3 on cell surfaces. Antibodies targeting GD2 are already in clinical use for therapy of neuroblastoma, a solid tumour of early childhood. Here, we set out to identify peptides with high affinity to human disialoganglioside GD2. To this end, we performed a combined in vivo and in vitro screen using a recombinant phage displayed peptide library. We isolated a phage displaying the peptide sequence WHWRLPS that specifically binds to the human disialoganglioside GD2. Binding specificity was confirmed by mutational scanning and by comparative analyses using structurally related disialogangliosides. In vivo, significant enrichment of phage binding to xenografts of human neuroblastoma cells in mice was observed. Tumour-specific phage accumulation could be blocked by intravenous coinjection of the corresponding peptide. Comparative pharmacokinetic analyses revealed higher specific accumulation of 68Ga-labelled GD2-binding peptide compared to 111In-labelled peptide in xenografts of human neuroblastoma. In contrast to 124I-MIBG, which is currently evaluated as a neuroblastoma marker in PET/CT, 68Ga-labelled GD2-specific peptide spared the thyroid but was enriched in the kidneys, which could be partially blocked by infusion of amino acids.In summary, we here report on a novel tumour-homing peptide that specifically binds to the disialoganglioside GD2, accumulates in xenografts of neuroblastoma cells in mice and bears the potential for tumour detection using PET/CT. Thus, this peptide may serve as a new scaffold for diagnosing GD2-positive tumours of neuroectodermal origin. PMID:27716771

  11. Identification of pro-opiomelanocortin and secretion of its peptide fragments in bovine adrenals

    SciTech Connect

    Tennov, A.V.; Dmitriev, A.D.; Kizim, E.A.; Ustinova, E.E.

    1986-01-01

    This paper describes the results of an investigation to show that biosynthesis of POMC, its proteolytic processing, an secretion of the peptide products of that processing take place in the bovine adrenals. Rabbit antisera against endorphins were obtained and used for radioimmunoassay of peptides. I 125-labeled peptides were obtained by the chloramine method and purified from free I 125 on Sephadex G-10 (0.7 x 5 cm, centrifugation for 10 min at 1500 g). To detect secretion of peptide fragments of POMC in the adrenals experiments were undertaken to determine the beta-endorphin content in perfusates obtained during retrograde perfusion of the bovine adrenals. It was found that immunoreactive compounds, indistinguishable in their immunochemical properties from beta-endorphin, are present in the perfusates, just as in the tissue extracts.

  12. Chemoenzymatic exchange of phosphopantetheine on protein and peptide

    PubMed Central

    Kosa, Nicolas M.; Pham, Kevin M.; Burkart, Michael D.

    2016-01-01

    Evaluation of new acyl carrier protein hydrolase (AcpH, EC 3.1.4.14) homologs from proteobacteria and cyanobacteria reveals significant variation in substrate selectivity and kinetic parameters for phosphopantetheine hydrolysis from carrier proteins. Evaluation with carrier proteins from both primary and secondary metabolic pathways reveals an overall preference for acyl carrier protein (ACP) substrates from type II fatty acid synthases, as well as variable activity for polyketide synthase ACPs and peptidyl carrier proteins (PCP) from non-ribosomal peptide synthases. We also demonstrate the kinetic parameters of these homologs for AcpP and the 11-mer peptide substrate YbbR. These findings enable the fully reversible labeling of all three classes of natural product synthase carrier proteins as well as full and minimal fusion protein constructs. PMID:26998215

  13. Phytosulfokine peptide signalling.

    PubMed

    Sauter, Margret

    2015-08-01

    Phytosulfokine (PSK) belongs to the group of plant peptide growth factors. It is a disulfated pentapeptide encoded by precursor genes that are ubiquitously present in higher plants, suggestive of universal functions. Processing of the preproprotein involves sulfonylation by a tyrosylprotein sulfotransferase in the trans-golgi and proteolytic cleavage in the apoplast. The secreted peptide is perceived at the cell surface by a membrane-bound receptor kinase of the leucine-rich repeat family. The PSK receptor PSKR1 from Arabidopsis thaliana is an active kinase and has guanylate cyclase activity resulting in dual-signal outputs. Receptor activity is regulated by calmodulin. While PSK may be an autocrine growth factor, it also acts non-cell autonomously by promoting growth of cells that are receptor-deficient. In planta, PSK has multiple functions. It promotes cell growth, acts in the quiescent centre cells of the root apical meristem, contributes to funicular pollen tube guidance, and differentially alters immune responses depending on the pathogen. It has been suggested that PSK integrates growth and defence signals to balance the competing metabolic costs of these responses. This review summarizes our current understanding of PSK synthesis, signalling, and activity.

