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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. Stoichiometry of phosphorylation to fluorescein 5-isothiocyanate binding in the Ca2+-ATPase of sarcoplasmic reticulum vesicles.

    PubMed

    Nakamura, S; Suzuki, H; Kanazawa, T

    1997-03-07

    In an attempt to establish the stoichiometry of phosphorylation in the Ca2+-ATPase of sarcoplasmic reticulum (SR) vesicles, phosphorylation by ATP (or Pi) or labeling by fluorescein 5-isothiocyanate (FITC) was performed with the SR vesicles under the conditions in which almost all the phosphorylation sites or FITC binding sites are phosphorylated or labeled. The resulting vesicles were solubilized in lithium dodecyl sulfate and then the Ca2+-ATPase was purified by size exclusion high performance liquid chromatography. Peptide mapping and sequencing of the tryptic digest of the purified enzyme showed that Lys-515 of the Ca2+-ATPase was exclusively labeled with FITC, in agreement with the previously reported findings. The content of the phosphoenzyme from ATP (4.57 nmol/mg of Ca2+-ATPase protein) or from Pi (4.94 nmol/mg of Ca2+-ATPase protein) in the purified enzyme was approximately half the content of the FITC binding site (8.17-8.25 nmol/mg of Ca2+-ATPase protein) and also half the content of the Ca2+-ATPase molecule (9.06 nmol/mg of Ca2+-ATPase protein) calculated from its molecular mass (110,331 Da). These results show that there is one specific FITC binding site per molecule of the Ca2+-ATPase (in agreement with the previously reported findings) and that the stoichiometry of phosphorylation to FITC binding is approximately 0. 5:1.0. All the above findings lead to the conclusion that only half of the Ca2+-ATPase molecules present in the SR vesicles can be phosphorylated. FITC binding completely inhibited the ATP-induced phosphorylation before the binding reached its maximum level. This finding indicates that FITC preferentially binds to a part of the Ca2+-ATPase molecules and that this binding is primarily responsible for the inhibition of phosphorylation, suggesting an intermolecular ATPase-ATPase interaction.

  3. Differential binding of tropomyosin isoforms to actin modified with m-maleimidobenzoyl-N-hydroxysuccinimide ester and fluorescein-5-isothiocyanate.

    PubMed

    Skórzewski, Radosław; Robaszkiewicz, Katarzyna; Jarzebińska, Justyna; Suder, Piotr; Silberring, Jerzy; Moraczewska, Joanna

    2009-11-01

    Differential interactions of tropomyosin (TM) isoforms with actin can be important for determination of the thin filament functions. A mechanism of tropomyosin binding to actin was studied by comparing interactions of five alphaTM isoforms with actin modified with m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) and with fluorescein-5-isothiocyanate (FITC). MBS attachment sites were revealed with mass spectrometry methods. We found that the predominant actin fraction was cross-linked by MBS within subdomain 3. A smaller fraction of the modified actin was cross-linked within subdomain 2 and between subdomains 2 and 1. Moreover, investigated actins carried single labels in subdomains 1, 2, and 3. Such extensive modification caused a large decrease in actin affinity for skeletal and smooth muscle tropomyosins, nonmuscle TM2, and chimeric TM1b9a. In contrast, binding of nonmuscle isoform TM5a was less affected. Isoform's affinity for actin modified in subdomain 2 by binding of FITC to Lys61 was intermediate between the affinity for native actin and MBS-modified actin except for TM5a, which bound to FITC-actin with similar affinity as to actin modified with MBS. The analysis of binding curves according to the McGhee-von Hippel model revealed that binding to an isolated site, as well as cooperativity of binding to a contiguous site, was affected by both actin modifications in a TM isoform-specific manner.

  4. An autoinhibitory peptide from the erythrocyte Ca-ATPase aggregates and inhibits both muscle Ca-ATPase isoforms.

    PubMed Central

    Reddy, L G; Shi, Y; Kutchai, H; Filoteo, A G; Penniston, J T; Thomas, D D

    1999-01-01

    We have studied the effects of C28R2, a basic peptide derived from the autoinhibitory domain of the plasma membrane Ca-ATPase, on enzyme activity, oligomeric state, and E1-E2 conformational equilibrium of the Ca-ATPase from skeletal and cardiac sarcoplasmic reticulum (SR). Time-resolved phosphorescence anisotropy (TPA) was used to determine changes in the distribution of Ca-ATPase among its different oligomeric species in SR. C28R2, at a concentration of 1-10 microM, inhibits the Ca-ATPase activity of both skeletal and cardiac SR (CSR). In skeletal SR, this inhibition by C28R2 is much greater at low (0.15 microM) than at high (10 microM) Ca2+, whereas in CSR the inhibition is the same at low and high Ca2+. The effects of the peptide on the rotational mobility of the Ca-ATPase correlated well with function, indicating that C28R2-induced protein aggregation and Ca-ATPase inhibition are much more Ca-dependent in skeletal than in CSR. In CSR at low Ca2+, phospholamban (PLB) antibody (functionally equivalent to PLB phosphorylation) increased the inhibitory effect of C28R2 slightly. Fluorescence of fluorescein 5-isothiocyanate-labeled SR suggests that C28R2 stabilizes the E1 conformation of the Ca-ATPase in skeletal SR, whereas in CSR it stabilizes E2. After the addition of PLB antibody, C28R2 still stabilizes the E2 conformational state of CSR. Therefore, we conclude that C28R2 affects Ca-ATPase activity, conformation, and self-association differently in cardiac and skeletal SR and that PLB is probably not responsible for the differences. PMID:10354431

  5. Neutron encoded labeling for peptide identification.

    PubMed

    Rose, Christopher M; Merrill, Anna E; Bailey, Derek J; Hebert, Alexander S; Westphall, Michael S; Coon, Joshua J

    2013-05-21

    Metabolic labeling of cells using heavy amino acids is most commonly used for relative quantitation; however, partner mass shifts also detail the number of heavy amino acids contained within the precursor species. Here, we use a recently developed metabolic labeling technique, NeuCode (neutron encoding) stable isotope labeling with amino acids in cell culture (SILAC), which produces precursor partners spaced ~40 mDa apart to enable amino acid counting. We implement large scale counting of amino acids through a program, "Amino Acid Counter", which determines the most likely combination of amino acids within a precursor based on NeuCode SILAC partner spacing and filters candidate peptide sequences during a database search using this information. Counting the number of lysine residues for precursors selected for MS/MS decreases the median number of candidate sequences from 44 to 14 as compared to an accurate mass search alone (20 ppm). Furthermore, the ability to co-isolate and fragment NeuCode SILAC partners enables counting of lysines in product ions, and when the information is used, the median number of candidates is reduced to 7. We then demonstrate counting leucine in addition to lysine results in a 6-fold decrease in search space, 43 to 7, when compared to an accurate mass search. We use this scheme to analyze a nanoLC-MS/MS experiment and demonstrate that accurate mass plus lysine and leucine counting reduces the number of candidate sequences to one for ~20% of all precursors selected, demonstrating an ability to identify precursors without MS/MS analysis.

  6. Neutron Encoded Labeling for Peptide Identification

    PubMed Central

    Rose, Christopher M.; Merrill, Anna E.; Bailey, Derek J.; Hebert, Alexander S.; Westphall, Michael S.; Coon, Joshua J.

    2013-01-01

    Metabolic labeling of cells using heavy amino acids is most commonly used for relative quantitation; however, partner mass shifts also detail the number of heavy amino acids contained within the precursor species. Here, we use a recently developed metabolic labeling technique, NeuCode (neutron encoding) stable isotope labeling with amino acids in cell culture (SILAC), which produces precursor partners spaced ~40 mDa apart to enable amino acid counting. We implement large scale counting of amino acids through a program, “Amino Acid Counter”, which determines the most likely combination of amino acids within a precursor based on NeuCode SILAC partner spacing and filters candidate peptide sequences during a database search using this information. Counting the number of lysine residues for precursors selected for MS/MS decreases the median number of candidate sequences from 44 to 14 as compared to an accurate mass search alone (20 ppm). Furthermore, the ability to co-isolate and fragment NeuCode SILAC partners enables counting of lysines in product ions, and when the information is used, the median number of candidates is reduced to 7. We then demonstrate counting leucine in addition to lysine results in a 6-fold decrease in search space, 43 to 7, when compared to an accurate mass search. We use this scheme to analyze a nanoLC-MS/MS experiment and demonstrate that accurate mass plus lysine and leucine counting reduces the number of candidate sequences to one for ~20% of all precursors selected, demonstrating an ability to identify precursors without MS/MS analysis. PMID:23638792

  7. 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.

  8. 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

  9. 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

  10. New Methods for Labeling RGD Peptides with Bromine-76

    PubMed Central

    Lang, Lixin; Li, Weihua; Jia, Hong-Mei; Fang, De-Cai; Zhang, Shushu; Sun, Xilin; Zhu, Lei; Ma, Ying; Shen, Baozhong; Kiesewetter, Dale O.; Niu, Gang; Chen, Xiaoyuan

    2011-01-01

    Direct bromination of the tyrosine residues of peptides and antibodies with bromine-76, to create probes for PET imaging, has been reported. For peptides that do not contain tyrosine residues, however, a prosthetic group is required to achieve labeling via conjugation to other functional groups such as terminal α-amines or lysine ε-amines. The goal of this study was to develop new strategies for labeling small peptides with Br-76 using either a direct labeling method or a prosthetic group, depending on the available functional group on the peptides. A new labeling agent, N-succinimidyl-3-[76Br]bromo-2,6-dimethoxybenzoate ([76Br]SBDMB) was prepared for cyclic RGD peptide labeling. N-succinimidyl-2, 6-dimethoxybenzoate was also used to pre-attach a 2, 6-dimethoxybenzoyl (DMB) moiety to the peptide, which could then be labeled with Br-76. A competitive cell binding assay was performed to determine the binding affinity of the brominated peptides. PET imaging of U87MG human glioblastoma xenografted mice was performed using [76Br]-BrE[c(RGDyK)]2 and [76Br]-BrDMB-E[c(RGDyK)]2. An ex vivo biodistribution assay was performed to confirm PET quantification. The mechanisms of bromination reaction between DMB-c(RGDyK) and the brominating agent CH3COOBr were investigated with the SCRF-B3LYP/6-31G* method with the Gaussian 09 program package. The yield for direct labeling of c(RGDyK) and E[c(RGDyK)]2 using chloramine-T and peracetic acid at ambient temperature was greater than 50%. The yield for [76Br]SBDMB was over 60% using peracetic acid. The conjugation yields for labeling c(RGDfK) and c(RGDyK) were over 70% using the prosthetic group at room temperature. Labeling yield for pre-conjugated peptides was over 60%. SDMB conjugation and bromination did not affect the binding affinity of the peptides with integrin receptors. Both [76Br]Br-E[c(RGDyK)]2 and [76Br]BrDMB-E[c(RGDyK)]2 showed high tumor uptake in U87MG tumor bearing mice. The specificity of the imaging tracers was

  11. NIPTL-Novo: Non-isobaric peptide termini labeling assisted peptide de novo sequencing.

    PubMed

    Zhang, Shen; Shan, Yichu; Zhang, Shurong; Sui, Zhigang; Zhang, Lihua; Liang, Zhen; Zhang, Yukui

    2017-02-10

    A simple and effective de novo sequencing strategy assisted by non-isobaric peptide termini labeling, NIPTL-Novo, was established. The y-series ions and b-series ions of peptides can be clearly distinguished according to the different mass tags incorporated in N-terminus and C-terminus. This is helpful for improving the accuracy of peptide sequencing and increasing the sequencing speed. For the spectra commonly identified by both de novo sequencing and database searching software (Mascot or Maxquant), NIPTL-Novo gave identical result to more than 85% of these spectra. Furthermore, the quantitative profiling of the sample can be performed simultaneously along with de novo sequencing. Finally, this strategy can be applied to discover the peptides with potential mutation sites by combining with mass-defect based isotopic labeling.

  12. 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.

  13. 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.

  14. Absolute peptide quantification by lutetium labeling and nanoHPLC-ICPMS with isotope dilution analysis.

    PubMed

    Rappel, Christina; Schaumlöffel, Dirk

    2009-01-01

    The need of analytical methods for absolute quantitative protein analysis spurred research on new developments in recent years. In this work, a novel approach was developed for accurate absolute peptide quantification based on metal labeling with lutetium diethylenetriamine pentaacetic acid (Lu-DTPA) and nanoflow high-performance liquid chromatography-inductively coupled plasma isotope dilution mass spectrometry (nanoHPLC-ICP-IDMS). In a two-step procedure peptides were derivatized at amino groups with diethylenetriamine pentaacetic anhydride (DTPAA) followed by chelation of lutetium. Electrospray ionization mass spectrometry (ESI MS) of the reaction product demonstrated highly specific peptide labeling. Under optimized nanoHPLC conditions the labeled peptides were baseline-separated, and the excess labeling reagent did not interfere. A 176Lu-labeled spike was continuously added to the column effluent for quantification by ICP-IDMS. The recovery of a Lu-DTPA-labeled standard peptide was close to 100% indicating high labeling efficiency and accurate absolute quantification. The precision of the entire method was 4.9%. The detection limit for Lu-DTPA-tagged peptides was 179 amol demonstrating that lutetium-specific peptide quantification was by 4 orders of magnitude more sensitive than detection by natural sulfur atoms present in cysteine or methionine residues. Furthermore, the application to peptides in insulin tryptic digest allowed the identification of interfering reagents decreasing the labeling efficiency. An additional advantage of this novel approach is the analysis of peptides, which do not naturally feature ICPMS-detectable elements.

  15. ZoomQuant: an application for the quantitation of stable isotope labeled peptides.

    PubMed

    Halligan, Brian D; Slyper, Ronit Y; Twigger, Simon N; Hicks, Wayne; Olivier, Michael; Greene, Andrew S

    2005-03-01

    The main goal of comparative proteomics is the quantitation of the differences in abundance of many proteins between two different biological samples in a single experiment. By differentially labeling the peptides from the two samples and combining them in a single analysis, relative ratios of protein abundance can be accurately determined. Protease catalyzed (18)O exchange is a simple method to differentially label peptides, but the lack of robust software tools to analyze the data from mass spectra of (18)O labeled peptides generated by common ion trap mass spectrometers has been a limitation. ZoomQuant is a stand-alone computational tool that analyzes the mass spectra of (18)O labeled peptides from ion trap instruments and determines relative abundance ratios between two samples. Starting with a filtered list of candidate peptides that have been successfully identified by Sequest, ZoomQuant analyzes the isotopic forms of the peptides using high-resolution zoom scan spectrum data. The theoretical isotope distribution is determined from the peptide sequence and is used to deconvolute the peak areas associated with the unlabeled, partially labeled, and fully labeled species. The ratio between the labeled and unlabeled peptides is then calculated using several different methods. ZoomQuant's graphical user interface allows the user to view and adjust the parameters for peak calling and quantitation and select which peptides should contribute to the overall abundance ratio calculation. Finally, ZoomQuant generates a summary report of the relative abundance of the peptides identified in the two samples.

  16. 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.

  17. 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.

  18. Monitoring membrane binding and insertion of peptides by two-color fluorescent label.

    PubMed

    Postupalenko, V Y; Shvadchak, V V; Duportail, G; Pivovarenko, V G; Klymchenko, A S; Mély, Y

    2011-01-01

    Herein, we developed an approach for monitoring membrane binding and insertion of peptides using a fluorescent environment-sensitive label of the 3-hydroxyflavone family. For this purpose, we labeled the N-terminus of three synthetic peptides, melittin, magainin 2 and poly-l-lysine capable to interact with lipid membranes. Binding of these peptides to lipid vesicles induced a strong fluorescence increase, which enabled to quantify the peptide-membrane interaction. Moreover, the dual emission of the label in these peptides correlated well with the depth of its insertion measured by the parallax quenching method. Thus, in melittin and magainin 2, which show deep insertion of their N-terminus, the label presented a dual emission corresponding to a low polar environment, while the environment of the poly-l-lysine N-terminus was rather polar, consistent with its location close to the bilayer surface. Using spectral deconvolution to distinguish the non-hydrated label species from the hydrated ones and two photon fluorescence microscopy to determine the probe orientation in giant vesicles, we found that the non-hydrated species were vertically oriented in the bilayer and constituted the best indicators for evaluating the depth of the peptide N-terminus in membranes. Thus, this label constitutes an interesting new tool for monitoring membrane binding and insertion of peptides.

  19. Development of a Small Peptide Tag for Covalent Labeling of Proteins

    PubMed Central

    Tanaka, Fujie; Fuller, Roberta; Asawapornmongkol, Lily; Warsinke, Axel; Gobuty, Sarah; Barbas, Carlos F.

    2008-01-01

    A 21-mer peptide that can be used to covalently introduce synthetic molecules into proteins has been developed. Phage displayed peptide libraries were subjected to reaction-based selection with 1,3-diketones. The peptide was further evolved by addition of a randomized region and reselection for improved binding. The resulting 21-mer peptide had a reactive amino group that formed an enaminone with 1,3-diketone and was used as a tag for labeling of maltose binding protein. Using this peptide tag and 1,3-diketone derivatives, a variety of molecules such as reporter probes and functionalities may be covalently introduced into proteins of interest. PMID:17602682

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. Photoaffinity labeling of Ras converting enzyme using peptide substrates that incorporate benzoylphenylalanine (Bpa) residues: improved labeling and structural implications.

    PubMed

    Kyro, Kelly; Manandhar, Surya P; Mullen, Daniel; Schmidt, Walter K; Distefano, Mark D

    2011-12-15

    Rce1p catalyzes the proteolytic trimming of C-terminal tripeptides from isoprenylated proteins containing CAAX-box sequences. Because Rce1p processing is a necessary component in the Ras pathway of oncogenic signal transduction, Rce1p holds promise as a potential target for therapeutic intervention. However, its mechanism of proteolysis and active site have yet to be defined. Here, we describe synthetic peptide analogues that mimic the natural lipidated Rce1p substrate and incorporate photolabile groups for photoaffinity-labeling applications. These photoactive peptides are designed to crosslink to residues in or near the Rce1p active site. By incorporating the photoactive group via p-benzoyl-l-phenylalanine (Bpa) residues directly into the peptide substrate sequence, the labeling efficiency was substantially increased relative to a previously-synthesized compound. Incorporation of biotin on the N-terminus of the peptides permitted photolabeled Rce1p to be isolated via streptavidin affinity capture. Our findings further suggest that residues outside the CAAX-box sequence are in contact with Rce1p, which has implications for future inhibitor design.