  14. Recognition of Bacterial Signal Peptides by Mammalian Formyl Peptide Receptors

    PubMed Central

    Bufe, Bernd; Schumann, Timo; Kappl, Reinhard; Bogeski, Ivan; Kummerow, Carsten; Podgórska, Marta; Smola, Sigrun; Hoth, Markus; Zufall, Frank

    2015-01-01

    Formyl peptide receptors (FPRs) are G-protein-coupled receptors that function as chemoattractant receptors in innate immune responses. Here we perform systematic structure-function analyses of FPRs from six mammalian species using structurally diverse FPR peptide agonists and identify a common set of conserved agonist properties with typical features of pathogen-associated molecular patterns. Guided by these results, we discover that bacterial signal peptides, normally used to translocate proteins across cytoplasmic membranes, are a vast family of natural FPR agonists. N-terminally formylated signal peptide fragments with variable sequence and length activate human and mouse FPR1 and FPR2 at low nanomolar concentrations, thus establishing FPR1 and FPR2 as sensitive and broad signal peptide receptors. The vomeronasal receptor mFpr-rs1 and its sequence orthologue hFPR3 also react to signal peptides but are much more narrowly tuned in signal peptide recognition. Furthermore, all signal peptides examined here function as potent activators of the innate immune system. They elicit robust, FPR-dependent calcium mobilization in human and mouse leukocytes and trigger a range of classical innate defense mechanisms, such as the production of reactive oxygen species, metalloprotease release, and chemotaxis. Thus, bacterial signal peptides constitute a novel class of immune activators that are likely to contribute to mammalian immune defense against bacteria. This evolutionarily conserved detection mechanism combines structural promiscuity with high specificity and enables discrimination between bacterial and eukaryotic signal sequences. With at least 175,542 predicted sequences, bacterial signal peptides represent the largest and structurally most heterogeneous class of G-protein-coupled receptor agonists currently known for the innate immune system. PMID:25605714

  15. Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

    PubMed Central

    Klingler, Diana; Hardt, Markus

    2013-01-01

    Stable isotopes are essential tools in biological mass spectrometry. Historically, 18O-stable isotopes have been extensively used to study the catalytic mechanisms of proteolytic enzymes1-3. With the advent of mass spectrometry-based proteomics, the enzymatically-catalyzed incorporation of 18O-atoms from stable isotopically enriched water has become a popular method to quantitatively compare protein expression levels (reviewed by Fenselau and Yao4, Miyagi and Rao5 and Ye et al.6). 18O-labeling constitutes a simple and low-cost alternative to chemical (e.g. iTRAQ, ICAT) and metabolic (e.g. SILAC) labeling techniques7. Depending on the protease utilized, 18O-labeling can result in the incorporation of up to two 18O-atoms in the C-terminal carboxyl group of the cleavage product3. The labeling reaction can be subdivided into two independent processes, the peptide bond cleavage and the carboxyl oxygen exchange reaction8. In our PALeO (protease-assisted labeling employing 18O-enriched water) adaptation of enzymatic 18O-labeling, we utilized 50% 18O-enriched water to yield distinctive isotope signatures. In combination with high-resolution matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS), the characteristic isotope envelopes can be used to identify cleavage products with a high level of specificity. We previously have used the PALeO-methodology to detect and characterize endogenous proteases9 and monitor proteolytic reactions10-11. Since PALeO encodes the very essence of the proteolytic cleavage reaction, the experimental setup is simple and biochemical enrichment steps of cleavage products can be circumvented. The PALeO-method can easily be extended to (i) time course experiments that monitor the dynamics of proteolytic cleavage reactions and (ii) the analysis of proteolysis in complex biological samples that represent physiological conditions. PALeO-TimeCourse experiments help identifying rate-limiting processing

  16. Homosexual Labeling by University Youths

    ERIC Educational Resources Information Center

    Nyberg, Kenneth L.; Alston, Jon P.

    1977-01-01

    Details the responses of young, urban, college-educated people on their attitudes toward homosexuals, specifically focusing on issues of public identification and negative labeling as it effects homosexual persons and their behaviors. (Author/RK)

  17. How to read food labels

    MedlinePlus

    ... 1 serving. You should also pay attention to trans fats on any food label. These fats raise "bad" ... foods and desserts. Many fast food restaurants use trans fats for frying. If a food has these fats, ...

  18. Dietary Supplement Label Database (DSLD)

    MedlinePlus

    ... Print Report Error T he Dietary Supplement Label Database (DSLD) is a joint project of the National ... participants in the latest survey in the DSLD database (NHANES): The search options: Quick Search, Browse Dietary ...

  19. Food Labels Tell the Story!

    MedlinePlus

    ... Environment Kids Health Topics Environment & Health Healthy Living Pollution Reduce, Reuse, Recycle Science – How It Works The ... Pay close attention to serving sizes. Products labeled "light" or "lite" must have 1/3 fewer calories ...

  20. Affinity Labeling of Highly Hydrophobic Integral Membrane Proteins for Proteome-Wide Analysis

    SciTech Connect

    Goshe, Michael B.; Blonder, Josip; Smith, Richard D.

    2003-03-01

    The ability to identify and quantify integral membrane proteins is an analytical challenge for mass spectrometry-based proteomics. The use of surfactants to solubilize and derivatize these proteins can suppress peptide ionization and interfere with chromatographic separations during microcapillary reversed-phase liquid chromatography-electrospray-tandem mass spectrometry. To circumvent the use of surfactants and increase proteome coverage, an affinity labeling method has been developed to target highly hydrophobic integral membrane proteins using organic-assisted extraction and solubilization followed by cysteinyl-specific labeling using biotinylation reagents. As demonstrated on the membrane subproteome of Deinococcus radiodurans, specific and quantitative labeling of integral membrane proteins was achieved using a 60% methanol-aqueous buffer system and (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine as the cysteinyl-alkylating reagent. From a total of 220 unique Cys-labeled peptides, 89 proteins were identified of which 40 were integral membrane proteins containing from 1 to 9 mapped transmembrane domains with a maximum positive GRAVY of 1.08. The protocol described can be used with other stable isotope labeling reagents (e.g. ICAT) to enable comparative measurements to be made on differentially expressed hydrophobic membrane proteins from various organisms (e.g. pathogenic bacteria) and cell types and provide a viable method for comparative proteome-wide analyses.

  1. Clinical uses of gut peptides.

    PubMed Central

    Geoghegan, J; Pappas, T N

    1997-01-01

    OBJECTIVE: The authors review clinical applications of gut-derived peptides as diagnostic and therapeutic agents. SUMMARY BACKGROUND DATA: An increasing number of gut peptides have been evaluated for clinical use. Earlier uses as diagnostic agents have been complemented more recently by increasing application of gut peptides as therapeutic agents. METHOD: The authors conducted a literature review. RESULTS: Current experience with clinical use of gut peptides is described. Initial clinical applications focused on using secretomotor effects of gut peptides in diagnostic tests, many of which have now fallen into disuse. More recently, attention has been directed toward harnessing these secretomotor effects for therapeutic use in a variety of disorders, and also using the trophic effects of gut peptides to modulate gut mucosal growth in benign and malignant disease. Gut peptides have been evaluated in a variety of other clinical situations including use as adjuncts to imaging techniques, and modification of behaviors such as feeding and panic disorder. CONCLUSIONS: Gut peptides have been used successfully in an increasing variety of clinical conditions. Further refinements in analogue and antagonist design are likely to lead to even more selective agents that may have important clinical applications. Further studies are needed to identity and evaluate these new agents. PMID:9065291

  2. Thermolysin: a peptide forming enzyme.

    PubMed

    Reddy, A V

    1991-02-01

    Thermolysin, a thermostable endopeptidase, is recognised as a potential peptide bond forming enzyme. The importance of structural properties and its stereospecific nature towards peptide bond formation is described. Thermolysin's use in the keystep of the preparation of an artificial sweetener 'aspartame' is highlighted.

  3. Urinary Peptides in Rett Syndrome.

    ERIC Educational Resources Information Center

    Solaas, K. M.; Skjeldal, O.; Gardner, M. L. G.; Kase, B. F.; Reichelt, K. L.

    2002-01-01

    A study found a significantly higher level of peptides in the urine of 53 girls with Rett syndrome compared with controls. The elevation was similar to that in 35 girls with infantile autism. Levels of peptides were lower in girls with classic Rett syndrome than those with congenital Rett syndrome. (Contains references.) (Author/CR)

  4. Electrothermal branding for embryo labeling.

    PubMed

    Wang, L; Beebe, D J; Williams, A R; Easley, K D

    1997-11-01

    A novel embryo labeling technique based on electrothermal branding is developed. Two types of micro branding irons are fabricated and tested. One utilizes 25 microns tungsten wire as the heating element. The other utilizes surface micromachining techniques to fabricate polysilicon branding irons. The thermal behavior of the branding irons and the heat distributions in the embryos are analytically modeled. Micron-scale labels on unfertilized bovine embryos are achieved.