  6. Photoaffinity Labeling of Ras Converting Enzyme using Peptide Substrates that Incorporate Benzoylphenylalanine (Bpa) Residues: Improved Labeling and Structural Implications

    PubMed Central

    Kyro, Kelly; Manandhar, Surya P.; Mullen, Daniel; Schmidt, Walter K.; Distefano, Mark D.

    2012-01-01

    Rce1p catalyzes the proteolytic trimming of C-terminal tripeptides from isoprenylated proteins containing CAAX-box sequences. Because Rce1p processing is a necessary component in the Ras pathway of oncogenic signal transduction, Rce1p holds promise as a potential target for therapeutic intervention. However, its mechanism of proteolysis and active site have yet to be defined. Here, we describe synthetic peptide analogues that mimic the natural lipidated Rce1p substrate and incorporate photolabile groups for photoaffinity-labeling applications. These photoactive peptides are designed to crosslink to residues in or near the Rce1p active site. By incorporating the photoactive group via p-benzoyl-L-phenylalanine (Bpa) residues directly into the peptide substrate sequence, the labeling efficiency was substantially increased relative to a previously-synthesized compound. Incorporation of biotin on the N-terminus of the peptides permitted photolabeled Rce1p to be isolated via streptavidin affinity capture. Our findings further suggest that residues outside the CAAX-box sequence are in contact with Rce1p, which has implications for future inhibitor design. PMID:22079863

  7. Selective ruthenium labeling of the tryptophan residue in the bee venom Peptide melittin.

    PubMed

    Perekalin, Dmitry S; Novikov, Valentin V; Pavlov, Alexander A; Ivanov, Igor A; Anisimova, Natalia Yu; Kopylov, Alexey N; Volkov, Dmitry S; Seregina, Irina F; Bolshov, Michail A; Kudinov, Alexander R

    2015-03-23

    Melittin is a membrane-active peptide from bee venom with promising antimicrobial and anticancer activity. Herein we report on a simple and selective method for labeling of the tryptophan residue in melittin by the organometallic fragment [(C5 H5 )Ru](+) in aqueous solution and in air. Ruthenium coordination does not disturb the secondary structure of the peptide (as verified by 2D NMR spectroscopy), but changes the pattern of its intermolecular interactions resulting in an 11-fold decrease of hemolytic activity. The high stability of the organometallic conjugate allowed the establishment of the biodistribution of the labeled melittin in mice by inductively coupled plasma MS analysis of ruthenium.

  8. 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

  9. Rapid biosynthesis of stable isotope-labeled peptides from a reconstituted in vitro translation system for targeted proteomics.

    PubMed

    Xian, Feng; Li, Shuwei; Liu, Siqi

    2015-01-01

    Stable isotope-labeled peptides are routinely used as internal standards (a.k.a. reference peptides) for absolute quantitation of proteins in targeted proteomics. These peptides can either be synthesized chemically on solid supports or expressed biologically by concatenating multiple peptides together to a large protein. Neither method, however, has required versatility, convenience, and economy for making a large number of reference peptides. Here, we describe the biosynthesis of stable isotope-labeled peptides from a reconstituted Escherichia coli in vitro translation system. We provide a detailed protocol on how to express these peptides with high purity and how to determine their concentrations with easiness. Our strategy offers a general, fast, and scalable approach for the easy preparation of labeled reference peptides, which will have broad application in both basic research and translational medicine.

  10. Metabolic flux analysis using 13C peptide label measurements

    Technology Transfer Automated Retrieval System (TEKTRAN)

    13C metabolic flux analysis (MFA) has become the experimental method of choice to investigate cellular metabolism. MFA has established flux maps of central metabolism for dozens of microbes, cell cultures, and plant seeds. Steady-state MFA utilizes isotopic labeling measurements of amino acids obtai...

  11. 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.

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

    PubMed

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

    2015-02-10

    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 35days. 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.

  13. 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.

  14. Photodamage of Lipid Bilayers by Irradiation of a Fluorescently Labeled Cell-Penetrating Peptide

    PubMed Central

    Meerovich, Igor; Muthukrishnan, Nandhini; Johnson, Gregory A.; Erazo-Oliveras, Alfredo; Pellois, Jean-Philippe

    2013-01-01

    Background Fluorescently labeled cell-penetrating peptides can translocate into cells by endocytosis and upon light irradiation, lyse the endocytic vesicles. This photo-inducible endosomolytic activity of Fl-CPPs can be used to efficiently deliver macromolecules such as proteins and nucleic acids and other small organic molecules into the cytosol of live cells. The requirement of a light trigger to induce photolysis provides a more spatial and temporal control to the intracellular delivery process. Methods In this report, we examine the molecular level mechanisms by which cell-penetrating peptides such as TAT when labeled with small organic fluorophore molecules acquire a photo-induced lytic activity using a simplified model of lipid vesicles. Results The peptide TAT labeled with 5(6)-carboxy-tetramethylrhodamine binds to negatively charged phospholipids, thereby bringing the fluorophore in close proximity to the membrane of liposomes. Upon light irradiation, the excited fluorophore produces reactive oxygen species at the lipid bilayer and oxidation of the membrane is achieved. In addition, the fluorescent peptide causes aggregation of photo-oxidized lipids, an activity that requires the presence of arginine residues in the peptide sequence. Conclusions These results suggest that the cell penetrating peptide plays a dual role. On one hand, TAT targets a conjugated fluorophore to membranes. On the other hand, TAT participates directly in the destabilization of photosensitized membranes. Peptide and fluorophore therefore appear to act in synergy to destroy membranes efficiently. General Significance Understanding the mechanism behind Fl-CPP mediated membrane photodamage will help to design optimally photo-endosomolytic compounds. PMID:24135456

  15. Peptides Labeled with Pyridinium Salts for Sensitive Detection and Sequencing by Electrospray Tandem Mass Spectrometry

    PubMed Central

    Waliczek, Mateusz; Kijewska, Monika; Rudowska, Magdalena; Setner, Bartosz; Stefanowicz, Piotr; Szewczuk, Zbigniew

    2016-01-01

    Mass spectrometric analysis of trace amounts of peptides may be problematic due to the insufficient ionization efficiency resulting in limited sensitivity. One of the possible ways to overcome this problem is the application of ionization enhancers. Herein we developed new ionization markers based on 2,4,6-triphenylpyridinium and 2,4,6-trimethylpyridinium salts. Using of inexpensive and commercially available pyrylium salt allows selective derivatization of primary amino groups, especially those sterically unhindered, such as ε-amino group of lysine. The 2,4,6-triphenylpyridinium modified peptides generate in MS/MS experiments an abundant protonated 2,4,6-triphenylpyridinium ion. This fragment is a promising reporter ion for the multiple reactions monitoring (MRM) analysis. In addition, the fixed positive charge of the pyridinium group enhances the ionization efficiency. Other advantages of the proposed ionization enhancers are the simplicity of derivatization of peptides and the possibility of convenient incorporation of isotopic labels into derivatized peptides. PMID:27892962

  16. Synthesis and characterization of a 'fluorous' (fluorinated alkyl) affinity reagent that labels primary amine groups in proteins/peptides.

    PubMed

    Qian, Jiang; Cole, Richard B; Cai, Yang

    2011-01-01

    Strong non-covalent interactions such as biotin-avidin affinity play critical roles in protein/peptide purification. A new type of 'fluorous' (fluorinated alkyl) affinity approach has gained popularity due especially to its low level of non-specific binding to proteins/peptides. We have developed a novel water-soluble fluorous labeling reagent that is reactive (via an active sulfo-N-hydroxylsuccinimidyl ester group) to primary amine groups in proteins/peptides. After fluorous affinity purification, the bulky fluorous tag moiety and the long oligoethylene glycol (OEG) spacer of this labeling reagent can be trimmed via the cleavage of an acid labile linker. Upon collision-induced dissociation, the labeled peptide ion yields a characteristic fragment that can be retrieved from the residual portion of the fluorous affinity tag, and this fragment ion can serve as a marker to indicate that the relevant peptide has been successfully labeled. As a proof of principle, the newly synthesized fluorous labeling reagent was evaluated for peptide/protein labeling ability in phosphate-buffered saline (PBS). Results show that both the aqueous environment protein/peptide labeling and the affinity enrichment/separation process were highly efficient.

  17. 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

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

    PubMed

    Yotapan, Nattawut; Charoenpakdee, Chayan; Wathanathavorn, Pawinee; Ditmangklo, Boonsong; Wagenknecht, Hans-Achim; Vilaivan, Tirayut

    2014-01-01

    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.

  19. Labeling and distribution of linear peptides identified using in vivo phage display selection for tumors.

    PubMed

    Kennel, S J; Mirzadeh, S; Hurst, G B; Foote, L J; Lankford, T K; Glowienka, K A; Chappell, L L; Kelso, J R; Davern, S M; Safavy, A; Brechbiel, M W

    2000-11-01

    To develop targeting molecules to be used for vascular targeting of short half-lived alpha-emitters for radioimmunotherapy, linear peptide phage display libraries were selected in vivo for binding to IC-12 rat tracheal tumors growing in severe combined immune deficient mice. After three rounds of selection, 15 phage clones were analyzed for DNA sequence, and the deduced translation products of cDNA inserts were compared. Three consensus sequences were chosen from three separate experimental selection series and peptides of these sequences with added -gly-gly-tyr were obtained. Peptides were radiolabeled on tyrosine with (125)I and the biodistribution in tumor-bearing mice was determined. The radioiodinated peptides were stable in vitro and when injected in tumor-bearing mice approximately 3.0 %ID/g accumulated in the tumor; however, much of the (125)I was found in the gastrointestinal tract and thyroid, indicative of dehalogenation of the labeled peptide. Radiolabeling peptide 2 with N-succinimidyl-3-(125)I-iodobenzoate resulted in faster excretion, which in turn resulted in lower levels in tumor and other organs, especially thyroid and gastrointestinal tract. Peptide 2 was derivatized with the bifunctional isothiocyanates of cyclohexyl-B diethylenetriaminepentaacetic acid (DTPA) or CHX-A" DTPA by direct conjugation or with a hydroxylamine derivative of 1B4M-DTPA (2-(p-[O-(carboxamylmethyl)hydroxylamine]benzyl)-6-methyl-diethylenetriamine-N,N,N',N",N"-pentaacetic acid ) coupled at the N-terminus. The primary molecular species in the conjugated products were shown by mass spectrometry to have one DTPA per peptide. Peptide chelate conjugates were radiolabeled with (213)Bi and the products tested for biodistribution in tumor-bearing mice. The data show that chelation of (213)Bi to peptides was accomplished by both the direct method of DTPA attachment and by the method using the linker at the N-terminus. Only small amounts of peptide accumulated at tumor sites. We

  20. Low-pH Solid-Phase Amino Labeling of Complex Peptide Digests with TMTs Improves Peptide Identification Rates for Multiplexed Global Phosphopeptide Analysis.

    PubMed

    Böhm, Gitte; Prefot, Petra; Jung, Stephan; Selzer, Stefan; Mitra, Vikram; Britton, David; Kuhn, Karsten; Pike, Ian; Thompson, Andrew H

    2015-06-05

    We present a novel tandem mass tag solid-phase amino labeling (TMT-SPAL) protocol using reversible immobilization of peptides onto octadecyl-derivatized (C18) solid supports. This method can reduce the number of steps required in complex protocols, saving time and potentially reducing sample loss. In our global phosphopeptide profiling workflow (SysQuant), we can cut 24 h from the protocol while increasing peptide identifications (20%) and reducing side reactions. Solid-phase labeling with TMTs does require some modification to typical labeling conditions, particularly pH. It has been found that complete labeling equivalent to standard basic pH solution-phase labeling for small and large samples can be achieved on C18 resins under slightly acidic buffer conditions. Improved labeling behavior on C18 compared to that with standard basic pH solution-phase labeling is demonstrated. We analyzed our samples for histidine, serine, threonine, and tyrosine labeling to determine the degree of overlabeling and observed higher than expected levels (25% of all peptide spectral matches (PSMs)) of overlabeling at all of these amino acids (predominantly at tyrosine and serine) in our standard solution-phase labeling protocol. Overlabeling at all of these sites is greatly reduced (4-fold, to 7% of all PSMs) by the low-pH conditions used in the TMT-SPAL protocol. Overlabeling seems to represent a so-far overlooked mechanism causing reductions in peptide identification rates with NHS-activated TMT labeling compared to that with label-free methods. Our results also highlight the importance of searching data for overlabeling when labeling methods are used.

  1. 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.

  2. 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.

  3. 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

  4. Imaging focal sites of bacterial infection in rats with indium-111-labeled chemotactic peptide analogs

    SciTech Connect

    Fischman, A.J.; Pike, M.C.; Kroon, D.; Fucello, A.J.; Rexinger, D.; ten Kate, C.; Wilkinson, R.; Rubin, R.H.; Strauss, H.W. )

    1991-03-01

    Four DTPA-derivatized chemotactic peptide analogs: ForNleLFNleYK-DTPA (P1), ForMLFNH(CH2)6NH-DTPA (P2), ForNleLFK(NH2)-DTPA (P3), and ForNleLFK-DTPA (P4), were synthesized and evaluated for in vitro bioactivity and receptor binding. The peptides were radiolabeled with 111In by transchelation and their biodistribution determined in rats at 5, 30, 60 and 120 min after injection. Localization at sites of infection was determined by scintillation camera imaging in animals with deep-thigh infection due to Escherichia coli. Images were recorded from 5 min to 2 hr after injection. All peptides maintained biologic activity (EC50 for O2-production by human PMN's: 3-150 nM) and the ability to bind to the oligopeptide chemoattractant receptor on human PMN's (EC50 for binding: 7.5-50 nM); biologic activity and receptor binding were highly correlated (r = 0.99). For all the peptides, blood clearance was rapid (half-lives: 21.5, 33.1, 31.6, and 28.7 min for P1, P2, P3, and P4, respectively). Biodistributions of the individual peptides were similar with low levels of accumulation in the heart, lung, liver, spleen, and gastrointestinal tract. In the kidney, P1 had much greater accumulation than other organs. All peptides yielded high quality images of the infection sites within 1 hr of injection. This study demonstrates that 111In-labeled chemotactic peptide analogs were effective agents for the external imaging of focal sites of infection.

  5. Metabolic flux analysis using ¹³C peptide label measurements.

    PubMed

    Mandy, Dominic E; Goldford, Joshua E; Yang, Hong; Allen, Doug K; Libourel, Igor G L

    2014-02-01

    ¹³C metabolic flux analysis (MFA) has become the experimental method of choice to investigate the cellular metabolism of microbes, cell cultures and plant seeds. Conventional steady-state MFA utilizes isotopic labeling measurements of amino acids obtained from protein hydrolysates. To retain spatial information in conventional steady-state MFA, tissues or subcellular fractions must be dissected or biochemically purified. In contrast, peptides retain their identity in complex protein extracts, and may therefore be associated with a specific time of expression, tissue type and subcellular compartment. To enable 'single-sample' spatially and temporally resolved steady-state flux analysis, we investigated the suitability of peptide mass distributions (PMDs) as an alternative to amino acid label measurements. PMDs are the discrete convolution of the mass distributions of the constituent amino acids of a peptide. We investigated the requirements for the unique deconvolution of PMDs into amino acid mass distributions (AAMDs), the influence of peptide sequence length on parameter sensitivity, and how AAMD and flux estimates that are determined through deconvolution compare to estimates from a conventional GC-MS measurement-based approach. Deconvolution of PMDs of the storage protein β-conglycinin of soybean (Glycine max) resulted in good AAMD and flux estimates if fluxes were directly fitted to PMDs. Unconstrained deconvolution resulted in inferior AAMD and flux estimates. PMD measurements do not include amino acid backbone fragments, which increase the information content in GC-MS-derived analyses. Nonetheless, the resulting flux maps were of comparable quality due to the precision of Orbitrap quantification and the larger number of peptide measurements.

  6. 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.

  7. Development of MAG3 p-nitrophenyl ester for technetium-99m and rhenium-188 labeling of amines and peptides

    SciTech Connect

    Guhlke, S.; Diekmann, D.; Biersack, H.J.; Zamora, P.O.; Knapp, F.F. Jr.

    1994-09-01

    Conjugate labeling by active ester chemistry is a well-established method for labeling peptides and proteins with technetium and rhenium. The easy preparation and high conjugations yields presented in this paper show that both {sup 188}Re and {sup 99m}Tc-MAG{sub 3} p-nitrophenyl esters are promising agents for labeling a wide range of biomolecules for radio therapy or diagnostic imaging.

  8. Synthesis of fluorescein-labelled O-mannosylated peptides as components for synthetic vaccines: comparison of two synthetic strategies.

    PubMed

    Brimble, Margaret A; Kowalczyk, Renata; Harris, Paul W R; Dunbar, P Rod; Muir, Victoria J

    2008-01-07

    Mannose-binding proteins on the surface of antigen-presenting cells (APCs) are capable of recognizing and internalizing foreign agents in the early stages of immune response. These receptors offer a potential target for synthetic vaccines, especially vaccines designed to stimulate T cells. We set out to synthesize a series of fluorescein-labelled O-mannosylated peptides using manual solid phase peptide synthesis (SPPS) on pre-loaded Wang resin, in order to test their ability to bind mannose receptors on human APCs in vitro. A flexible and reliable method for the synthesis of fluorescein-labelled O-mannosylated glycopeptides was desired in order to study their lectin-binding properties using flow cell cytometry. Two synthetic strategies were investigated: incorporation of a fluorescein label into the peptide chain via a lysine side chain epsilon-amino group at the final stage of standard Fmoc solid phase peptide synthesis or attachment of the fluorescein label to the N(alpha)-amino group of a lysine with further incorporation of a mannosylated peptide unit through the side chain N(epsilon)-amino group. The latter strategy proved more effective in that it facilitated SPPS by positioning the growing mannosylated peptide chain further removed from the fluorescein label.

  9. Fluorescence-labeled peptides as isoelectric point (pI) markers in capillary isoelectric focusing with fluorescence detection.

    PubMed

    Shimura, K; Kasai, K

    1995-08-01

    Commercially available peptides, mostly angiotensin derivatives, were labeled at their N-terminal amino group with 5-carboxytetramethylrhodamine succinimidyl ester, to obtain fluorescent pI markers for capillary isoelectric focusing with fluorescence detection. The labeled peptides were purified by reversed-phase chromatography. They were well separated on isoelectric focusing in a polyacrylamide gel slab and their pIs were determined by comigration with protein-pI markers. The fluorescent markers could be detected as sharp peaks in capillary isoelectric focusing with laser-induced fluorescence detection (He-Ne laser, 1 mW, 543.5 nm). The detection limit was found to be around 3 x 10(-12) M (0.8 amol). Tetramethylrhodamine-labeled pea lectin (3 pg) was subjected to capillary isoelectric focusing and the pIs of the fluorescent derivatives of the lectin were determined by using the fluorescence-labeled peptides as pI markers.