  5. Peptides in headlock – a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy

    PubMed Central

    Braun, Michael B.; Traenkle, Bjoern; Koch, Philipp A.; Emele, Felix; Weiss, Frederik; Poetz, Oliver; Stehle, Thilo; Rothbauer, Ulrich

    2016-01-01

    Nanobodies are highly valuable tools for numerous bioanalytical and biotechnical applications. Here, we report the characterization of a nanobody that binds a short peptide epitope with extraordinary affinity. Structural analysis reveals an unusual binding mode where the extended peptide becomes part of a β-sheet structure in the nanobody. This interaction relies on sequence-independent backbone interactions augmented by a small number of specificity-determining side chain contacts. Once bound, the peptide is fastened by two nanobody side chains that clamp it in a headlock fashion. Exploiting this unusual binding mode, we generated a novel nanobody-derived capture and detection system. Matrix-coupled nanobody enables the fast and efficient isolation of epitope-tagged proteins from prokaryotic and eukaryotic expression systems. Additionally, the fluorescently labeled nanobody visualizes subcellular structures in different cellular compartments. The high-affinity-binding and modifiable peptide tag of this system renders it a versatile and robust tool to combine biochemical analysis with microscopic studies. PMID:26791954

  6. Endotoxin (lipopolysaccharide) neutralization by innate immunity host-defense peptides. Peptide properties and plausible modes of action.

    PubMed

    Rosenfeld, Yosef; Papo, Niv; Shai, Yechiel

    2006-01-20

    Binding of lipopolysaccharide (LPS) to macrophages results in proinflammatory cytokine secretion. In extreme cases it leads to endotoxic shock. A few innate immunity antimicrobial peptides (AMPs) neutralize LPS activity. However, the underlying mechanism and properties of the peptides are not yet clear. Toward meeting this goal we investigated four AMPs and their fluorescently labeled analogs. These AMPs varied in composition, length, structure, and selectivity toward cells. The list included human LL-37 (37-mer), magainin (24-mer), a 15-mer amphipathic alpha-helix, and its D,L-amino acid structurally altered analog. The peptides were investigated for their ability to inhibit LPS-mediated cytokine release from RAW264.7 and bone marrow-derived primary macrophages, to bind LPS in solution, and when LPS is already bound to macrophages (fluorescence spectroscopy and confocal microscopy), to compete with LPS for its binding site on the CD14 receptor (flow cytometry) and affect LPS oligomerization. We conclude that a strong binding of a peptide to LPS aggregates accompanied by aggregate dissociation prevents LPS from binding to the carrier protein lipopolysaccharide-binding protein, or alternatively to its receptor, and hence inhibits cytokine secretion.

  7. Rat MHC-linked peptide transporter alleles strongly influence peptide binding by HLA-B27 but not B27-associated inflammatory disease.

    PubMed

    Simmons, W A; Leong, L Y; Satumtira, N; Butcher, G W; Howard, J C; Richardson, J A; Slaughter, C A; Hammer, R E; Taurog, J D

    1996-02-15

    Rats transgenic for the human MHC molecule HLA-B27 were used to study the effect of two alleles, cima and cimb, which are associated with peptide transport by the MHC-encoded Tap2 transporter, on the function of HLA-B27 as a restriction element for CTL recognition of the male H-Y minor H Ag and on the multisystem inflammatory disease characteristic of B27 transgenic rats. Anti-H-Y CTL generated in cima B27 transgenic rats lysed male B27 cimb/b targets significantly less well than cima/a or cima/b targets. Addition of exogenous H-Y peptides to male B27 cimb/b targets increased susceptibility to lysis to the level of cima/a targets. Male B27 cimb/b cells were less efficient than cima/a cells in competitively inhibiting CTL lysis of female B27 cima/a targets sensitized with exogenous H-Y peptides. 3H-Labeled peptides eluted from B27 molecules of lymphoblasts from rats of two cimb and three cima RT1 haplotypes showed that the cimb peptide pool favors comparatively longer and/or more hydrophobic peptides. These results indicate that RT1-linked Tap2 polymorphism in the rat strongly influences peptide loading of HLA-B27. Nonetheless, the prevalence and severity of multisystem inflammatory lesions were comparable in backcross rats bearing either cima/b or cimb/b. It thus appears either that binding of specific peptides to B27 is unimportant in the pathogenesis of B27-associated disease or that the critical peptides, unlike H-Y and many others, are not influenced by Tap transporter polymorphism. PMID:8568273

  8. Rat MHC-linked peptide transporter alleles strongly influence peptide binding by HLA-B27 but not B27-associated inflammatory disease.

    PubMed

    Simmons, W A; Leong, L Y; Satumtira, N; Butcher, G W; Howard, J C; Richardson, J A; Slaughter, C A; Hammer, R E; Taurog, J D

    1996-02-15

    Rats transgenic for the human MHC molecule HLA-B27 were used to study the effect of two alleles, cima and cimb, which are associated with peptide transport by the MHC-encoded Tap2 transporter, on the function of HLA-B27 as a restriction element for CTL recognition of the male H-Y minor H Ag and on the multisystem inflammatory disease characteristic of B27 transgenic rats. Anti-H-Y CTL generated in cima B27 transgenic rats lysed male B27 cimb/b targets significantly less well than cima/a or cima/b targets. Addition of exogenous H-Y peptides to male B27 cimb/b targets increased susceptibility to lysis to the level of cima/a targets. Male B27 cimb/b cells were less efficient than cima/a cells in competitively inhibiting CTL lysis of female B27 cima/a targets sensitized with exogenous H-Y peptides. 3H-Labeled peptides eluted from B27 molecules of lymphoblasts from rats of two cimb and three cima RT1 haplotypes showed that the cimb peptide pool favors comparatively longer and/or more hydrophobic peptides. These results indicate that RT1-linked Tap2 polymorphism in the rat strongly influences peptide loading of HLA-B27. Nonetheless, the prevalence and severity of multisystem inflammatory lesions were comparable in backcross rats bearing either cima/b or cimb/b. It thus appears either that binding of specific peptides to B27 is unimportant in the pathogenesis of B27-associated disease or that the critical peptides, unlike H-Y and many others, are not influenced by Tap transporter polymorphism.

  9. Peptide and non-peptide HIV fusion inhibitors.

    PubMed

    Jiang, Shibo; Zhao, Qian; Debnath, Asim K

    2002-01-01

    Fusion of the HIV envelope with the target cell membrane is a critical step of HIV entry into the target cell. The HIV envelope glycoprotein gp41 plays an important role in the fusion of viral and target cell membranes and serves as an attractive target for development of HIV fusion inhibitors. The extracellular domain of gp41 contains three important functional regions, i.e. fusion peptide (FP), N- and C-terminal heptad repeats (NHR and CHR, respectively). The FP region is composed of hydrophobic, glycine-rich residues that are essential for the initial penetration of the target cell membrane. NHR and CHR regions consist of hydrophobic residues, which have the tendency to form alpha-helical coiled coils. During the process of fusion of HIV or HIV-infected cells with uninfected cells, FP inserts into the target cell membrane and subsequently the NHR and CHR regions change conformations and associate with each other to form a fusion-active gp41 core. Peptides derived from NHR and CHR regions, designated N- and C-peptides, respectively, have potent inhibitory activity against HIV fusion by binding to the CHR and NHR regions, respectively, to prevent the formation of the fusion-active gp41 core. C-peptide may also bind to FP, thereby blocking its insertion into the target cell membrane. One of the C-peptides, T-20, which is in the phase III clinical trials, has potent in vivo activity against HIV infection and is expected to become the first peptide HIV fusion inhibitory drug in the near future. However, this peptide HIV fusion inhibitor lacks oral availability and is sensitive to the proteolytic digestion. Therefore, it is essential to develop small molecular non-peptide HIV fusion inhibitors having a mechanism of action similar to the C-peptides. One of the approaches in identifying the inhibitors is to use an immunological assay to screen chemical libraries for the compounds that potentially block the interaction between the NHR and CHR regions to form a fusion

  10. Multi-focus cluster labeling.

    PubMed

    Eikvil, Line; Jenssen, Tor-Kristian; Holden, Marit

    2015-06-01

    Document collections resulting from searches in the biomedical literature, for instance, in PubMed, are often so large that some organization of the returned information is necessary. Clustering is an efficient tool for organizing search results. To help the user to decide how to continue the search for relevant documents, the content of each cluster can be characterized by a set of representative keywords or cluster labels. As different users may have different interests, it can be desirable with solutions that make it possible to produce labels from a selection of different topical categories. We therefore introduce the concept of multi-focus cluster labeling to give users the possibility to get an overview of the contents through labels from multiple viewpoints. The concept for multi-focus cluster labeling has been established and has been demonstrated on three different document collections. We illustrate that multi-focus visualizations can give an overview of clusters along axes that general labels are not able to convey. The approach is generic and should be applicable to any biomedical (or other) domain with any selection of foci where appropriate focus vocabularies can be established. A user evaluation also indicates that such a multi-focus concept is useful.