  10. Integrated microchip-device for the digestion, separation and postcolumn labeling of proteins and peptides.

    PubMed

    Gottschlich, N; Culbertson, C T; McKnight, T E; Jacobson, S C; Ramsey, J M

    2000-08-04

    A microchip device was demonstrated that integrated enzymatic reactions, electrophoretic separation of the reactants from the products and post-separation labeling of proteins and peptides prior to detection. A tryptic digestion of oxidized insulin B-chain was performed in 15 min under stopped flow conditions in a heated channel, and the separation was completed in 1 min. Localized thermal control of the reaction channel was achieved using a resistive heating element. The separated reaction products were then labeled with naphthalene-2,3-dicarboxaldehyde (NDA) and detected by laser-induced fluorescence. A second reaction at elevated temperatures was also demonstrated for the on-chip reduction of disulfide bridges using insulin as a model protein. This device represents one of the highest levels, to date, of monolithic integration of chemical processes on a microchip.

  11. Methodology for Labeling Proteins and Peptides with Lead-212 (212Pb)

    PubMed Central

    Baidoo, Kwamena E.; Milenic, Diane E.; Brechbiel, Martin W.

    2013-01-01

    Introduction Alpha particles possess an exquisite degree of cytotoxicity when employed for targeted α–particle therapy (TAT) or radioimmunotherapy (RIT). 212Pb, which acts as an in vivo generator of the α-emitting nuclide 212Bi has shown great promise in pre-clinical studies when used to label the HER2 binding antibody, trastuzumab. Currently, the first RIT clinical trial employing 212Pb radiolabeled trastuzumab is in progress. This report provides detailed current protocol operations and steps that were generated for use in the clinical trial as well as the relevant pre-clinical experimentation, and describes in detail the labeling of proteins or peptides with 212Pb as provided via a 224Ra based generator system. Methods 212Pb was eluted from the 224Ra/212Pb generator using hydrochloric acid (2 M). The generator eluate was evaporated and digested with nitric acid (8M) followed by extraction of the 212Pb with dilute nitric acid (0.1 M). The dilute nitric acid solution of 212Pb was used to label the immunoconjugate Trastuzumab-TCMC (2-(4-isothiocyanatobenzyl-1,4,7,10-tetraaza-1,4,7,10,tetra-(2-carbamonylmethyl)-cyclododecane) at pH 5.5. Results Elution of 212Pb from the generator was efficient yielding > 90% of available 212Pb. Trastuzumab-TCMC was efficiently labeled with a radiochemical yield of 94 +/− 4% (n = 7) by ITLC and an isolated yield of 73 +/− 3 % (n = 7). Conclusions The results show the feasibility of generating radioimmunoconjugates and peptide conjugates for use as in vivo α generator systems in the clinic. The technology holds promise in applications involving the treatment of minimal disease such as micrometastases and residual tumor after surgical debulking, hematological cancers, infections, and compartmental cancers, such as ovarian cancer. PMID:23602604

  12. 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

    2016-09-02

    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.

  13. 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.

  14. A novel approach to infection imaging using a synthetic Tc-99m-labeled leukotactic peptide

    SciTech Connect

    Som, P.; Oster, Z.H.; Sharma, S. ||

    1996-05-01

    RMT1, a synthetic peptide binding to PMN and macrophage receptors was labeled with Tc-99m and investigated as a potential imaging agent for abscess and inflammation. Experimental abscesses were induced in rabbits and dogs by turpentine and E.coli injection. After injection of Tc-99m-RMT1 two and twelve day old abscesses were visualized within 20 min. In initial studies, a dose of 30 {mu}g of peptide/3 mCi was used. This amount was subsequently reduced to 1.5 {mu}g peptide with same amount of Tc-99m yielding similar imaging results. Technetium-99m-IgG and Tc-99m-MAG-3 were used as positive and negative controls, respectively. After injection of IgG abscesses were visualized but activity in blood was always higher than in abscess. When using Tc-99m RMT1 rapid abscess visualization and faster blood clearance was observed. The accumulation of RMT1 was monophasic, i.e., following the initial visualization, activity continued to build up continuously for 1{1/2} hr. Tc-99m-MAG3 accumulated initially in abscess, but activity washed out. In dogs, RMT1 blood clearance showed three components: a fast component with t{1/2}=1.9 min, 73%, intermediate t{1/2}=22 min, 24.5% and slow component, t{1/2}=115, 9.5% with 3 hours cumulative urine excretion of 40-51%. RMT1 appears to be more advantageous than currently available methods because of rapidity of imaging, simpler preparation before injection and will probably be less expensive and time consuming compared to labeled WBC. These results indicate that clinical experiments are warranted.

  15. Affinity fluorescence-labeled peptides for the early detection of cancer in Barrett's esophagus

    NASA Astrophysics Data System (ADS)

    Li, Meng; Lu, Shaoying; Piraka, Cyrus; Appelman, Henry; Kwon, Rich; Soetikno, Roy; Kaltenbach, Tonya; Wang, Thomas D.

    2009-02-01

    Fluorescence-labeled peptides that affinity bind to neoplastic mucsosa are promising for use as a specific contrast agent in the detection of pre-malignant tissue in the esophagus. This method is can be used to identify expression of biological markers associated with dysplasia on endoscopic imaging as a guide for biopsy and represents a novel method for the early detection and prevention of cancer. We demonstrate the use of phage display to select affinity peptides and identify the sequence "ASYNYDA" that binds with high target-to-background ratio to dysplastic esophageal mucosa compared to that of intestinal metaplasia. Validation of preferential binding is demonstrated for neoplasia in the setting of Barrett's esophagus. An optimal tradeoff between sensitivity and specificity of 82% and 85% was found at the relative threshold of 0.60 with a target-to-background ratio of 1.81 and an area under the ROC curve of 0.87. Peptides are a novel class of ligand for targeted detection of pre-malignant mucosa for purposes of screening and surveillance.

  16. Affinity labelling of proteinases with tryptic specificity by peptides with C-terminal lysine chloromethyl ketone

    PubMed Central

    Coggins, John R.; Kray, William; Shaw, Elliott

    1974-01-01

    Methods are described for the synthesis of peptides terminating in Lys-CH2Cl. The products were examined as affinity labels for several enzymes of trypsin-like specificity which are resistant to Tos-Lys-CH2Cl. In part, the inertness of the latter may be due to the sulphonamide group, since Z-Lys-CH2Cl was more effective. However, a number of tripeptides with C-terminal Lys-CH2Cl were superior in their ability to inactivate subtilisin, thrombin and plasma kallikrein. The possibility of developing enzyme-specific reagents selective for members within the trypsin-like group is demonstrated by Ala-Phe-Lys-CH2Cl, which readily inactivates plasma kallikrein but not thrombin. PMID:4422496

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. Peptide labeling with photoactivatable trifunctional cadaverine derivative and identification of interacting partners by biotin transfer.

    PubMed

    App, Christine; Knop, Jana; Huff, Thomas; Seebahn, Angela; Becker, Cord-Michael; Iavarone, Federica; Castagnola, Massimo; Hannappel, Ewald

    2014-07-01

    A new photoactivatable trifunctional cross-linker, cBED (cadaverine-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3'-dithiopropionate), was synthesized by chemical conversion of sulfo-SBED (sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3'-dithiopropionate) with cadaverine. This cross-linker was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) and characterized using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. cBED is based on sulfo-SBED that has a photoactivatable azido group, a cleavable disulfide bond for label transfer methods, and a biotin moiety for highly sensitive biotin/avidin detection. By ultraviolet (UV) light, the azido group is converted to a reactive nitrene, transforming transient bindings of interacting structures to covalent bonds. In contrast to the sulfo-N-hydroxysuccinimide (sulfo-NHS) moiety of sulfo-SBED, which attaches quite unspecifically to amino groups, cBED includes a cadaverine moiety that can be attached by transglutaminase more specifically to certain glutamine residues. For instance, thymosin β4 can be labeled with cBED using tissue transglutaminase. By high-resolution HPLC/ESI-MS (electrospray ionization-mass spectrometry) and tandem MS (MS/MS) of the trypsin digest, it was established that glutamine residues at positions 23 and 36 were labeled, whereas Q39 showed no reactivity. The covalent binding of cBED to thymosin β4 did not influence its G-actin sequestering activity, and the complex could be used to identify new interaction partners. Therefore, cBED can be used to better understand the multifunctional role of thymosin β4 as well as of other proteins and peptides.

  2. Quantitative Analysis of Single Amino Acid Variant Peptides Associated with Pancreatic Cancer in Serum by an Isobaric Labeling Quantitative Method

    PubMed Central

    2015-01-01

    Single amino acid variations are highly associated with many human diseases. The direct detection of peptides containing single amino acid variants (SAAVs) derived from nonsynonymous single nucleotide polymorphisms (SNPs) in serum can provide unique opportunities for SAAV associated biomarker discovery. In the present study, an isobaric labeling quantitative strategy was applied to identify and quantify variant peptides in serum samples of pancreatic cancer patients and other benign controls. The largest number of SAAV peptides to date in serum including 96 unique variant peptides were quantified in this quantitative analysis, of which five variant peptides showed a statistically significant difference between pancreatic cancer and other controls (p-value < 0.05). Significant differences in the variant peptide SDNCEDTPEAGYFAVAVVK from serotransferrin were detected between pancreatic cancer and controls, which was further validated by selected reaction monitoring (SRM) analysis. The novel biomarker panel obtained by combining α-1-antichymotrypsin (AACT), Thrombospondin-1 (THBS1) and this variant peptide showed an excellent diagnostic performance in discriminating pancreatic cancer from healthy controls (AUC = 0.98) and chronic pancreatitis (AUC = 0.90). These results suggest that large-scale analysis of SAAV peptides in serum may provide a new direction for biomarker discovery research. PMID:25393578

  3. 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

    PubMed Central

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

    2014-01-01

    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. PMID:25462949

  4. 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.

  5. 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

    2012-12-04

    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.

  6. Label-Free Fluorescent Detection of Trypsin Activity Based on DNA-Stabilized Silver Nanocluster-Peptide Conjugates

    PubMed Central

    Zhuo, Cai-Xia; Wang, Li-Hui; Feng, Jing-Jing; Zhang, Yao-Dong

    2016-01-01

    Trypsin is important during the regulation of pancreatic exocrine function. The detection of trypsin activity is currently limited because of the need for the substrate to be labeled with a fluorescent tag. A label-free fluorescent method has been developed to monitor trypsin activity. The designed peptide probe consists of six arginine molecules and a cysteine terminus and can be conjugated to DNA-stabilized silver nanoclusters (DNA-AgNCs) by Ag-S bonding to enhance fluorescence. The peptide probe can also be adsorbed to the surface of graphene oxide (GO), thus resulting in the fluorescence quenching of DNA-AgNCs-peptide conjugate because of Förster resonance energy transfer. Once trypsin had degraded the peptide probe into amino acid residues, the DNA-AgNCs were released from the surface of GO, and the enhanced fluorescence of DNA-AgNCs was restored. Trypsin can be determined with a linear range of 0.0–50.0 ng/mL with a concentration as low as 1 ng/mL. This label-free method is simple and sensitive and has been successfully used for the determination of trypsin in serum. The method can also be modified to detect other proteases. PMID:27834849

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

    PubMed

    Dong, Maoqing; Miller, Laurence J

    2013-08-01

    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 analog probes with sites of covalent attachment extending from position 26 through 32 were characterized, with the highest affinity analogs 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.

  8. Photoaffinity labeling of Ras converting enzyme 1 (Rce1p) using a benzophenone-containing peptide substrate.

    PubMed

    Kyro, Kelly; Manandhar, Surya P; Mullen, Daniel; Schmidt, Walter K; Distefano, Mark D

    2010-08-01

    Isoprenylation is a post-translational modification that increases protein hydrophobicity and helps target certain proteins to membranes. Ras converting enzyme 1 (Rce1p) is an endoprotease that catalyzes the removal of a three residue fragment from the C-terminus of isoprenylated proteins. To obtain structural information about this membrane protein, photoaffinity labeling agents are being prepared and employed. Here, we describe the synthesis of a benzophenone-containing peptide substrate analogue for Rce1p. Using a continuous spectrofluorometric assay, this peptide was shown to be a substrate for Rce1p. Mass spectrometry was performed to confirm the site of cleavage and structure of the processed probe. Photolysis of the biotinylated compound in the presence of membranes containing Rce1p followed by streptavidin pull-down and Western blot analysis indicated that Rce1p had been labeled by the probe. Photolysis in the presence of both the biotinylated, benzophenone-containing probe and a farnesylated peptide competitor reduced the extent of labeling, suggesting that labeling is occurring in the active site.

  9. Photoaffinity Labeling of Ras Converting Enzyme 1 (Rce1p) using a Benzophenone-Containing Peptide Substrate

    PubMed Central

    Kyro, Kelly; Manandhar, Surya P.; Mullen, Daniel; Schmidt, Walter K.; Distefano, Mark D.

    2010-01-01

    Isoprenylation is a post-translational modification that increases protein hydrophobicity and helps target certain proteins to membranes. Ras Converting Enzyme 1 (Rce1p) is an endoprotease that catalyzes the removal of a three residue fragment from the C-terminus of isoprenylated proteins. To obtain structural information about this membrane protein, photoaffinity labeling agents are being prepared and employed. Here, we describe the synthesis of a benzophenone-containing peptide substrate analogue for Rce1p. Using a continuous spectrofluorometric assay, this peptide was shown to be a substrate for Rce1p. Mass spectrometry was performed to confirm the site of cleavage and structure of the processed probe. Photolysis of the biotinylated compound in the presence of membranes containing Rce1p followed by streptavidin pull-down and Western blot analysis indicated that Rce1p had been labeled by the probe. Photolysis in the presence of both the biotinylated, benzophenone-containing probe and a farnesylated peptide competitor reduced the extent of labeling, suggesting that labeling is occurring in the active site. PMID:20619662

  10. Lutetium-177-labeled gastrin releasing peptide receptor binding analogs: a novel approach to radionuclide therapy.

    PubMed

    Panigone, S; Nunn, A D

    2006-12-01

    Optimization of therapy for individual patients remains a goal of clinical practice. Radionuclide imaging can identify those patients who may benefit from subsequent targeted therapy by providing regional information on the distribution of the target. An ideal situation may be when the imaging and the therapeutic compounds are the same agent. Two antibodies ([ [90Y]ibritumomab, [131I]tositumomab) are now approved for the systemic radiotherapy of non-Hodgkin's lymphoma. The main hurdle is to deliver higher absorbed doses to the more refractory solid tumors paying particular regard to the bone marrow toxicity. The low dose is thought to be a result of the large size of antibodies slowing delivery to the target. Peptides having high affinity to receptors expressed on cancer cells are a promising alternative. They are usually rapidly excreted from the body through renal and/or hepatobiliary excretion thus creating a prolonged accumulation of the radioactivity in the kidneys, which represents a recognized issue for systemic radiotherapy. The first radiopeptide developed was a somatostatin analogue, which led to a major breakthrough in the field. Beside the kidney issue, somatostatin use remains limited to few cancers that express receptors in sufficiently large quantities, mainly neuroendocrine tumors. The gastrin releasing peptide (GRP) receptor is an attractive target for development of new radiopeptides with diagnostic and therapeutic potential. This is based upon the functional expression of GRP receptors in several of the more prevalent cancers including prostate, breast, and small cell lung cancer. This review covers the efforts currently underway to develop new and clinically promising GRP-receptor specific molecules labeled with imageable and therapeutic radionuclides.

  11. 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

  12. Novel indium-111 labeled gastrin peptide analogues (MG-CL1-4): synthesis and quality control.

    PubMed

    Naqvi, Syed Ali-Raza; Khan, Zulfiqar Ali; Nagra, Saeed Ahmad; Yar, Muhammad; Sherazi, Tauqir A; Shahzad, Sohail Shahzad; Shah, Syed Qaiser; Mahmood, Nasir; Ishfaq, Malik Muhammad; Mather, Stephen John

    2013-03-01

    Radiolabeled neuropeptides are widely investigated to diagnose and therapy of tumors. These peptides get internalization after binding with particular receptors at the surface of cells and finally move to lysosome. Internalization into tumor cells helps in mapping the infected site. Minigastrin peptide analogues (MG-CL1-4) were synthesised and labeled with 111-In radioisotope under different sets of conditions for imaging CCk-2 receptor bearing tumors. Different parameters such as temperature (80-100°C), pH (4-12), incubation time (5-30 minutes) and dilution effect were investigated to get the maximum labeling yield and stability. The results indicated that MG-CL1-4 is successfully labeled with indium-111 at pH 4.5 with heating at 98°C for 15 minute. At these conditions i.e. heating, pH and incubation minimum oxidized and maximum labeling yield, more than 94 %, was obtained. The labeling stability was studied by incubating the radiolabeled complex for predefined time points in PBSA and blood serum. Results show that more than 90% radiolabeled MG-CL1-4 remained intact.

  13. Label-free quantitative phosphoproteomic profiling of cellular response induced by an insect cytokine paralytic peptide.

    PubMed

    Song, Liang; Wang, Fei; Dong, Zhaoming; Hua, Xiaoting; Xia, Qingyou

    2017-02-10

    Paralytic peptide (PP) participates in diverse physiological processes as an insect cytokine, such as immunity control, paralysis induction, regulation of cell morphology and proliferation. To investigate the molecular mechanism underlying those physiological activities, we systematically investigated the global phosphorylation events in fat body of silkworm larvae induced by PP through label-free quantitative phosphoproteomics. 2534 phosphosites were finally identified, of which the phosphorylation level of 620 phosphosites on 244 proteins was significantly up-regulated and 67 phosphosites on 43 proteins was down-regulated. Among those proteins, 13 were protein kinases (PKs), 13 were transcription factors (TFs) across 10 families and 17 were metabolism related enzymes. Meanwhile, Motif-X analysis of the phosphorylation sites showed that 16 motifs are significantly enriched, including 8 novel phosphorylation motifs. In addition, KEGG and functional interacting network analysis revealed that phosphorylation cascades play the crucial regulation roles in PP-dependent signaling pathways, and highlighted the potential central position of the mitogen-activated protein kinases (MAPKs) in them. These analyses provide direct insights into the molecule mechanisms of cellular response induced by PP.

  14. [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.