  11. Enhanced Aqueous Suzuki–Miyaura Coupling Allows Site-Specific Polypeptide 18F-Labeling

    PubMed Central

    2013-01-01

    The excesses of reagents used in protein chemistry are often incompatible with the reduced or even inverse stoichiometries used for efficient radiolabeling. Analysis and screening of aqueous Pd(0) ligand systems has revealed the importance of a guanidine core and the discovery of 1,1-dimethylguanidine as an enhanced ligand for aqueous Suzuki–Miyaura cross-coupling. This novel Pd catalyst system has now allowed the labeling of small molecules, peptides, and proteins with the fluorine-18 prosthetic [18F]4-fluorophenylboronic acid. These findings now enable site-specific protein 18F-labeling under biologically compatible conditions using a metal-triggered reaction. PMID:23991754

  12. Peptides and peptidomimetics as immunomodulators

    PubMed Central

    Gokhale, Ameya S; Satyanarayanajois, Seetharama

    2014-01-01

    Peptides and peptidomimetics can function as immunomodulating agents by either blocking the immune response or stimulating the immune response to generate tolerance. Knowledge of B- or T-cell epitopes along with conformational constraints is important in the design of peptide-based immunomodulating agents. Work on the conformational aspects of peptides, synthesis and modified amino acid side chains have contributed to the development of a new generation of therapeutic agents for autoimmune diseases and cancer. The design of peptides/peptidomimetics for immunomodulation in autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, systemic lupus and HIV infection is reviewed. In cancer therapy, peptide epitopes are used in such a way that the body is trained to recognize and fight the cancer cells locally as well as systemically. PMID:25186605

  13. Isotope labeled internal standards (ILIS) as a basis for quality control in clinical studies using plasma samples.

    PubMed

    Rezeli, Melinda; Végvári, Akos; Marko-Varga, György; Laurell, Thomas

    2010-04-18

    For clinical proteomic studies, the quality of the biofluid samples such as human blood plasma is extremely important. In this study we have investigated the stability of human plasma samples by spiking stable isotope-labeled peptides into the plasma and monitoring their degradation under different storage conditions. FPA-1, C4A and C3f were synthesized with isotopically labeled amino acids, and used as reference peptides. The mixture of internal calibrants was spiked into plasma at the starting point of investigation, mimicking the time of collection for future biobanking efforts, and their qualitative and quantitative changes were analyzed over time by using both MALDI-MS (LTQ Orbitrap XL) and nanoLC-ESI-MS (LTQ XL ETD). We have found that all three synthetic peptides were stable in plasma at -20 and -80 degrees C during the examined 2-month period. However, different proteolytic degradation profiles of the peptides were observed at room temperature. We anticipate that the use of these isotope-labeled peptides as internal standards (ILIS) provides a quality control for long-term storage and proteomic plasma analysis.

  14. Antibacterial peptides isolated from insects.

    PubMed

    Otvos, L

    2000-10-01

    Insects are amazingly resistant to bacterial infections. To combat pathogens, insects rely on cellular and humoral mechanisms, innate immunity being dominant in the latter category. Upon detection of bacteria, a complex genetic cascade is activated, which ultimately results in the synthesis of a battery of antibacterial peptides and their release into the haemolymph. The peptides are usually basic in character and are composed of 20-40 amino acid residues, although some smaller proteins are also included in the antimicrobial repertoire. While the proline-rich peptides and the glycine-rich peptides are predominantly active against Gram-negative strains, the defensins selectively kill Gram-positive bacteria and the cecropins are active against both types. The insect antibacterial peptides are very potent: their IC50 (50% of the bacterial growth inhibition) hovers in the submicromolar or low micromolar range. The majority of the peptides act through disintegrating the bacterial membrane or interfering with membrane assembly, with the exception of drosocin, apidaecin and pyrrhocoricin which appear to deactivate a bacterial protein in a stereospecific manner. In accordance with their biological function, the membrane-active peptides form ordered structures, e.g. alpha-helices or beta-pleated sheets and often cast permeable ion-pores. Their cytotoxic properties were exploited in in vivo studies targeting tumour progression. Although the native peptides degrade quickly in biological fluids other than insect haemolymph, structural modifications render the peptides resistant against proteases without sacrificing biological activity. Indeed, a pyrrhocoricin analogue shows lack of toxicity in vitro and in vivo and protects mice against experimental Escherichia coli infection. Careful selection of lead molecules based on the insect antibacterial peptides may extend their utility and produce viable alternatives to the conventional antimicrobial compounds for mammalian therapy.

  15. Earle K. Plyler Prize for Molecular Spectroscopy and Dynamics Lecture: 2D IR Spectroscopy of Peptide Conformation

    NASA Astrophysics Data System (ADS)

    Tokmakoff, Andrei

    2012-02-01

    Descriptions of protein and peptide conformation are colored by the methods we use to study them. Protein x-ray and NMR structures often lead to impressions of rigid or well-defined conformations, even though these are dynamic molecules. The conformational fluctuations and disorder of proteins and peptides is more difficult to quantify. This presentation will describe an approach toward characterizing and quantifying structural heterogeneity and disorder in peptides using 2D IR spectroscopy. Using amide I vibrational spectroscopy, isotope labeling strategies, and computational modeling based on molecular dynamics simulations and Markov state models allows us to characterize distinct peptide conformers and conformational variation. The examples illustrated include the beta-hairpin tripzip2 and elastin-like peptides.

  16. Identification of a peptide binding protein that plays a role in antigen presentation

    SciTech Connect

    Lakey, E.K.; Margoliash, E.; Pierce, S.K.

    1987-03-01

    The helper T-cell response to globular proteins appears, in general, to require intracellular processing of the antigen, such that a peptide fragment containing the T-cell antigenic determinant is released and transported to and held on the surface of an Ia-expressing, antigen-presenting cell. However, the molecular details underlying these phenomena are largely unknown. The means by which antigenic peptides are anchored on the antigen-presenting cell surface was investigated. A cell surface protein is identified that was isolated by it ability to bind to a 24-amino acid peptide fragment of pigeon cytochrome c, residues 81-104, containing the major antigenic determinant for B10.A mouse T cells. This peptide binding protein, purified from (/sup 35/S)methionine-labeled cells, appears as two discrete bands of approx. =72 and 74 kDa after NaDodSO/sub 4//PAGE. The protein can be eluted from the peptide affinity column with equivalent concentrations of either the antigenic pigeon cytochrome c peptide or the corresponding nonantigenic peptide of mouse cytochrome c. However, it does not bind to the native cytochromes c, either of pigeon or mouse, and thus the protein appears to recognize some structure available only in the free peptides. This protein plays a role in antigen presentation. Its expression is not major histocompatibility complex-restricted in that the blocking activity of the antisera can be absorbed on spleen cells from mice of different haplotypes. This peptide binding protein can be isolated from a variety of cell types, including B cells, T cells, and fibroblasts. The anchoring of processed peptides on the cell surface by such a protein may play a role in antigen presentation.

  17. Analysis of peptides in prohormone convertase 1/3 null mouse brain using quantitative peptidomics

    PubMed Central

    Wardman, Jonathan H.; Zhang, Xin; Gagnon, Sandra; Castro, Leandro M.; Zhu, Xiaorong; Steiner, Donald F.; Day, Robert; Fricker, Lloyd D.

    2010-01-01

    Neuropeptides are produced from larger precursors by limited proteolysis, first by endopeptidases and then by carboxypeptidases. Major endopeptidases required for these cleavages include prohormone convertase (PC) 1/3 and PC2. In the present study, quantitative peptidomics analysis was used to characterize the specific role PC1/3 plays in this process. Peptides isolated from hypothalamus, amygdala, and striatum of PC1/3 null mice were compared to those from heterozygous and wild-type mice. Extracts were labeled with stable isotopic tags and fractionated by HPLC, after which relative peptide levels were determined using tandem mass spectrometry. In total, 92 peptides were found, of which 35 were known neuropeptides or related peptides derived from 15 distinct secretory pathway proteins: 7B2, chromogranin A and B, cocaine- and amphetamine-regulated transcript, procholecystokinin, proenkephalin, promelanin concentrating hormone, proneurotensin, propituitary adenylate cyclase-activating peptide, proSAAS, prosomatosatin, provasoactive intestinal peptide, provasopressin, secretogranin III, and VGF. Among the peptides derived from these proteins, ~1/3 were decreased in the PC1/3 null mice relative to wild-type mice, ~1/3 showed no change, and ~1/3 increased in PC1/3 null. Cleavage sites were analyzed in peptides that showed no change or that decreased in PC1/3 mice, and these results were compared with peptides that showed no change or decreased in previous peptidomic studies with PC2 null mice. Analysis of these sites showed that while PC1/3 and PC2 have overlapping substrate preferences, there are particular cleavage site residues that distinguish peptides preferred by each PC. PMID:20412386