  15. The Effect of Superparamagnetic Iron Oxide with iRGD Peptide on the Labeling of Pancreatic Cancer Cells In Vitro: A Preliminary Study

    PubMed Central

    Zuo, Hou Dong; Yao, Wei Wu; Chen, Tian Wu; Zhu, Jiang; Zhang, Juan Juan; Pu, Yu; Liu, Gang; Zhang, Xiao Ming

    2014-01-01

    The iRGD peptide loaded with iron oxide nanoparticles for tumor targeting and tissue penetration was developed for targeted tumor therapy and ultrasensitive MR imaging. Binding of iRGD, a tumor homing peptide, is mediated by integrins, which are widely expressed on the surface of cells. Several types of small molecular drugs and nanoparticles can be transfected into cells with the help of iRGD peptide. Thus, we postulate that SPIO nanoparticles, which have good biocompatibility, can also be transfected into cells using iRGD. Despite the many kinds of cell labeling studies that have been performed with SPIO nanoparticles and RGD peptide or its analogues, only a few have applied SPIO nanoparticles with iRGD peptide in pancreatic cancer cells. This paper reports our preliminary findings regarding the effect of iRGD peptide (CRGDK/RGPD/EC) combined with SPIO on the labeling of pancreatic cancer cells. The results suggest that SPIO with iRGD peptide can enhance the positive labeling rate of cells and the uptake of SPIO. Optimal functionalization was achieved with the appropriate concentration or concentration range of SPIO and iRGD peptide. This study describes a simple and economical protocol to label panc-1 cells using SPIO in combination with iRGD peptide and may provide a useful method to improve the sensitivity of pancreatic cancer imaging. PMID:24977163

  16. The effect of superparamagnetic iron oxide with iRGD peptide on the labeling of pancreatic cancer cells in vitro: a preliminary study.

    PubMed

    Zuo, Hou Dong; Yao, Wei Wu; Chen, Tian Wu; Zhu, Jiang; Zhang, Juan Juan; Pu, Yu; Liu, Gang; Zhang, Xiao Ming

    2014-01-01

    The iRGD peptide loaded with iron oxide nanoparticles for tumor targeting and tissue penetration was developed for targeted tumor therapy and ultrasensitive MR imaging. Binding of iRGD, a tumor homing peptide, is mediated by integrins, which are widely expressed on the surface of cells. Several types of small molecular drugs and nanoparticles can be transfected into cells with the help of iRGD peptide. Thus, we postulate that SPIO nanoparticles, which have good biocompatibility, can also be transfected into cells using iRGD. Despite the many kinds of cell labeling studies that have been performed with SPIO nanoparticles and RGD peptide or its analogues, only a few have applied SPIO nanoparticles with iRGD peptide in pancreatic cancer cells. This paper reports our preliminary findings regarding the effect of iRGD peptide (CRGDK/RGPD/EC) combined with SPIO on the labeling of pancreatic cancer cells. The results suggest that SPIO with iRGD peptide can enhance the positive labeling rate of cells and the uptake of SPIO. Optimal functionalization was achieved with the appropriate concentration or concentration range of SPIO and iRGD peptide. This study describes a simple and economical protocol to label panc-1 cells using SPIO in combination with iRGD peptide and may provide a useful method to improve the sensitivity of pancreatic cancer imaging.

  17. 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

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

    PubMed Central

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

    2016-01-01

    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 15N isotope-labeled Aβ42 for structural studies by NMR. The protocol involves utilization of an auto induction system with 15N isotope labeled medium, for high-level expression of Aβ42 as a fusion with IFABP. After the over-expression of the 15N isotope-labeled IFABP-Aβ42 fusion protein in the inclusion bodies, pure 15N 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 15N isotope-labeled Aβ42 peptide. Mass spectrometry and 1H–15N 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 15N and 13C in Aβ42 peptide as well as its other variants including any Aβ42 peptide mutants. PMID:26231074

  19. New strategy for the preparation of clickable peptides and labeling with 1-(azidomethyl)-4-[(18)F]-fluorobenzene for PET.

    PubMed

    Thonon, David; Kech, Cécile; Paris, Jérôme; Lemaire, Christian; Luxen, André

    2009-04-01

    The alkyne-azide Cu(I)-catalyzed Huisgen cycloaddition, a click type reaction was used to label a peptide with fluorine-18. A novel solid phase synthesis approach for the preparation of clickable peptides has been developed and has also permitted the straightforward preparation of reference compounds. A complementary azide labeling agent (1-(azidomethyl)-4-[(18)F]-fluorobenzene) has been produced in a four step procedure in 75 min with a 34% radiochemical yield (decay corrected). Conjugation of [(18)F]fluoroazide with a model alkyne-neuropeptide produced the desired (18)F-radiolabeled peptide in less than 15 min with a yield of 90% and excellent radiochemical purity.

  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. sup 18 F-labeled insulin: A prosthetic group methodology for incorporation of a positron emitter into peptides and proteins

    SciTech Connect

    Shai, Y.; Kirk, K.L.; Channing, M.A.; Dunn, B.B.; Lesniak, M.A.; Eastman, R.C.; Finn, R.D.; Roth, J.; Jacobson, K.A. )

    1989-05-30

    In the present study we synthesize {sup 18}F-labeled insulin of high specific radioactivity. A new prosthetic group methodology, in which ({sup 18}F)fluoride displaces a bromide group of 4-(bromomethyl)-benzoylamine intermediates, was used. The 4-(fluoromethyl)benzoyl product was chemically stable. {sup 18}F-Labeled insulin retains the essential biological properties of native insulin, as measured in vitro by binding to insulin receptors on human cells and stimulation of glucose metabolism in rat adipocytes. The overall process can be carried out speedily to yield a product of sufficient purity to permit in vivo studies. The method appears to be applicable to a wide variety of peptides.

  2. Detection of alpha-helical coiled-coil dimer formation by spin-labeled synthetic peptides: a model parallel coiled-coil peptide and the antiparallel coiled coil formed by a replica of the ProP C-terminus.

    PubMed

    Hillar, Alexander; Tripet, Brian; Zoetewey, David; Wood, Janet M; Hodges, Robert S; Boggs, Joan M

    2003-12-30

    Electron paramagnetic resonance spectroscopy was used to determine relative peptide orientation within homodimeric, alpha-helical coiled-coil structures. Introduction of cysteine (Cys) residues into peptides/proteins for spin labeling allows detection of their oligomerization from exchange broadening or dipolar interactions between residues within 25 A of each other. Two synthetic peptides containing Cys substitutions were used: a 35-residue model peptide and the 30-residue ProP peptide. The model peptide is known to form a stable, parallel homodimeric coiled coil, which is partially destabilized by Cys substitutions at heptad a and d positions (peptides C30a and C33d). The ProP peptide, a 30-residue synthetic peptide, corresponds to residues 468-497 of osmoregulatory transporter ProP from Escherichia coli. It forms a relatively unstable, homodimeric coiled coil that is predicted to be antiparallel in orientation. Cys was introduced in heptad g positions of the ProP peptide, near the N-terminus (K473C, creating peptide C473g) or closer to the center of the sequence (E480C, creating peptide C480g). In contrast to the destabilizing effect of Cys substitution at the core heptad a or d positions of model peptides C30a and C33d, circular dichroism spectroscopy showed that Cys substitutions at the heptad g positions of the ProP peptide had little or no effect on coiled-coil stability. Thermal denaturation analysis showed that spin labeling increased the stability of the coiled coil for all peptides. Strong exchange broadening was detected for both C30a and C33d, in agreement with a parallel structure. EPR spectra of C480g had a large hyperfine splitting of about 90 G, indicative of strong dipole-dipole interactions and a distance between spin-labeled residues of less than 9 A. Spin-spin interactions were much weaker for C473g. These results supported the hypothesis that the ProP peptide primarily formed an antiparallel coiled coil, since formation of a parallel dimer

  3. 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.

  4. 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.

  5. A 99mTc-tricine-HYNIC-labeled Peptide Targeting the Melanocortin-1 Receptor for Melanoma Imaging

    PubMed Central

    Shamshirian, Danial; Erfani, Mostafa; Beiki, Davood; Hajiramazanali, Maliheh; Fallahi, Babak

    2016-01-01

    Melanocortin-1 (MC1) receptor is an attractive melanoma-specific target for the development of α-MSH peptide based imaging and therapeutic agents. In this work a new lactam bridge α-MSH analogue was synthesized and radiolabeled with 99mTc via HYNIC chelator and tricine as co-ligand. Also, stability in human serum, receptor bound internalization and tissue biodistribution in tumor bearing nude mice were thoroughly investigated. Radiolabeling with 99mTc was performed at high specific activities (163MBq/nmol) with an acceptable labeling yield (>98%). The radioligand showed specific internalization into B16/F10 cells (13.35 ± 0.9% at 4 h). In biodistribution studies, a receptor-specific uptake was observed in MC1 receptor positive organ so that after 4 h the tumor uptake was 4.51 ± 0.11 % ID/g. Predominant renal excretion pathway with a highest accumulation of activity in tumor was observed for this radiopeptide. Obtained results show that the new designed labeled peptide conjugate can be a suitable candidate for diagnosis of metastatic melanomas. PMID:27980570

  6. 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.

  7. Orientation of the antimicrobial peptide PGLa in lipid membranes determined from 19F-NMR dipolar couplings of 4-CF3-phenylglycine labels.

    PubMed

    Glaser, Ralf W; Sachse, Carsten; Dürr, Ulrich H N; Wadhwani, Parvesh; Ulrich, Anne S

    2004-05-01

    A highly sensitive solid state (19)F-NMR strategy is described to determine the orientation and dynamics of membrane-associated peptides from specific fluorine labels. Several analogues of the antimicrobial peptide PGLa were synthesized with the non-natural amino acid 4-trifluoromethyl-phenylglycine (CF(3)-Phg) at different positions throughout the alpha-helical peptide chain. A simple 1-pulse (19)F experiment allows the simultaneous measurement of both the anisotropic chemical shift and the homonuclear dipolar coupling within the rotating CF(3)-group in a macroscopically oriented membrane sample. The value and sign of the dipolar splitting determines the tilt of the CF(3)-rotational axis, which is rigidly attached to the peptide backbone, with respect to the external magnetic field direction. Using four CF(3)-labeled peptide analogues (with L-CF(3)-Phg at Ile9, Ala10, Ile13, and Ala14) we confirmed that PGLa is aligned at the surface of lipid membranes with its helix axis perpendicular to the bilayer normal at a peptide:lipid ratio of 1:200. We also determined the azimuthal rotation angle of the helix, which agrees well with the orientation expected from its amphiphilic character. Peptide analogues with a D-CF(3)-Phg label resulting from racemization of the amino acid during synthesis were separately collected by HPLC. Their spectra provide additional information about the PGLa structure and orientation but allow only to discriminate qualitatively between multiple solutions. The structural and functional characterization of the individual CF(3)-labeled peptides by circular dichroism and antimicrobial assays showed only small effects for our four substitutions on the hydrophobic face of the helix, but a significant disturbance was observed in a fifth analogue where Ala8 on the hydrophilic face had been replaced. Even though the hydrophobic CF(3)-Phg side chain cannot be utilized in all positions, it allows highly sensitive NMR measurements over a wide range of

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. A New Strategy for Early Diagnosis of Type 2 Diabetes by Standard-Free, Label-Free LC-MS/MS Quantification of Glycated Peptides

    PubMed Central

    Zhang, Mei; Xu, Wei; Deng, Yulin

    2013-01-01

    The early diagnosis of diabetes, one of the top three chronic incurable diseases, is becoming increasingly important. Here, we investigated the applicability of an 18O-labeling technique for the development of a standard-free, label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the early diagnosis of type 2 diabetes mellitus (T2DM). Rather than attempting to identify quantitative differences in proteins as biomarkers, glycation of the highest abundance protein in human plasma, human serum albumin (HSA), was monitored through quantitative analysis of HSA characteristic peptides using the 18O-labeling technique. Eight glucose-sensitive peptides and one glucose-insensitive peptide were discovered. The glucose-insensitive peptide served as the internal standard, and a standard-free, label-free LC-MS/MS method was developed. This method was then used to select putative biomarkers for T2DM in a clinical trial with 389 human plasma samples. As a result, three of the eight glucose-sensitive peptides (FKDLGEENFK, LDELRDEGK, and KVPQVSTPTLVEVSR) were selected and could be used as potential biomarkers for the early diagnosis of T2DM. PMID:23894188

  14. Label-free mass spectrometry exploits dozens of detected peptides to quantify lamins in wildtype and knockdown cells.

    PubMed

    Swift, Joe; Harada, Takamasa; Buxboim, Amnon; Shin, Jae-Won; Tang, Hsin-Yao; Speicher, David W; Discher, Dennis E

    2013-01-01

    Label-free quantitation and characterization of proteins by mass spectrometry (MS) is now feasible, especially for moderately expressed structural proteins such as lamins that typically yield dozens of tryptic peptides from tissue cells. Using standard cell culture samples, we describe general algorithms for quantitative analysis of peptides identified in liquid chromatography tandem mass spectrometry (LC-MS/MS). The algorithms were foundational to the discovery that the absolute stoichiometry of A-type to B-type lamins scales with tissue stiffness (Swift et al., Science 2013). Isoform dominance helps make sense of why mutations and changes with age of mechanosensitive lamin-A,C only affect "stiff" tissues such as heart, muscle, bone, or even fat, but not brain. A Peak Ratio Fingerprinting (PRF) algorithm is elaborated here through its application to lamin-A,C knockdown. After demonstrating the large dynamic range of PRF using calibrated mixtures of human and mouse lysates, we validate measurements of partial knockdown with standard cell biology analyses using quantitative immunofluorescence and immunoblotting. Optimal sets of MS-detected peptides as determined by PRF demonstrate that the strongest peptide signals are not necessarily the most reliable for quantitation. After lamin-A,C knockdown, PRF computes an invariant set of "housekeeping" proteins as part of a broader proteomic analysis that also shows the proteome of mesenchymal stem cells (MSCs) is more broadly perturbed than that of a human epithelial cancer line (A549s), with particular variation in nuclear and cytoskeletal proteins. These methods offer exciting prospects for basic and clinical studies of lamin-A,C as well as other MS-detectable proteins.

  15. 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

  16. Prediction of Impending Type 1 Diabetes through Automated Dual-Label Measurement of Proinsulin:C-Peptide Ratio

    PubMed Central

    Balti, Eric V.; Keymeulen, Bart; Gillard, Pieter; Lapauw, Bruno; De Block, Christophe; Abrams, Pascale; Weber, Eric; Vermeulen, Ilse; De Pauw, Pieter; Pipeleers, Daniël; Weets, Ilse; Gorus, Frans K.

    2016-01-01

    Background The hyperglycemic clamp test, the gold standard of beta cell function, predicts impending type 1 diabetes in islet autoantibody-positive individuals, but the latter may benefit from less invasive function tests such as the proinsulin:C-peptide ratio (PI:C). The present study aims to optimize precision of PI:C measurements by automating a dual-label trefoil-type time-resolved fluorescence immunoassay (TT-TRFIA), and to compare its diagnostic performance for predicting type 1 diabetes with that of clamp-derived C-peptide release. Methods Between-day imprecision (n = 20) and split-sample analysis (n = 95) were used to compare TT-TRFIA (AutoDelfia, Perkin-Elmer) with separate methods for proinsulin (in-house TRFIA) and C-peptide (Elecsys, Roche). High-risk multiple autoantibody-positive first-degree relatives (n = 49; age 5–39) were tested for fasting PI:C, HOMA2-IR and hyperglycemic clamp and followed for 20–57 months (interquartile range). Results TT-TRFIA values for proinsulin, C-peptide and PI:C correlated significantly (r2 = 0.96–0.99; P<0.001) with results obtained with separate methods. TT-TRFIA achieved better between-day %CV for PI:C at three different levels (4.5–7.1 vs 6.7–9.5 for separate methods). In high-risk relatives fasting PI:C was significantly and inversely correlated (rs = -0.596; P<0.001) with first-phase C-peptide release during clamp (also with second phase release, only available for age 12–39 years; n = 31), but only after normalization for HOMA2-IR. In ROC- and Cox regression analysis, HOMA2-IR-corrected PI:C predicted 2-year progression to diabetes equally well as clamp-derived C-peptide release. Conclusions The reproducibility of PI:C benefits from the automated simultaneous determination of both hormones. HOMA2-IR-corrected PI:C may serve as a minimally invasive alternative to the more tedious hyperglycemic clamp test. PMID:27907006

  17. 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.

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

    PubMed Central

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

    2015-01-01

    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. Furthermore, the circular dichroism (CD) spectrum of untreated Aβ shows a continuous, progressive change over a 24-hour period, while the spectrum of Aβ treated with SLF remains relatively constant following initial incubation. These findings suggest the conformation of Aβ within the oligomer provides a complementary determinant of Aβ toxicity in addition to oligomer growth and size. Although SLF does not produce a dominant state of secondary structure in Aβ, it does induce a net reduction in beta secondary content compared to untreated samples of Aβ. The FCS results, combined with electron paramagnetic resonance spectroscopy and CD spectroscopy, demonstrate SLFs can inhibit the growth of Aβ oligomers and disrupt existing oligomers, while retaining Aβ as a population of smaller, yet largely disordered oligomers. PMID:26374940

  19. 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.

  20. 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.

  1. Extensive Peptide Fractionation and y1 Ion-Based Interference Detection Method for Enabling Accurate Quantification by Isobaric Labeling and Mass Spectrometry.

    PubMed

    Niu, Mingming; Cho, Ji-Hoon; Kodali, Kiran; Pagala, Vishwajeeth; High, Anthony A; Wang, Hong; Wu, Zhiping; Li, Yuxin; Bi, Wenjian; Zhang, Hui; Wang, Xusheng; Zou, Wei; Peng, Junmin

    2017-02-22

    Isobaric labeling quantification by mass spectrometry (MS) has emerged as a powerful technology for multiplexed large-scale protein profiling, but measurement accuracy in complex mixtures is confounded by the interference from coisolated ions, resulting in ratio compression. Here we report that the ratio compression can be essentially resolved by the combination of pre-MS peptide fractionation, MS2-based interference detection, and post-MS computational interference correction. To recapitulate the complexity of biological samples, we pooled tandem mass tag (TMT)-labeled Escherichia coli peptides at 1:3:10 ratios and added in ∼20-fold more rat peptides as background, followed by the analysis of two-dimensional liquid chromatography (LC)-MS/MS. Systematic investigation shows that quantitative interference was impacted by LC fractionation depth, MS isolation window, and peptide loading amount. Exhaustive fractionation (320 × 4 h) can nearly eliminate the interference and achieve results comparable to the MS3-based method. Importantly, the interference in MS2 scans can be estimated by the intensity of contaminated y1 product ions, and we thus developed an algorithm to correct reporter ion ratios of tryptic peptides. Our data indicate that intermediate fractionation (40 × 2 h) and y1 ion-based correction allow accurate and deep TMT profiling of more than 10 000 proteins, which represents a straightforward and affordable strategy in isobaric labeling proteomics.