  18. Radiolabeled Peptide Scaffolds for PET/SPECT - Optical in Vivo Imaging of Carbohydrate-Lectin Interactions

    SciTech Connect

    Deutscher, Susan

    2014-09-30

    The objective of this research is to develop phage display-selected peptides into radio- and fluoresecently- labeled scaffolds for the multimodal imaging of carbohydrate-lectin interactions. While numerous protein and receptor systems are being explored for the development of targeted imaging agents, the targeting and analysis of carbohydrate-lectin complexes in vivo remains relatively unexplored. Antibodies, nanoparticles, and peptides are being developed that target carbohydrate-lectin complexes in living systems. However, antibodies and nanoparticles often suffer from slow clearance and toxicity problems. Peptides are attractive alternative vehicles for the specific delivery of radionuclides or fluorophores to sites of interest in vivo, although, because of their size, uptake and retention may be less than antibodies. We have selected high affinity peptides that bind a specific carbohydrate-lectin complex involved in cell-cell adhesion and cross-linking using bacteriophage (phage) display technologies (1,2). These peptides have allowed us to probe the role of these antigens in cell adhesion. Fluorescent versions of the peptides have been developed for optical imaging and radiolabeled versions have been used in single photon emission computed tomography (SPECT) and positron emission tomography (PET) in vivo imaging (3-6). A benefit in employing the radiolabeled peptides in SPECT and PET is that these imaging modalities are widely used in living systems and offer deep tissue sensitivity. Radiolabeled peptides, however, often exhibit poor stability and high kidney uptake in vivo. Conversely, optical imaging is sensitive and offers good spatial resolution, but is not useful for deep tissue penetration and is semi-quantitative. Thus, multimodality imaging that relies on the strengths of both radio- and optical- imaging is a current focus for development of new in vivo imaging agents. We propose a novel means to improve the efficacy of radiolabeled and fluorescently

  19. Recent trends in the analysis of bioactive peptides in milk and dairy products.

    PubMed

    Capriotti, Anna Laura; Cavaliere, Chiara; Piovesana, Susy; Samperi, Roberto; Laganà, Aldo

    2016-04-01

    Food-derived constituents represent important sources of several classes of bioactive compounds. Among them peptides have gained great attention in the last two decades thanks to the scientific evidence of their beneficial effects on health in addition to their established nutritional value. Several functionalities for bioactive peptides have been described, including antioxidative, antihypertensive, anti-inflammatory, immunomodulatory, and antimicrobial activity. They are now considered as novel and potential dietary ingredients to promote human health, though in some cases they may also have detrimental effects on health. Bioactive peptides can be naturally occurring, produced in vitro by enzymatic hydrolysis, and formed in vivo during gastrointestinal digestion of proteins. Thus, the need to gain a better understanding of the positive health effects of food peptides has prompted the development of analytical strategies for their isolation, separation, and identification in complex food matrices. Dairy products and milk are potential sources of bioactive peptides: several of them possess extra-nutritional physiological functions that qualify them to be classified under the functional food label. In this trends article we briefly describe the state-of-the-art of peptidomics methods for the identification and discovery of bioactive peptides, also considering recent progress in their analysis and highlighting the difficulty in the analysis of short amino acid sequences and endogenous peptides.

  20. Measurement of beta-amyloid peptides in specific cells using a photo thin-film transistor

    NASA Astrophysics Data System (ADS)

    Kim, Chang-Beom; Chae, Cheol-Joo; Shin, Hye-Rim; Song, Ki-Bong

    2012-01-01

    The existence of beta-amyloid [Aβ] peptides in the brain has been regarded as the most archetypal biomarker of Alzheimer's disease [AD]. Recently, an early clinical diagnosis has been considered a great importance in identifying people who are at high risk of AD. However, no microscale electronic sensing devices for the detection of Aβ peptides have been developed yet. In this study, we propose an effective method to evaluate a small quantity of Aβ peptides labeled with fluorescein isothiocyanate [FITC] using a photosensitive field-effect transistor [p-FET] with an on-chip single-layer optical filter. To accurately evaluate the quantity of Aβ peptides within the cells cultured on the p-FET device, we measured the photocurrents which resulted from the FITC-conjugated Aβ peptides expressed from the cells and measured the number of photons of the fluorochrome in the cells using a photomultiplier tube. Thus, we evaluated the correlation between the generated photocurrents and the number of emitted photons. We also evaluated the correlation between the number of emitted photons and the amount of FITC by measuring the FITC volume using AFM. Finally, we estimated the quantity of Aβ peptides of the cells placed on the p-FET sensing area on the basis of the binding ratio between FITC molecules and Aβ peptides.