  2. Rapid 'de novo' peptide sequencing by a combination of nanoelectrospray, isotopic labeling and a quadrupole/time-of-flight mass spectrometer.

    PubMed

    Shevchenko, A; Chernushevich, I; Ens, W; Standing, K G; Thomson, B; Wilm, M; Mann, M

    1997-01-01

    Protein microanalysis usually involves the sequencing of gel-separated proteins available in very small amounts. While mass spectrometry has become the method of choice for identifying proteins in databases, in almost all laboratories 'de novo' protein sequencing is still performed by Edman degradation. Here we show that a combination of the nanoelectrospray ion source, isotopic end labeling of peptides and a quadrupole/ time-of-flight instrument allows facile read-out of the sequences of tryptic peptides. Isotopic labeling was performed by enzymatic digestion of proteins in 1:1 16O/18O water, eliminating the need for peptide derivatization. A quadrupole/time-of-flight mass spectrometer was constructed from a triple quadrupole and an electrospray time-of-flight instrument. Tandem mass spectra of peptides were obtained with better than 50 ppm mass accuracy and resolution routinely in excess of 5000. Unique and error tolerant identification of yeast proteins as well as the sequencing of a novel protein illustrate the potential of the approach. The high data quality in tandem mass spectra and the additional information provided by the isotopic end labeling of peptides enabled automated interpretation of the spectra via simple software algorithms. The technique demonstrated here removes one of the last obstacles to routine and high throughput protein sequencing by mass spectrometry.

  3. Diagnosis of osteomyelitis and soft tissue infection using a Tc-99m labeled tuftsin-analog peptide

    SciTech Connect

    Som, P.; Oster, Z.H.; Sharma, S.

    1997-05-01

    The localization of infection sites and of osteomyelitis is still an ongoing diagnostic challenge. In joints affected by arthritis, complicated fractures and around prosthetic devices, three-phase bone scans are non-diagnostic because the underlying condition will cause the third phase scan to be positive, while surrounding soft tissue inflammation may cause the first and second phase scans to be positive as well. Currently, the method of choice in these situations is to use radiolabeled white blood cell scans involving lengthy and expensive procedure and need for delayed imaging. We describe a method using a Tc-99m labeled leukotactic peptide for imaging osteomyelitis and soft tissue infections, which appears to be simpler, and enabling fast diagnosis. Abscesses, clean fractures and infected fractures simulating osteomyelitis were induced in rabbits as described earlier. RMT-1, a tuftsin-mimetic synthetic tetrapeptide labeled with Tc-99m was used. Blood clearance, urine excretion and whole body timed scintigraphy were carried out in normal dogs and evaluation of the compound was performed in dogs and rabbits with soft tissue chemical and bacterial abscesses and in rabbits with clean fractures and experimental osteomyelitis.

  4. F-18 Labeled Vasoactive Intestinal Peptide Analogue in the PET Imaging of Colon Carcinoma in Nude Mice

    PubMed Central

    Liu, Yuxia; Shen, Hua; Pang, Lifang; Yin, Duanzhi; Wang, Yongxian; Li, Shanqun; Shi, Hongcheng

    2013-01-01

    As large amount of vasoactive intestinal peptide (VIP) receptors are expressed in various tumors and VIP-related diseases, radiolabeled VIP provides a potential PET imaging agent for VIP receptor. However, structural modification of VIP is required before being radiolabeled and used for VIP receptor imaging due to its poor in vivo stability. As a VIP analogue, [R8, 15, 21, L17]-VIP exhibited improved stability and receptor specificity in preliminary studies. In this study, F-18 labeled [R8,15,21, L17]-VIP was produced with the radiochemical yield being as high as 33.6% ± 3% (decay-for-corrected, n = 5) achieved within 100 min, a specific activity of 255 GBq/μmol, and a radiochemical purity as high as 99% as characterized by radioactive HPLC, TLC, and SDS-Page radioautography. A biodistribution study in normal mice also demonstrated fast elimination of F-18 labeled [R8,15,21, L17]-VIP in the blood, liver, and gastrointestinal tracts. A further micro-PET imaging study in C26 colon carcinoma bearing mice confirmed the high tumor specificity, with the tumor/muscle radioactivity uptake ratio being as high as 3.03 at 60 min following injection, and no apparent radioactivity concentration in the intestinal tracts. In addition, blocking experiment and Western Blot test further confirmed its potential in PET imaging of VIP receptor-positive tumor. PMID:24459669

  5. 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.

  6. 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.

  7. 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.

  8. Phage display evolution of a peptide substrate for yeast biotin ligase and application to two-color quantum dot labeling of cell surface proteins.

    PubMed

    Chen, Irwin; Choi, Yoon-Aa; Ting, Alice Y

    2007-05-23

    Site-specific protein labeling with Escherichia coli biotin ligase (BirA) has been used to introduce fluorophores, quantum dots (QDs), and photocross-linkers onto recombinant proteins fused to a 15-amino acid acceptor peptide (AP) substrate for BirA and expressed on the surface of living mammalian cells. Here, we used phage display to engineer a new and orthogonal biotin ligase-AP pair for site-specific protein labeling. Yeast biotin ligase (yBL) does not recognize the AP, but we discovered a new 15-amino acid substrate for yBL called the yeast acceptor peptide (yAP), using two generations of phage display selection from 15-mer peptide libraries. The yAP is not recognized by BirA, and thus, we were able to specifically label AP and yAP fusion proteins coexpressed in the same cell with differently colored QDs. We fused the yAP to a variety of recombinant proteins and demonstrated biotinylation by yBL at the N-terminus, C-terminus, and within a flexible internal region. yBL is extremely sequence-specific, as endogenous proteins on the surface of yeast and HeLa cells are not biotinylated. This new methodology expands the scope of biotin ligase labeling to two-color imaging and yeast-based applications.

  9. Sulfonation of Tyrosine as a Method to Improve Biodistribution of Peptide-Based Radiotracers: Novel (18)F-Labelled Cyclic RGD Analogues.

    PubMed

    Haskali, Mohammad Baqir; Denoyer, Delphine; Noonan, Wayne; Cullinane, Carleen; Rangger, Christine; Pouliot, Normand; Haubner, Roland; Roselt, Peter D; Hicks, Rodney J; Hutton, Craig A

    2017-02-13

    The labeling of peptides with positron emitting radionuclides has long held the promise of a wide range of PET agents possessing high affinity and selectivity. Not surprisingly, controlling the biodistribution of these agents has proven to be a major challenge in their successful application. Modification of peptide hydrophilicity in order to increase renal clearance has been a common endeavor to improve overall biodistribution. Herein, we examine the effect of site-specific sulfonation of tyrosine moieties in cyclic(RGDyK) peptides as a means to enhance their hydrophilicity and improve their biodistribution. The novel sulfonated cyclic(RGDyK) peptides were conjugated directly to 4-nitrophenyl 2-[18F]fluoropropionate and the biodistribution of the radiolabeled peptides was compared with that of their non-sulfonated, clinically relevant counterparts, [18F]GalactoRGD and [18F]FPPRGD2. Site-specific sulfonation of the tyrosine residues was shown to increase hydrophilicity and improve biodistribution of the RGD peptides, despite contributing just 79 Da towards the MW, compared with 189 Da for both the 'Galacto' and mini-PEG moieties, suggesting this may be a broadly applicable approach to enhancing biodistribution of radiolabelled peptides.

  10. 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

  11. A convenient method for europium-labeling of a recombinant chimeric relaxin family peptide R3/I5 for receptor-binding assays.

    PubMed

    Zhang, Wei-Jie; Jiang, Qian; Wang, Xin-Yi; Song, Ge; Shao, Xiao-Xia; Guo, Zhan-Yun

    2013-06-01

    Relaxin family peptides have important biological functions, and so far, four G-protein-coupled receptors have been identified as their receptors (RXFP1-4). A chimeric relaxin family peptide R3/I5, containing the B-chain of relaxin-3 and the A-chain of INSL5, is a selective agonist for both RXFP3 and RXFP4. In a previous study, europium-labeled R3/I5, as a nonradioactive and low-background receptor-binding tracer, was prepared through a chemical synthesis approach. In the present study, we established a convenient alternative approach for preparing the europium-labeled R3/I5 tracer based on a recombinant R3/I5 designed to carry a solubilizing tag at the A-chain N-terminus and a pyroglutamate residue at the B-chain N-terminus. Because of the presence of a single primary amine moiety, the recombinant R3/I5 peptide was site-specifically mono-labeled at the A-chain N-terminus by a diethylenetriaminepentaacetic acid/europium moiety through a convenient one-step procedure. The diethylenetriaminepentaacetic acid/Eu3+-labeled R3/I5 bound both receptors RXFP3 and RXFP4 with high binding affinities and low nonspecific binding. Thus, we have presented a valuable nonradioactive tracer for future interaction studies on RXFP3 and RXFP4 with various natural or designed ligands. The present approach could also be adapted for preparing and labeling of other chimeric relaxin family peptides.

  12. 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

  13. microPET Imaging of Glioma Integrin (alpha-v, beta-3) Expression Using Cu-64-Labeled Tetrameric RGD Peptide

    SciTech Connect

    Wu, Yun; Zhang, , Xianzhong; Xiong, , Zhengming; Cheng, Zhen; Fisher, Darrell R.; Liu, Shu-hong; Gambhir, Sanjiv S.; Chen, Xiaoyuan

    2005-10-01

    Integrins ?v?3 and ?v?5 play a critical role in tumor-induced angiogenesis and metastasis, and have become promising diagnostic indicators and therapeutic targets of tumors. Radiolabeled RGD peptides that are integrin-specific may be used for non-invasive imaging of integrin expression level as well as for integrin-targeted radionuclide therapy. We previously conjugated a series of mono- and dimeric RGD peptides with 1,4,7,10-tetraazacyclododecane-N, N?,N??,N???-tetraacetic acid (DOTA) and labeled these with copper-64 for microPET imaging in various mouse xenograft models. The copper-64 tracers showed ?v?3-selective tumor uptake, but the magnitude of tumor uptake was relatively low, the tumor washout was rapid, and non-target organ/tissue retention was high. In this study we developed a tetrameric RGD peptide tracer 64Cu-DOTA-E{l_brace}E[c(RGDfK)]2{r_brace}2 for positron emission tomography (PET) imaging of integrin ?v?3 expression in a subcutaneous U87MG glioma xenograft model in female athymic nude mice. The RGD tetramer showed significantly higher integrin binding affinity than the corresponding mono- and dimeric RGD analogs, most likely due to polyvalency effect. The radiolabeled peptide showed rapid blood clearance (0.61 ? 0.01%ID/g at 30 min and 0.21 ? 0.01 %ID/g at 4 h postinjection (p.i.), respectively) and predominantly renal excretion. Tumor uptake was rapid and high and the tumor washout was slow (9.93 ? 1.05 %ID/g at 30 min p.i. and 4.56 ? 0.51 %ID/g at 24 h post-injection). The metabolic stability of 64Cu-DOTA-E{l_brace}E[c(RGDfK)]2{r_brace}2 was determined in mouse blood, urine, and liver and kidney homogenates at different times after tracer injection. The average fractions of intact tracer in these organs at 1 h were approximately 70, 58, 51 and 26 percent, respectively. Non-invasive microPET imaging studies showed significant tumor uptake and good contrast in the subcutaneous tumor-bearing mice, which agreed well with the biodistribution results

  14. Membrane position of a basic aromatic peptide that sequesters phosphatidylinositol 4,5 bisphosphate determined by site-directed spin labeling and high-resolution NMR.

    PubMed

    Ellena, Jeffrey F; Moulthrop, Jason; Wu, Jing; Rauch, Michelle; Jaysinghne, Sajith; Castle, J David; Cafiso, David S

    2004-11-01

    The membrane interactions and position of a positively charged and highly aromatic peptide derived from a secretory carrier membrane protein (SCAMP) are examined using magnetic resonance spectroscopy and several biochemical methods. This peptide (SCAMP-E) is shown to bind to membranes containing phosphatidylinositol 4,5-bisphosphate, PI(4,5)P2, and sequester PI(4,5)P2 within the plane of the membrane. Site-directed spin labeling of the SCAMP-E peptide indicates that the position and structure of membrane bound SCAMP-E are not altered by the presence of PI(4,5)P2, and that the peptide backbone is positioned within the lipid interface below the level of the lipid phosphates. A second approach using high-resolution NMR was used to generate a model for SCAMP-E bound to bicelles. This approach combined oxygen enhancements of nuclear relaxation with a computational method to dock the SCAMP-E peptide at the lipid interface. The model for SCAMP generated by NMR is consistent with the results of site-directed spin labeling and places the peptide backbone in the bilayer interfacial region and the aromatic side chains within the lipid hydrocarbon region. The charged side chains of SCAMP-E lie well within the interface with two arginine residues lying deeper than a plane defined by the position of the lipid phosphates. These data suggest that SCAMP-E interacts with PI(4,5)P2 through an electrostatic mechanism that does not involve specific lipid-peptide contacts. This interaction may be facilitated by the position of the positively charged side chains on SCAMP-E within a low-dielectric region of the bilayer interface.

  15. Measuring affinity constants of 1450 monoclonal antibodies to peptide targets with a microarray-based label-free assay platform.

    PubMed

    Landry, J P; Ke, Yaohuang; Yu, Guo-Liang; Zhu, X D

    2015-02-01

    Monoclonal antibodies (mAbs) are major reagents for research and clinical diagnosis. For their inherently high specificities to intended antigen targets and thus low toxicity in general, they are pursued as one of the major classes of new drugs. Yet binding properties of most monoclonal antibodies are not well characterized in terms of affinity constants and how they vary with presentations and/or conformational isomers of antigens, buffer compositions, and temperature. We here report a microarray-based label-free assay platform for high-throughput measurements of monoclonal antibody affinity constants to antigens immobilized on solid surfaces. Using this platform we measured affinity constants of over 1410 rabbit monoclonal antibodies and 46 mouse monoclonal antibodies to peptide targets that are immobilized through a terminal cysteine residue to a glass surface. The experimentally measured affinity constants vary from 10 pM to 200 pM with the median value at 66 pM. We compare the results obtained from the microarray-based platform with those from a benchmarking surface-plasmon-resonance-based (SPR) sensor (Biacore 3000).

  16. Differential T cell receptor photoaffinity labeling among H-2Kd restricted cytotoxic T lymphocyte clones specific for a photoreactive peptide derivative. Labeling of the alpha-chain correlates with J alpha segment usage

    PubMed Central

    1993-01-01

    Using a direct binding assay based on photoaffinity labeling, we studied the interaction of T cell receptor (TCR) with a Kd-bound photoreactive peptide derivative on living cells. The Kd-restricted Plasmodium berghei circumsporozoite (PbCS) peptide 253-260 (YIPSAEKI) was reacted NH2-terminally with biotin and at the TCR contact residue Lys259 with photoreactive iodo, 4-azido salicylic acid (IASA) to make biotin-YIPSAEK(IASA)I. Cytotoxic T lymphocyte (CTL) clones derived from mice immunized with this derivative recognized this conjugate, but not a related one lacking the IASA group nor the parental PbCS peptide. The clones were Kd restricted. Recognition experiments with variant conjugates, lacking substituents from IASA, revealed a diverse fine specificity pattern and indicated that this group interacted directly with the TCR. The TCR of four clones could be photoaffinity labeled by biotin-YIPSAEK(125IASA)I. This labeling was dependent on the conjugates binding to the Kd molecule and was selective for the TCR alpha (2 clones) or beta chain (1 clone), or was common for both chains (1 clone). TCR sequence analysis showed a preferential usage of J alpha TA28 containing alpha chains that were paired with V beta 1 expressing beta chains. The TCR that were photoaffinity labeled at the alpha chain expressed these J alpha and V beta segments. The tryptophan encoded by the J alpha TA28 segment is rarely found in other J alpha segments. Moreover, we show that the IASA group interacts preferentially with tryptophan in aqueous solution. We thus propose that for these CTL clones, labeling of the alpha chain occurs via the J alpha-encoded tryptophan residue. PMID:8478607

  17. UV Raman spatially resolved melting dynamics of isotopically labeled polyalanyl peptide: slow alpha-helix melting follows 3(10)-helices and pi-bulges premelting.

    PubMed

    Mikhonin, Aleksandr V; Asher, Sanford A; Bykov, Sergei V; Murza, Adrian

    2007-03-29

    We used UV resonance Raman (UVRR) to examine the spatial dependence of the T-jump secondary structure relaxation of an isotopically labeled 21-residue mainly Ala peptide, AdP. The AdP penultimate Ala residues were perdeuterated, leaving the central residues hydrogenated, to allow separate monitoring of melting of the middle versus the end peptide bonds. For 5 to 30 degrees C T-jumps, the central peptide bonds show a approximately 2-fold slower relaxation time (189 +/- 31 ns) than do the exterior peptide bonds (97 +/- 15 ns). In contrast, for a 20 to 40 degrees C T-jump, the central peptide bond relaxation appears to be faster (56 +/- 6 ns) than that of the penultimate peptide bonds (131 +/- 46 ns). We show that, if the data are modeled as a two-state transition, we find that only exterior peptide bonds show anti-Arrhenius folding behavior; the middle peptide bonds show both normal Arrhenius-like folding and unfolding. This anti-Arrhenius behavior results from the involvement of pi-bulges/helices and 3(10)-helix states in the melting. The unusual temperature dependence of the (un)folding rates of the interior and exterior peptide bonds is due to the different relative (un)folding rates of 3(10)-helices, alpha-helices, and pi-bulges/helices. Pure alpha-helix unfolding rates are approximately 12-fold slower (approximately 1 micros) than that of pi-bulges and 3(10)-helices. In addition, we also find that the alpha-helix is most stable at the AdP N-terminus where eight consecutive Ala occur, whereas the three hydrophilic Arg located in the middle and at the C-terminus destabilize the alpha-helix in these regions and induce defects such as pi-bulges and 3(10)-helices.

  18. Studies on gonococcus infection. XVIII. 125I-labeled peptide mapping of the major protein of the gonococcal cell wall outer membrane.

    PubMed Central

    Swanson, J

    1979-01-01

    The major outer membrane proteins from 10 gonococcal strains were examined after 125I-labeling of the proteins as single bands resolved by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. These 125I-proteins were then treated with either trypsin or alpha-chymotrypsin, and the resultant 125I-peptides were visualized by autoradiography after two-dimensional electrophoretic and chromatographic separation on thin-layer cellulose sheets. Several 125I-peptides were present in all the major outer membrane proteins examined. The presence and absence of additional 125I-peptides segregated the major proteins into two pattern groups. One group consisted of major outer membranes with molecular weights of 34,000 or 33,000; major proteins with molecular weights of 32,000 constituted the other group. Two beta-lactamase-producing gonococcal isolates were examined. Their major outer membrane proteins were identical in apparent molecular weights and alpha-chymotryptic 125I-peptide fingerprints; these proteins contained 125I-peptides not found in other gonococcal major proteins. No 125I-peptide differences were found among the major outer membrane proteins of strain F62 gonococci that exhibited differences in piliation and/or colony opacity characteristics. Images PMID:110681

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

    PubMed

    Hippeläinen, E; Tenhunen, M; Sohlberg, A

    2015-09-07

    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

  20. 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

  1. Preferential labeling of alpha-amino N-terminal groups in peptides by biotin: application to the detection of specific anti-peptide antibodies by enzyme immunoassays.

    PubMed

    Sélo, I; Négroni, L; Créminon, C; Grassi, J; Wal, J M

    1996-12-15

    Experimental conditions (pH 6.5, 24 h reaction, peptide:biotin ratio 1:5) were defined for preferential incorporation of the biotin molecule in the N-terminal alpha-amino group of peptides. This strategy could be helpful in numerous applications when an entire peptide chain must remain accessible for antibody or receptor binding. We illustrate this advantage in a solid-phase enzyme immunoassay designed to detect antibodies specific for bovine beta-lactoglobulin present in rabbit or human sera. This test involves synthetic peptides biotinylated in different positions and immobilized on a solid phase. The use of biotin/streptavidin interactions permitted more efficient detection of specific anti-peptide antibodies than solid phases prepared using conventional passive-adsorption techniques. The highest levels of antibody binding were measured when biotinylation occurred at the N-terminal extremity of immobilized peptides.

  2. 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.

  3. Gaining efficiency by parallel quantification and identification of iTRAQ-labeled peptides using HCD and decision tree guided CID/ETD on an LTQ Orbitrap.

    PubMed

    Mischerikow, Nikolai; van Nierop, Pim; Li, Ka Wan; Bernstein, Hans-Gert; Smit, August B; Heck, Albert J R; Altelaar, A F Maarten

    2010-10-01

    Isobaric stable isotope labeling of peptides using iTRAQ is an important method for MS based quantitative proteomics. Traditionally, quantitative analysis of iTRAQ labeled peptides has been confined to beam-type instruments because of the weak detection capabilities of ion traps for low mass ions. Recent technical advances in fragmentation techniques on linear ion traps and the hybrid linear ion trap-orbitrap allow circumventing this limitation. Namely, PQD and HCD facilitate iTRAQ analysis on these instrument types. Here we report a method for iTRAQ-based relative quantification on the ETD enabled LTQ Orbitrap XL, which is based on parallel peptide quantification and peptide identification. iTRAQ reporter ion generation is performed by HCD, while CID and ETD provide peptide identification data in parallel in the LTQ ion trap. This approach circumvents problems accompanying iTRAQ reporter ion generation with ETD and allows quantitative, decision tree-based CID/ETD experiments. Furthermore, the use of HCD solely for iTRAQ reporter ion read out significantly reduces the number of ions needed to obtain informative spectra, which significantly reduces the analysis time. Finally, we show that integration of this method, both with existing CID and ETD methods as well as with existing iTRAQ data analysis workflows, is simple to realize. By applying our approach to the analysis of the synapse proteome from human brain biopsies, we demonstrate that it outperforms a latest generation MALDI TOF/TOF instrument, with improvements in both peptide and protein identification and quantification. Conclusively, our work shows how HCD, CID and ETD can be beneficially combined to enable iTRAQ-based quantification on an ETD-enabled LTQ Orbitrap XL.

  4. 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.

  5. 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.

  6. 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.

  7. Preparation of 18F-labeled peptides using the copper(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition.

    PubMed

    Gill, Herman S; Marik, Jan

    2011-10-13

    An optimized procedure for preparing fluorine-18 ((18)F)-labeled peptides by the copper-catalyzed azide-alkyne 1,3-dipolar cyloaddition (CuAAC) is presented here. The two-step radiosynthesis begins with the microwave-assisted nucleophilic (18)F-fluorination of a precursor containing a terminal p-toluenesulfonyl, terminal azide and polyethylene glycol backbone. The resulting (18)F-fluorinated azide-containing building block is coupled to an alkyne-decorated peptide by the CuAAC. The reaction is accelerated by the copper(I)-stabilizing ligand bathophenanthroline disulfonate and can be performed in either reducing or nonreducing conditions (e.g., to preserve disulfide bonds). After an HPLC purification, (18)F-labeled peptide can be obtained with a 31 ± 6% radiochemical yield (n = 4, decay-corrected from (18)F-fluoride elution) and a specific activity of 39.0 ± 12.4 Ci μmol(-1) within 77 ± 4 min.

  8. Melanoma targeting with [(99m)Tc(N)(PNP3)]-labeled α-melanocyte stimulating hormone peptide analogs: Effects of cyclization on the radiopharmaceutical properties.

    PubMed

    Carta, Davide; Salvarese, Nicola; Morellato, Nicolò; Gao, Feng; Sihver, Wiebke; Pietzsch, Hans Jurgen; Biondi, Barbara; Ruzza, Paolo; Refosco, Fiorenzo; Carpanese, Debora; Rosato, Antonio; Bolzati, Cristina

    2016-12-01

    The purpose of this study was to evaluate the effect of cyclization on the biological profile of a [(99m)Tc(N)(PNP3)]-labeled α-melanocyte stimulating hormone peptide analog. A lactam bridge-cyclized H-Cys-Ahx-βAla(3)-c[Lys(4)-Glu-His-D-Phe-Arg-Trp-Glu(10)]-Arg(11)-Pro-Val-NH2 (NAP-NS2) and the corresponding linear H-Cys-Ahx-βAla-Nle-Asp-His-D-Phe-Arg-Trp-Gly-NH2 (NAP-NS1) peptide were synthetized, characterized by ESI-MS spectroscopy and their melanocortin-1 receptor (MC1R) binding affinity was determined in B16/F10 melanoma cells. The consistent [(99m)Tc(N)(PNP3)]-labeled compounds were readily obtained in high specific activity and their stability and biological properties were assessed. As an example, the chemical identity of [(99m)Tc(N)(NAP-NS1)(PNP3)](+) was confirmed by carrier added experiments supported by radio/UV HPLC analysis combined with ESI(+)-MS. Compared with the linear peptide, cyclization negatively affected the biological properties of NAP-NS2 peptide by reducing its binding affinity for MC1R and by decreasing the overall excretion rate of the corresponding [(99m)Tc(N)(PNP3)]-labeled peptide from the body as well as its in vivo stability. [(99m)Tc(N)(NAP-NS1)(PNP3)](+) was evaluated for its potential as melanoma imaging probe in murine melanoma model. Data from in vitro and in vivo studies on B16/F10 melanoma model of [(99m)Tc(N)(NAP-NS1)(PNP3)](+) clearly evidenced that the radiolabeled linear peptide keeps its biological properties up on the conjugation to the [(99m)Tc(N)(PNP3)]-building block. The progressive increase of the tumor-to-nontarget ratios over the time indicates a quite stable interaction between the radio-complex and the MC1R.

  9. 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

  10. A paired ions scoring algorithm based on Morpheus for simultaneous identification and quantification of proteome samples prepared by isobaric peptide termini labeling strategies.

    PubMed

    Zhang, Shen; Wu, Qi; Shan, Yichu; Sui, Zhigang; Zhang, Lihua; Zhang, Yukui

    2015-06-01

    The isobaric peptide termini labeling (IPTL) method is a promising strategy in quantitative proteomics for its high accuracy, while the increased complexity of MS2 spectra originated from the paired b, y ions has adverse effect on the identification and the coverage of quantification. Here, a paired ions scoring algorithm (PISA) based on Morpheus, a database searching algorithm specifically designed for high-resolution MS2 spectra, was proposed to address this issue. PISA was first tested on two 1:1 mixed IPTL datasets, and increases in peptide to spectrum matchings, distinct peptides and protein groups compared to Morpheus itself and MASCOT were shown. Furthermore, the quantification is simultaneously performed and 100% quantification coverage is achieved by PISA since each of the identified peptide to spectrum matchings has several pairs of fragment ions which could be used for quantification. Then the PISA was applied to the relative quantification of human hepatocellular carcinoma cell lines with high and low metastatic potentials prepared by an IPTL strategy.

  11. [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.

  12. 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

  13. N-formyl peptide receptors in human neutrophils display distinct membrane distribution and lateral mobility when labeled with agonist and antagonist

    PubMed Central

    1993-01-01

    Receptors for bacterial N-formyl peptides are instrumental for neutrophil chemotactic locomotion and activation at sites of infection. As regulatory mechanisms for signal transduction, both rapid coupling of the occupied receptor to cytoskeletal components, and receptor lateral redistribution, have been suggested (Jesaitis et al., 1986, 1989). To compare the distribution and lateral diffusion of the nonactivated and activated neutrophil N-formyl-peptide receptor, before internalization, we used a new fluorescent N-formyl-peptide receptor antagonist, tertbutyloxycarbonyl-Phe(D)-Leu-Phe(D)-Leu-Phe-OH (Boc- FLFLF, 0.1-1 microM), and the fluorescent receptor agonist formyl-Nle- Leu-Phe-Nle-Tyr-Lys (fnLLFnLYK, 0.1-1 microM). Fluorescent Boc-FLFLF did not elicit an oxidative burst in the neutrophil at 37 degrees C, as assessed by chemiluminescence and reduction of p-nitroblue tetrazolium chloride, but competed efficiently both with formyl-methionyl-leucyl- phenylalanine (fMLF) and fnLLFnLYK. It was not internalized, as evidenced by confocal microscopy and acid elution of surface bound ligand. The lateral mobility characteristics of the neutrophil fMLF receptor were investigated with the technique of FRAP. The diffusion coefficient (D) was similar for antagonist- and agonist-labeled receptors (D approximately 5 x 10(-10) cm2/s), but the fraction of mobile receptors was significantly lower in agonist- compared to antagonist-labeled cells, approximately 40% in contrast to approximately 60%. This reduction in receptor mobile fraction was slightly counteracted, albeit not significantly, by dihydrocytochalasin B (dhcB, 5 microM). To block internalization of agonist-labeled receptors, receptor mobility measurements were done at 14 degrees C. At this temperature, confocal microscopy revealed clustering of receptors in response to agonist binding, compared to a more uniform receptor distribution in antagonist-labeled cells. The pattern of agonist- induced receptor clustering was

  14. Hybridization-modulated ion fluxes through peptide-nucleic-acid- functionalized gold nanotubes. A new approach to quantitative label-free DNA analysis.

    PubMed

    Jágerszki, Gyula; Gyurcsányi, Róbert E; Höfler, Lajos; Pretsch, Ernö

    2007-06-01

    The inner walls of gold nanotubes, prepared by template synthesis in the nanopores of polycarbonate track etch membranes, have been chemically modified with peptide nucleic acid (PNA) and used for label-free quantification of complementary DNA sequences. Selective binding of DNA to the PNA-modified nanotubes is shown to decrease the flux of optically detected anionic markers through the nanotubes in a concentration-dependent manner. The strong dependence of the biorecognition-modulated ion transport through the nanopores on the ionic strength suggests a dominantly electrostatic exclusion mechanism of the ion flux decrease as a result of DNA binding to the PNA-modified nanopores.

  15. In Vitro Assessment of a Peptide Nucleic Acid (PNA) - Peptide Conjugate Labeled With an Auger-Emitting Radionuclide for Prostate Cell Killing

    DTIC Science & Technology

    2005-02-01

    synthesis of a peptide nucleic acid (PNA) that has an Auger-emitter (1-125) incorporated. By design the PNA will bind with mRNA and DNA associated with...bind with cell surface gastrin -releasing peptide receptors and be internalized (3). Binding with mRNA and nuclear DNA specific to the insulin-like...route proposed to prepare 10 is shown in Figure 1 (compounds 1-10). This synthesis began with the preparation of the base-reactive intermediate 5

  16. Effects of amino acids on melanoma targeting and clearance properties of Tc-99m-labeled Arg-X-Asp-conjugated α-melanocyte stimulating hormone peptides.

    PubMed

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

    2013-11-14

    The purpose of this study was to examine the effects of amino acids on melanoma targeting and clearance properties of new (99m)Tc-labeled Arg-X-Asp-conjugated α-melanocyte stimulating hormone (α-MSH) peptides. RSD-Lys-(Arg(11))CCMSH {c[Arg-Ser-Asp-DTyr-Asp]-Lys-Cys-Cys-Glu-His-dPhe-Arg-Trp-Cys-Arg-Pro-Val-NH2}, RNleD-Lys-(Arg(11))CCMSH, RPheD-Lys-(Arg(11))CCMSH, and RdPheD-Lys-(Arg(11))CCMSH peptides were synthesized and evaluated for their melanocortin-1 (MC1) receptor binding affinities in B16/F1 melanoma cells. The biodistribution of (99m)Tc-RSD-Lys-(Arg(11))CCMSH, (99m)Tc-RFD-Lys-(Arg(11))CCMSH, and (99m)Tc-RfD-Lys-(Arg(11))CCMSH were determined in B16/F1 melanoma-bearing C57 mice. The substitution of Gly with Ser, Phe, and dPhe increased the MC1 receptor binding affinities of the peptides, whereas the substitution of Gly with Nle decreased the MC1 receptor binding affinity of the peptide. (99m)Tc-RSD-Lys-(Arg(11))CCMSH exhibited the highest melanoma uptake (18.01 ± 4.22% ID/g) and the lowest kidney and liver uptake among these (99m)Tc-peptides. The B16/F1 melanoma lesions could be clearly visualized by SPECT/CT using (99m)Tc-RSD-Lys-(Arg(11))CCMSH as an imaging probe. It is desirable to reduce the renal uptake of (99m)Tc-RSD-Lys-(Arg(11))CCMSH to facilitate its potential therapeutic application.

  17. “Click” cyclized gallium-68 labeled peptides for molecular imaging and therapy: Synthesis and preliminary in vitro and in vivo evaluation in a melanoma model system

    PubMed Central

    Martin, Molly E.; O'Dorisio, M. Sue; Leverich, Whitney M.; Kloepping, Kyle C.; 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 disufide bond formation. While these techniques have been demonstrated to be effective, orthogonal protection/deprotection steps can limit achievable product yields. 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) represents 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) MC1R to melanoma cells in vitro, high stability in human serum, and produced high contrast PET/CT images of tumor xenografts. Gallium-68 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 radiometals 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. PMID:22918759

  18. Continuous on-chip fluorescence labelling, free-flow isoelectric focusing and marker-free isoelectric point determination of proteins and peptides.

    PubMed

    Herzog, Christin; Poehler, Elisabeth; Peretzki, Andrea J; Borisov, Sergey M; Aigner, Daniel; Mayr, Torsten; Nagl, Stefan

    2016-04-26

    We present a microfluidic platform that contains a micro flow reactor for on-chip biomolecule labelling that is directly followed by a separation bed for continuous free-flow electrophoresis and has an integrated hydrogel-based near-infrared fluorescent pH sensor layer. Using this assembly, labelling of protein and peptide mixtures, their separation via free-flow isoelectric focusing and the determination of the isoelectric point (pI) of the separated products via the integrated sensor layer could be carried out within typically around 5 minutes. Spatially-resolved immobilization of fluidic and sensing structures was carried out via multistep photolithography. The assembly was characterized and optimized with respect to their fluidic and pH sensing properties and applied in the IEF of model proteins, peptides and a tryptic digest from physalaemine. We have therefore realized continuous sample preparation and preparative separation, analyte detection, process observation and analyte assignment capability based on pI on a single platform the size of a microscope slide.

  19. 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.

  20. 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

  1. 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...

  2. High sensitive and selective electrochemical biosensor: Label-free detection of human norovirus using affinity peptide as molecular binder.

    PubMed

    Hwang, Hye Jin; Ryu, Myung Yi; Park, Chan Young; Ahn, Junki; Park, Hyun Gyu; Choi, Changsun; Ha, Sang-Do; Park, Tae Jung; Park, Jong Pil

    2017-01-15

    Norovirus is known as the major cause of highly infection for gastrointestinal tracts. In this study, robust and highly sensitive biosensors for detecting human norovirus by employing a recognition affinity peptide-based electrochemical platform were described. A series of amino acid-substituted and cysteine-incorporated recognition peptides isolated from evolutionary phage display technique was chemically synthesized and immobilized to a gold sensor layer, the detection performance of the gold-immobilized synthetic peptide-based sensor system was assessed using QCM, CV and EIS. Using EIS, the limit of detection with Noro-1 as a molecular binder was found to be 99.8nM for recombinant noroviral capsid proteins (rP2) and 7.8copies/mL for human norovirus, thereby demonstrating a high degree of sensitivity for their corresponding targets. These results suggest that a biosensor which consists of affinity peptides as a molecular binder and miniaturized microdevices as diagnostic tool could be served as a new type of biosensing platform for point-of-care testing.

  3. Distance measurements in the borderline region of applicability of CW EPR and DEER: A model study on a homologous series of spin-labelled peptides

    NASA Astrophysics Data System (ADS)

    Banham, J. E.; Baker, C. M.; Ceola, S.; Day, I. J.; Grant, G. H.; Groenen, E. J. J.; Rodgers, C. T.; Jeschke, G.; Timmel, C. R.

    2008-04-01

    Inter-spin distances between 1 nm and 4.5 nm are measured by continuous wave (CW) and pulsed electron paramagnetic resonance (EPR) methods for a series of nitroxide-spin-labelled peptides. The upper distance limit for measuring dipolar coupling by the broadening of the CW spectrum and the lower distance limit for the present optimally-adjusted double electron electron resonance (DEER) set-up are determined and found to be both around 1.6-1.9 nm. The methods for determining distances and corresponding distributions from CW spectral line broadening are reviewed and further developed. Also, the work shows that a correction factor is required for the analysis of inter-spin distances below approximately 2 nm for DEER measurements and this is calculated using the density matrix formalism.

  4. Homogeneous time-resolved fluorescence assays for the detection of activity and inhibition of phosphatase enzymes employing phosphorescently labeled peptide substrates.

    PubMed

    O'Shea, Desmond J; O'Riordan, Tomás C; O'Sullivan, Paul J; Papkovsky, Dmitri B

    2007-02-05

    A rapid, homogenous, antibody-free assay for phosphatase enzymes was developed using the phosphorescent platinum (II)-coproporphyrin label (PtCP) and time-resolved fluorescent detection. An internally quenched decameric peptide substrate containing a phospho-tyrosine residue, labeled with PtCP-maleimide and dabcyl-NHS at its termini was designed. Phosphatase catalysed dephosphorylation of the substrate resulted in a minor increase in PtCP signal, while subsequent cleavage by chymotrypsin at the dephosphorylated Tyr-Leu site provided a 3.5 fold enhancement of PtCP phosphorescence. This phosphorescence phosphatase enhancement assay was optimized to a 96 well plate format with detection on a commercial TR-F plate reader, and applied to measure the activity and inhibition of alkaline phosphatase, recombinant human CD45, and tyrosine phosphatases in Jurkat cell lysates within 40 min. Parameters of these enzymatic reactions such as Km's, limits of detection (L.O.D's) and IC50 values for the non-specific inhibitor sodium orthovanadate were also determined.

  5. 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.

  6. A novel Tc-99 m and fluorescence labeled peptide as a multimodal imaging agent for targeting angiogenesis in a murine tumor model.

    PubMed

    Kim, Myoung Hyoun; Kim, Chang Guhn; Kim, Seul-Gi; Kim, Dae-Weung

    2016-11-01

    The serine-aspartic acid-valine (SDV) peptide binds specifically to integrin αV β3 . In the present study, we successfully developed a TAMRA-GHEG-ECG-SDV peptide labeled with both Tc-99 m and TAMRA to target the integrin αV β3 of tumor cells; furthermore, we evaluated the diagnostic performance of Tc-99 m TAMRA-GHEG-ECG-SDV as a dual-modality imaging agent for tumor of the murine model. TAMRA-GHEG-ECG-SDV was synthesized using Fmoc solid-phase peptide synthesis. Radiolabeling of TAMRA-GHEG-ECG-SDV with Tc-99 m was done using ligand exchange methods. Labeling stability and cytotoxicity studies were performed. Gamma camera imaging, biodistribution and ex vivo imaging studies were performed in murine models with HT-1080 and HT-29 tumors. A tumor tissue slide was prepared and analyzed using confocal microscopy. After radiolabeling procedures with Tc-99 m, the Tc-99 m TAMRA-GHEG-ECG-SDV complexes were prepared in high yield (>99%). In the gamma camera imaging study, a substantial uptake of Tc-99 m TAMRA-GHEG-ECG-SDV into HT-1080 tumor (integrin αV β3 positive) and low uptake of Tc-99 m TAMRA-GHEG-ECG-SDV into HT-29 tumor (integrin αV β3 negative) were demonstrated. A competition study revealed that HT-1080 tumor uptake was effectively blocked by the co-injection of an excess concentration of SDV. Specific uptake of Tc-99 m TAMRA-GHEG-ECG-SDV was confirmed by biodistribution, ex vivo imaging and confocal microscopy studies. Our in vivo and in vitro studies revealed substantial uptake of Tc-99 m TAMRA-GHEG-ECG-SDV in the integrin αV β3 -positive tumor. Tc-99 m TAMRA-GHEG-ECG-SDV could be a good candidate for a dual-modality imaging agent targeting tumor angiogenesis. Copyright © 2016 John Wiley & Sons, Ltd.

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

    PubMed

    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 Å(2) and 446 Å(2); the [M + 3H](3+) ions also yield two different conformer types having collision cross sections of 340 Å(2) and 367 Å(2). 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).

  8. 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).

  9. 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

  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. 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.

  12. Molecular Targeting of Papillary Thyroid Carcinoma With Fluorescently Labeled Ratiometric Activatable Cell Penetrating Peptides in a Transgenic Murine Model

    PubMed Central

    OROSCO, RYAN K.; SAVARIAR, ELAMPRAKASH N.; WEISSBROD, PHILIP A.; DIAZ-PEREZ, JULIO A.; BOUVET, MICHAEL; TSIEN, ROGER Y.; NGUYEN, QUYEN T.

    2016-01-01

    Background and Objectives Molecularly targeted fluorescent molecules may help detect tumors that are unseen by traditional white-light surgical techniques. We sought to evaluate a fluorescent ratiometric activatable cell penetrating peptide (RACPP) for tumor detection in a transgenic model of PTC. Methods Thirteen BRAFV600E mice with PTC were studied—seven injected intravenously with RACPP, four controls with saline. Total thyroidectomy was performed with microscopic white-light visualization. Fluorescent imaging of post-thyroidectomy fields was performed, and tissue with increased signal was removed and evaluated for PTC. Final samples were analyzed by a pathologist blinded to conditions. Vocal cord function was evaluated postoperatively with video laryngoscopy. Results The average in situ ratiometric (Cy5/Cy7) thyroid tumor-to-background contrast ratio was 2.27 +/−0.91. Fluorescence-guided clean-up following thyroidectomy identified additional tumor in 2 of 7 RACPP animals (smallest dimension 1.2 mm), and decreased the number of animals with residual tumor from 4 to 3. All retained tumor foci on final pathology were smaller than 0.76 mm. Intact vocal abduction was present in all of the RACPP animals. Conclusions RACPPs successfully targeted PTC in a transgenic thyroidectomy model, and allowed for residual tumor detection that reduced positive margins beyond what was possible with white-light surgery alone. PMID:26799257

  13. 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.

  14. Construction of a viral T2A-peptide based knock-in mouse model for enhanced Cre recombinase activity and fluorescent labeling of podocytes.

    PubMed

    Koehler, Sybille; Brähler, Sebastian; Braun, Fabian; Hagmann, Henning; Rinschen, Markus M; Späth, Martin R; Höhne, Martin; Wunderlich, F Thomas; Schermer, Bernhard; Benzing, Thomas; Brinkkoetter, Paul T

    2017-02-07

    Podocyte injury is a key event in glomerular disease leading to proteinuria and opening the path toward glomerular scarring. As a consequence, glomerular research strives to discover molecular mechanisms and signaling pathways affecting podocyte health. The hNphs2.Cre mouse model has been a valuable tool to manipulate podocyte-specific genes and to label podocytes for lineage tracing and purification. Here we designed a novel podocyte-specific tricistronic Cre mouse model combining codon improved Cre expression and fluorescent cell labeling with mTomato under the control of the endogenous Nphs2 promoter using viral T2A-peptides. Independent expression of endogenous podocin, codon improved Cre, and mTomato was confirmed by immunofluorescence, fluorescent activated cell sorting and protein analyses. Nphs2(pod.T2A.ciCre.T2A.mTomato/wild-type) mice developed normally and did not show any signs of glomerular disease or off-target effects under basal conditions and in states of disease. Nphs2(pod.T2A.ciCre.T2A.mTomato/wild-type)-mediated gene recombination was superior to conventional hNphs2.Cre mice-mediated gene recombination. Last, we compared Cre efficiency in a disease model by mating Nphs2(pod.T2A.ciCre.T2A.mTomato/wild-type) and hNphs2.Cre mice to Phb2(fl/fl) mice. The podocyte-specific Phb2 knockout by Nphs2(pod.T2A.ciCre.T2A.mTomato/wild-type) mice resulted in an aggravated glomerular injury as compared to a podocyte-specific Phb2 gene deletion triggered by hNphs2.Cre. Thus, we generated the first tricistronic podocyte mouse model combining enhanced Cre recombinase efficiency and fluorescent labeling in podocytes without the need for additional matings with conventional reporter mouse lines.

  15. Absolute quantification of Pru av 2 in sweet cherry fruit by liquid chromatography/tandem mass spectrometry with the use of a stable isotope-labelled peptide.

    PubMed

    Ippoushi, Katsunari; Sasanuma, Motoe; Oike, Hideaki; Kobori, Masuko; Maeda-Yamamoto, Mari

    2016-08-01

    Pru av 2, a pathogenesis-related (PR) protein present in the sweet cherry (Prunus avium L.) fruit, is the principal allergen of cherry and one of the chief causes of pollen food syndrome (oral allergy syndrome). In this study, a quantitative assay for this protein was developed with the use of the protein absolute quantification (AQUA) method, which consists of liquid chromatography/tandem mass spectrometry (LC/MS/MS) employing TGC[CAM]STDASGK[(13)C6,(15)N2], a stable isotope-labelled internal standard (SIIS) peptide. This assay gave a linear relationship (r(2)>0.99) in a concentration range (2.3-600fmol/μL), and the overall coefficient of variation (CV) for multiple tests was 14.6%. Thus, the contents of this allergenic protein in sweet cherry products could be determined using this assay. This assay should be valuable for allergological investigations of Pru av 2 in sweet cherry and detection of protein contamination in foods.

  16. Differential peptide labeling (iTRAQ) in LC-MS/MS based proteomics in Daphnia reveal mechanisms of an antipredator response.

    PubMed

    Effertz, Christoph; Müller, Stefan; Elert, Eric von

    2015-02-06

    Daphnia, an important model organism for studies on ecology and evolution, has become a textbook example for inducible defenses against predators. Inducible defenses are widespread in nature, and the underlying molecular mechanisms for this plasticity in general and in particular in Daphnia are not fully understood. Here, we provide for the first time a combination of established life-history changes (LHC), which are induced by chemical cues of a predator (fish kairomones), in Daphnia with differential peptide labeling (iTRAQ) in LC-MS/MS based proteomics. The aim of the present study is the elucidation of proteins involved in specific antipredator responses in a predator-prey system of ecological relevance by high-throughput proteomics. To obtain a highly specific antifish response of Daphnia, highly purified fish kairomones were applied in the presence or absence of light. We were able to identify a set of functional proteins, which are likely to explain the kairomone-mediated and light-dependent LHC in Daphnia.

  17. In vivo pharmacokinetic analysis for fluorescently labeled RGD peptide targeted to the αvβ3 integrin in Kaposi"s sarcoma

    NASA Astrophysics Data System (ADS)

    Kwon, Sunkuk; Ke, Shi; Houston, Jessica P.; Wang, Wei; Wu, Qingping; Li, Chun; Sevick Muraca, Eva M.

    2005-04-01

    The dose dependence of near-infrared (NIR) fluorescent labeled RGD peptide targeted to the αvβ3 integrin was assessed from xenografts bearing a subcutaneous human Kaposi"s sarcoma (KS1767) with dynamic NIR fluorescence optical imaging. The three-compartment pharmacokinetic (PK) model was used to determine PK parameters from fluorescence images acquired with an intensified charge-coupled device (ICCD) system. Dynamic imaging of Kaposi"s sarcoma bearing animals was conducted with i.v. administration of Cy5.5-c(KRGDf) at doses of 0.75 to 6 nmol/animal and at the doses of 300 or 600 nmol of c(KRGDf) administered 1 hour before the injection of 3 nmol dose of the conjugate. The results show early and rapid uptake of Cy5.5-c(KRGDf), which was mediated by the administration of c(KRGDf) 1 hour before administration at the conjugate agent. From the results we found a linear increase in PK uptake rates at doses of 0.75 to 1.5 nmol, reflecting unsaturated binding to the integrin receptor. However, the results show the dose independence at large dose amounts from 3 to 6 nmol per animal. The effects of cancer treatments as well as diagnostics may be evaluated by in vivo PK analysis with NIR fluorescence optical imaging.

  18. 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

  19. 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

  20. 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.

  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.

  2. Radioiodinated Exendin-4 Is Superior to the Radiometal-Labelled Glucagon-Like Peptide-1 Receptor Probes Overcoming Their High Kidney Uptake

    PubMed Central

    Läppchen, Tilman; Tönnesmann, Roswitha; Eersels, Jos; Meyer, Philipp T.; Maecke, Helmut R.; Rylova, Svetlana N.

    2017-01-01

    GLP-1 receptors are ideal targets for preoperative imaging of benign insulinoma and for quantifying the beta cell mass. The existing clinical tracers targeting GLP-1R are all agonists with low specific activity and very high kidney uptake. In order to solve those issues we evaluated GLP-1R agonist Ex-4 and antagonist Ex(9–39) radioiodinated at Tyr40 side by side with [Nle14,Lys40(Ahx-DOTA-68Ga)NH2]Ex-4 (68Ga-Ex-4) used in the clinic. The Kd, Bmax, internalization and binding kinetics of [Nle14,125I-Tyr40-NH2]Ex-4 and [Nle14,125I-Tyr40-NH2]Ex(9–39) were studied in vitro using Ins-1E cells. Biodistribution and imaging studies were performed in nude mice bearing Ins-1E xenografts. In vitro evaluation demonstrated high affinity binding of the [Nle14,125I-Tyr40-NH2]Ex-4 agonist to the Ins-1E cells with fast internalization kinetics reaching a plateau after 30 min. The antagonist [Nle14,125I-Tyr40-NH2]Ex(9–39) did not internalize and had a 4–fold higher Kd value compared to the agonist. In contrast to [Nle14,125I-Tyr40-NH2]Ex(9–39), which showed low and transient tumor uptake, [Nle14,125I-Tyr40-NH2]Ex-4 demonstrated excellent in vivo binding properties with tumor uptake identical to that of 68Ga-Ex-4, but substantially lower kidney uptake resulting in a tumor-to-kidney ratio of 9.7 at 1 h compared to 0.3 with 68Ga-Ex-4. Accumulation of activity in thyroid and stomach for both peptides, which was effectively blocked by irenat, confirms that in vivo deiodination is the mechanism behind the low kidney retention of iodinated peptides. The 124I congener of [Nle14,125I-Tyr40-NH2]Ex-4 demonstrated a similar favourable biodistribution profile in the PET imaging studies in contrast to the typical biodistribution pattern of [Nle14,Lys40(Ahx-DOTA-68Ga)NH2]Ex-4. Our results demonstrate that iodinated Ex-4 is a very promising tracer for imaging of benign insulinomas. It solves the problem of high kidney uptake of the radiometal-labelled tracers by improving the tumor

  3. Evaluation of new Tc-99m-labeled Arg-X-Asp-conjugated α-melanocyte stimulating hormone peptides for melanoma imaging.

    PubMed

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

    2013-09-03

    The purpose of this study was to examine the melanoma targeting and imaging properties of two new (99m)Tc-labeled Arg-X-Asp-conjugated α-melanocyte stimulating hormone (α-MSH) peptides. RTD-Lys-(Arg(11))CCMSH {c[Asp-Arg-Thr-Asp-DTyr]-Lys-Cys-Cys-Glu-His-DPhe-Arg-Trp-Cys-Arg-Pro-Val-NH2} and RVD-Lys-(Arg(11))CCMSH peptides were synthesized, and their melanocortin-1 (MC1) receptor binding affinities were determined in B16/F1 melanoma cells. The biodistribution and melanoma imaging properties of (99m)Tc-RTD-Lys-(Arg(11))CCMSH and (99m)Tc-RVD-Lys-(Arg(11))CCMSH were determined in B16/F1 melanoma-bearing C57 mice. The IC50 values of RTD-Lys-(Arg(11))CCMSH and RVD-Lys-(Arg(11))CCMSH were 0.7 ± 0.07 and 1.0 ± 0.3 nM in B16/F1 melanoma cells. Both (99m)Tc-RTD-Lys-(Arg(11))CCMSH and (99m)Tc-RVD-Lys-(Arg(11))CCMSH displayed high melanoma uptake. (99m)Tc-RTD-Lys-(Arg(11))CCMSH exhibited the highest tumor uptake of 18.77 ± 5.13% ID/g at 2 h postinjection, whereas (99m)Tc-RVD-Lys-(Arg(11))CCMSH reached the highest tumor uptake of 19.63 ± 4.68% ID/g at 4 h postinjection. Both (99m)Tc-RTD-Lys-(Arg(11))CCMSH and (99m)Tc-RVD-Lys-(Arg(11))CCMSH showed low accumulation in normal organs (<1.7% ID/g) except for the kidneys at 2 h postinjection. The renal uptake of (99m)Tc-RTD-Lys-(Arg(11))CCMSH and (99m)Tc-RVD-Lys-(Arg(11))CCMSH was 135.14 ± 23.62 and 94.01 ± 18.31% ID/g at 2 h postinjection, respectively. The melanoma lesions were clearly visualized by single-photon emission computed tomography (SPECT)/CT using either (99m)Tc-RTD-Lys-(Arg(11))CCMSH or (99m)Tc-RVD-Lys-(Arg(11))CCMSH as an imaging probe at 2 h postinjection. Overall, the introduction of Thr or Val residue retained high melanoma uptake of (99m)Tc-RTD-Lys-(Arg(11))CCMSH and (99m)Tc-RVD-Lys-(Arg(11))CCMSH. However, high renal uptake of (99m)Tc-RTD-Lys-(Arg(11))CCMSH and (99m)Tc-RVD-Lys-(Arg(11))CCMSH need to be reduced to facilitate their future applications.

  4. Impact of Multiple Negative Charges on Blood Clearance and Biodistribution Characteristics of 99mTc-Labeled Dimeric Cyclic RGD Peptides

    PubMed Central

    2015-01-01

    This study sought to evaluate the impact of multiple negative charges on blood clearance kinetics and biodistribution properties of 99mTc-labeled RGD peptide dimers. Bioconjugates HYNIC-P6G-RGD2 and HYNIC-P6D-RGD2 were prepared by reacting P6G-RGD2 and P6D-RGD2, respectively, with excess HYNIC-OSu in the presence of diisopropylethylamine. Their IC50 values were determined to be 31 ± 5 and 41 ± 6 nM, respectively, against 125I-echistatin bound to U87MG glioma cells in a whole-cell displacement assay. Complexes [99mTc(HYNIC-P6G-RGD2)(tricine)(TPPTS)] (99mTc-P6G-RGD2) and [99mTc(HYNIC-P6D-RGD2)(tricine)(TPPTS)] (99mTc-P6D-RGD2) were prepared in high radiochemical purity (RCP > 95%) and specific activity (37–110 GBq/μmol). They were evaluated in athymic nude mice bearing U87MG glioma xenografts for their biodistribution. The most significant difference between 99mTc-P6D-RGD2 and 99mTc-P6G-RGD2 was their blood radioactivity levels and tumor uptake. The initial blood radioactivity level for 99mTc-P6D-RGD2 (4.71 ± 1.00%ID/g) was ∼5× higher than that of 99mTc-P6G-RGD2 (0.88 ± 0.05%ID/g), but this difference disappeared at 60 min p.i. 99mTc-P6D-RGD2 had much lower tumor uptake (2.20–3.11%ID/g) than 99mTc-P6G-RGD2 (7.82–9.27%ID/g) over a 2 h period. Since HYNIC-P6D-RGD2 and HYNIC-P6G-RGD2 shared a similar integrin αvβ3 binding affinity (41 ± 6 nM versus 31 ± 5 nM), the difference in their blood activity and tumor uptake is most likely related to the nine negative charges and high protein binding of 99mTc-P6D-RGD2. Despite its low uptake in U87MG tumors, the tumor uptake of 99mTc-P6D-RGD2 was integrin αvβ3-specific. SPECT/CT studies were performed using 99mTc-P6G-RGD2 in athymic nude mice bearing U87MG glioma and MDA-MB-231 breast cancer xenografts. The SPECT/CT data demonstrated the tumor-targeting capability of 99mTc-P6G-RGD2, and its tumor uptake depends on the integrin αvβ3 expression levels on tumor cells and neovasculature. It was concluded that

  5. 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.

  6. 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.

  7. 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

  8. Effectiveness of quenchers to reduce radiolysis of (111)In- or (177)Lu-labelled methionine-containing regulatory peptides. Maintaining radiochemical purity as measured by HPLC.

    PubMed

    de Blois, Erik; Chan, Ho Sze; Konijnenberg, Mark; de Zanger, Rory; Breeman, Wouter A P

    2012-01-01

    An overview how to measure and to quantify radiolysis by the addition of quenchers and to maintain Radio-Chemical Purity (RCP) of vulnerable methionine-containing regulatory peptides is presented. High RCP was only achieved with a combination of quenchers. However, quantification of RCP is not standardized, and therefore comparison of radiolabelling and RCP of regulatory peptides between different HPLC-systems and between laboratories is cumbersome. Therefore we suggest a set of standardized requirements to quantify RCP by HPLC for radiolabelled DTPA- or DOTA-peptides. Moreover, a dosimetry model was developed to calculate the doses in the reaction vials during radiolabelling and storage of the radiopeptides, and to predict RCP in the presence and absence of quenchers. RCP was measured by HPLC, and a relation between radiation dose and radiolysis of RCP was established. The here described quenchers are tested individually as ƒ(concentration) to investigate efficacy to reduce radiolysis of radiolabelled methionine-containing regulatory peptides.

  9. 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.

  10. 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.

  11. Using infrared spectroscopy of a nitrile labeled phenylalanine and tryptophan fluorescence to probe the α-MSH peptide's side-chain interactions with a micelle model membrane

    NASA Astrophysics Data System (ADS)

    Gonzalez, Javier D.; Levonyak, Nicholas S.; Schneider, Sydney C.; Smith, Matthew J.; Cremeens, Matthew E.

    2014-01-01

    The interactions of α-MSH (Ac-SYSMEHFRWGKPV-NH2) side-chains were biophysically characterized with a micelle model membrane and in model intracellular bacterial conditions using infrared (IR) spectroscopy of a nitrile labeled α-MSH analogue, circular dichroism (CD), and tryptophan fluorescence. Local changes detected by the tryptophan and a nitrile-labeled phenylalanine using fluorescence and infrared spectroscopies, respectively, suggest that the Trp9 side-chain in the conserved core (HisPheArgTrp) of α-MSH is buried in an SDS micellar environment, while Phe(CN)7 does not appear to be buried.

  12. 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.

    PubMed

    Herndon, C S; Hartman, F C

    1984-03-10

    A new affinity label for ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum, 2-(4-bromoacetamido)anilino-2-deoxypentitol 1,5-bisphosphate, has been prepared, Reductive amination of ribulose-P2 with p-phenylenediamine in the presence of sodium cyanoborohydride yielded an epimeric mixture which was resolved by chromatography on quaternary aminoethyl-Sephadex. 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 Ki values of 705 and 104 microM, 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 CO2 and Mg2+, however, resulted in an irreversible, time-dependent loss of activity, with a Kinact of 125 microM and a minimal half-time of 7.3 min. Covalent incorporation of [14C]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 [14C]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. Automated Edman degradation established the sequence as (sequence in text) which is located near the NH2 terminus

  13. 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.

  14. Local injection of the 90Y-labelled peptidic vector DOTATOC to control gliomas of WHO grades II and III: an extended pilot study.

    PubMed

    Schumacher, T; Hofer, S; Eichhorn, K; Wasner, M; Zimmerer, S; Freitag, P; Probst, A; Gratzl, O; Reubi, J-C; Maecke, R; Mueller-Brand, J; Merlo, A

    2002-04-01

    We have previously presented preliminary observations on targeting somatostatin receptor-positive malignant gliomas of all grades by local injection of the radiolabelled peptidic vector 90Y-DOTATOC. We now report on our more thorough clinical experience with this novel compound, focussing on low-grade and anaplastic gliomas. Small peptidic vectors have the potential to target invisible infiltrative disease within normal surrounding brain tissue, thereby opening a window of opportunity for early intervention. Five progressive gliomas of WHO grades II and III and five extensively debulked low-grade gliomas were treated with varying fractions of 90Y-DOTATOC. The vectors were locally injected into the resection cavity or into solid tumour. The activity per single injection ranged from 555 to 1,875 MBq, and the cumulative activity from 555 to 7,030 MBq, according to tumour volumes and eloquence of the affected brain area, yielding dose estimates from 76+/-15 to 312+/-62 Gy. Response was assessed by the clinical status, by steroid dependence and, every 4-6 months, by magnetic resonance imaging and fluorine-18 fluorodeoxyglucose positron emission tomography. In the five progressive gliomas, lasting responses were obtained for at least 13-45 months without the need for steroids. Radiopeptide brachytherapy had been the only modality applied to counter tumour progression. Interestingly, we observed the slow transformation of a solid, primarily inoperable anaplastic astrocytoma into a resectable multi-cystic lesion 2 years after radiopeptide brachytherapy. Based on these observations, we also assessed the feasibility of local radiotherapy following extensive debulking, which was well tolerated. Targeted beta-particle irradiation based on diffusible small peptidic vectors appears to be a promising modality for the treatment of malignant gliomas.

  15. Feasibility of imaging of epidermal growth factor receptor expression with ZEGFR:2377 affibody molecule labeled with 99mTc using a peptide-based cysteine-containing chelator

    PubMed Central

    Andersson, Ken G.; Oroujeni, Maryam; Garousi, Javad; Mitran, Bogdan; Ståhl, Stefan; Orlova, Anna; Löfblom, John; Tolmachev, Vladimir

    2016-01-01

    The epidermal growth factor receptor (EGFR) is overexpressed in a number of malignant tumors and is a molecular target for several specific anticancer antibodies and tyrosine kinase inhibitors. The overexpression of EGFR is a predictive biomarker for response to several therapy regimens. Radionuclide molecular imaging might enable detection of EGFR overexpression by a non-invasive procedure and could be used repeatedly. Affibody molecules are engineered scaffold proteins, which could be selected to have a high affinity and selectivity to predetermined targets. The anti-EGFR ZEGFR:2377 affibody molecule is a potential imaging probe for EGFR detection. The use of the generator-produced radionuclide 99mTc should facilitate clinical translation of an imaging probe due to its low price, availability and favorable dosimetry of the radionuclide. In the present study, we evaluated feasibility of ZEGFR:2377 labeling with 99mTc using a peptide-based cysteine-containing chelator expressed at the C-terminus of ZEGFR:2377. The label was stable in vitro under cysteine challenge. In addition, 99mTc-ZEGFR:2377 was capable of specific binding to EGFR-expressing cells with high affinity (274 pM). Studies in BALB/C nu/nu mice bearing A431 xenografts demonstrated that 99mTc-ZEGFR:2377 accumulates in tumors in an EGFR-specific manner. The tumor uptake values were 3.6±1 and 2.5±0.4% ID/g at 3 and 24 h after injection, respectively. The corresponding tumor-to-blood ratios were 1.8±0.4 and 8±3. The xenografts were clearly visualized at both time-points. This study demonstrated the potential of 99mTc-labeled ZEGFR:2377 for imaging of EGFR in vivo. PMID:27748899

  16. High-level secretion and very efficient isotopic labeling of tick anticoagulant peptide (TAP) expressed in the methylotrophic yeast, Pichia pastoris.

    PubMed

    Laroche, Y; Storme, V; De Meutter, J; Messens, J; Lauwereys, M

    1994-11-01

    Tick anticoagulant peptide (TAP) is a potent and specific inhibitor of the blood coagulation protease Factor Xa. We designed and assembled a synthetic TAP-encoding gene (tapo) based on codons preferentially observed in the highly expressed Pichia pastoris alcohol oxidase 1 gene (AOX1), and fused it to a novel hybrid secretory prepro leader sequence. Expression from this gene yielded biologically active rTAP, which was correctly processed at the amino-terminal fusion site, and accumulated in the medium to approximately 1.7 g/l. This corresponds to a molar concentration of 0.24 mM, and is the highest yet described for a recombinant product secreted from P. pastoris. It also represents a seven-fold improvement in productivity compared to rTAP secretion from Saccharomyces cerevisiae, making P. pastoris an attractive host for the industrial-scale production of this potential therapeutic agent. This system was also used to prepare 21 mg 15N-rTAP, 11 mg 13C-rTAP and 27 mg 15N/13C-rTAP, with isotope incorporation levels higher than 98%, and purities sufficient to allow their use in determining the solution structure of the tick anticoagulant peptide using high field NMR.

  17. Synthesis and evaluation of bombesin derivatives on the basis of pan-bombesin peptides labeled with indium-111, lutetium-177, and yttrium-90 for targeting bombesin receptor-expressing tumors.

    PubMed

    Zhang, Hanwen; Chen, Jianhua; Waldherr, Christian; Hinni, Karin; Waser, Beatrice; Reubi, Jean Claude; Maecke, Helmut R

    2004-09-15

    Bombesin receptors are overexpressed on a variety of human tumors like prostate, breast, and lung cancer. The aim of this study was to develop radiolabeled (Indium-111, Lutetium-177, and Yttrium-90) bombesin analogues with affinity to the three bombesin receptor subtypes for targeted radiotherapy. The following structures were synthesized: diethylenetriaminepentaacetic acid-gamma-aminobutyric acid-[D-Tyr6, beta-Ala11, Thi13, Nle14] bombesin (6-14) (BZH1) and 1,4,7,10-tetraazacyclododecane-N,N',N",N"' -tetraacetic acid-gamma-aminobutyric acid-[D-Tyr6, beta-Ala11, Thi13, Nle14] bombesin (6-14) (BZH2). [111In]-BZH1 and in particular [90Y]-BZH2 were shown to have high affinity to all three human bombesin receptor subtypes with binding affinities in the nanomolar range. In human serum metabolic cleavage was found between beta-Ala11 and His12 with an approximate half-life of 2 hours. The metabolic breakdown was inhibited by EDTA and beta-Ala11-His12 (carnosine) indicating that carnosinase is the active enzyme. Both 111In-labeled peptides were shown to internalize into gastrin-releasing peptide-receptor-positive AR4-2J and PC-3 cells with similar high rates, which were independent of the radiometal. The biodistribution studies of [111In]-BZH1 and [111In]-BZH2 ([177Lu]-BZH2) in AR4-2J tumor-bearing rats showed specific and high uptake in gastrin-releasing peptide-receptor-positive organs and in the AR4-2J tumor. A fast clearance from blood and all of the nontarget organs except the kidneys was found. These radiopeptides were composed of the first pan-bombesin radioligands, which show great promise for the early diagnosis of tumors bearing not only gastrin-releasing peptide-receptors but also the other two bombesin receptor subtypes and may be of use in targeted radiotherapy of these tumors.

  18. 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

  19. Substitution of Gly with Ala enhanced the melanoma uptake of technetium-99m-labeled Arg-Ala-Asp-conjugated alpha-melanocyte stimulating hormone peptide.

    PubMed

    Yang, Jianquan; Miao, Yubin

    2012-02-15

    The purpose of this study was to determine the melanoma targeting property of (99m)Tc-RAD-Lys-(Arg(11))CCMSH in B16/F1 melanoma-bearing C57 mice and compare with (99m)Tc-RGD-Lys-(Arg(11))CCMSH we previously reported. (99m)Tc-RAD-Lys-(Arg(11))CCMSH exhibited rapid and high tumor uptake (19.91±4.02% ID/g at 2h post-injection) in B16/F1 melanoma-bearing C57 mice. The tumor uptake of (99m)Tc-RAD-Lys-(Arg(11))CCMSH was 1.51, 1.34 and 1.43 times the tumor uptake of (99m)Tc-RGD-Lys-(Arg(11))CCMSH at 0.5, 2 and 4h post-injection, respectively. Flank B16/F1 melanoma lesions were clearly imaged at 2h post-injection using (99m)Tc-RAD-Lys-(Arg(11))CCMSH as an imaging probe. The substitution of Gly with Ala significantly enhanced the melanoma uptake of (99m)Tc-RAD-Lys-(Arg(11))CCMSH compared to (99m)Tc-RGD-Lys-(Arg(11))CCMSH in B16/F1 melanoma-bearing C57 mice, providing a new insight into the design of α-MSH peptides for melanoma targeting.

  20. 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.

  1. Replacement of the Lys linker with an Arg linker resulting in improved melanoma uptake and reduced renal uptake of Tc-99m-labeled Arg-Gly-Asp-conjugated alpha-melanocyte stimulating hormone hybrid peptide.

    PubMed

    Yang, Jianquan; Guo, Haixun; Padilla, R Steve; Berwick, Marianne; Miao, Yubin

    2010-09-15

    The purpose of this study was to reduce the non-specific renal uptake of Arg-Gly-Asp (RGD)-conjugated alpha-melanocyte stimulating hormone (alpha-MSH) hybrid peptide through structural modification or L-lysine co-injection. The RGD motif {cyclic(Arg-Gly-Asp-DTyr-Asp)} was coupled to [Cys(3,4,10), D-Phe7, Arg11] alpha-MSH3-13 {(Arg11)CCMSH} through the Arg linker (substituting the Lys linker) to generate a novel RGD-Arg-(Arg11)CCMSH hybrid peptide. The melanoma targeting and pharmacokinetic properties of 99mTc-RGD-Arg-(Arg11)CCMSH were determined in B16/F1 melanoma-bearing C57 mice. The effect of L-lysine co-injection on the renal uptake was determined through the co-injection of L-lysine with 99mTc-RGD-Arg-(Arg11)CCMSH or 99mTc-RGD-Lys-(Arg11)CCMSH. Replacement of the Lys linker with an Arg linker exhibited a profound effect in reducing the non-specific renal uptake of 99mTc-RGD-Arg-(Arg11)CCMSH, as well as increasing the tumor uptake of 99mTc-RGD-Arg-(Arg11)CCMSH compared to 99mTc-RGD-Lys-(Arg11)CCMSH. 99mTc-RGD-Arg-(Arg11)CCMSH exhibited high tumor uptake (21.41+/-3.74% ID/g at 2 h post-injection) and prolonged tumor retention (6.81+/-3.71% ID/g at 24 h post-injection) in B16/F1 melanoma-bearing mice. The renal uptake values of 99mTc-RGD-Arg-(Arg11)CCMSH were 40.14-64.08% of those of 99mTc-RGD-Lys-(Arg11)CCMSH (p<0.05) at 0.5, 2, 4 and 24 h post-injection. Co-injection of L-lysine was effective in decreasing the renal uptakes of 99mTc-RGD-Arg-(Arg11)CCMSH by 27.7% and 99mTc-RGD-Lys-(Arg11)CCMSH by 52.1% at 2 h post-injection. Substitution of the Lys linker with an Arg linker dramatically improved the melanoma uptake and reduced the renal uptake of 99mTc-RGD-Arg-(Arg11)CCMSH, warranting the further evaluation of 188Re-labeled RGD-Arg-(Arg11)CCMSH as a novel MC1 receptor-targeting therapeutic peptide for melanoma treatment in the future.

  2. Experimental pretargeting studies of cancer with a humanized anti-CEA x murine anti-[In-DTPA] bispecific antibody construct and a (99m)Tc-/(188)Re-labeled peptide.

    PubMed

    Karacay, H; McBride, W J; Griffiths, G L; Sharkey, R M; Barbet, J; Hansen, H J; Goldenberg, D M

    2000-01-01

    The aim of this study was to localize (99m)Tc and (188)Re radionuclides to tumors, using a bispecific antibody (bsMAb) in a two-step approach where the radionuclides are attached to novel peptides incorporating moieties recognized by one arm of the bsMAb. A chemically cross-linked human/murine bsMAb, hMN-14 x 734 (Fab' x Fab'), anti-carcinoembryonic antigen [CEA] x anti-indium-DTPA was prepared as a prelude to constructing a fully humanized bsMAb for future clinical application. N,N'-o-Phenylenedimaleimide was used to cross-link the Fab' fragments of the two antibodies at their hinge regions. This construct was shown to be >92% pure and fully reactive with CEA and a divalent (indium)DTPA-peptide. For pretargeting purposes, a peptide, IMP-192 [Ac-Lys(In-DTPA)-Tyr-Lys(In-DTPA)-Lys(TscG-Cys-)-NH(2) ¿TscG = 3-thiosemicarbazonylglyoxyl¿], with two indium-DTPAs and a chelate for selectively binding (99m)Tc or (188)Re, was synthesized. IMP-192 was formulated in a "single dose" kit and later radiolabeled with (99m)Tc (94-99%) at up to 1836 Ci/mmol and with (188)Re (97%) at 459-945 Ci/mmol of peptide. [(99m)Tc]IMP-192 was shown to be stable by extensive in vitro and in vivo testing and had no specific uptake in the tumor with minimal renal uptake. The biodistribution of the hMN-14 x murine 734 bsMAb was compared alone and in a pretargeting setting to a fully murine anti-CEA (F6) x 734 bsMAb that was reported previously [Gautherot, E., Bouhou, J., LeDoussal, J.-M., Manetti, C., Martin, M., Rouvier, E., and Barbet, J. (1997) Therapy for colon carcinoma xenografts with bispecific antibody-targeted, iodine-131-labeled bivalent hapten. Cancer 80 (Suppl.), 2618-2623]. Both bsMAbs maintained their integrity and dual binding specificity in vivo, but the hMN-14 x m734 was cleared more rapidly from the blood. This coincided with an increased uptake of the hMN-14 x m734 bsMAb in the liver and spleen, suggesting an active reticuloendothelial cell recognition mechanism of this mixed

  3. New insights into the coordination of Cu(II) by the amyloid-B 16 peptide from Fourier transform IR spectroscopy and isotopic labeling.

    PubMed

    El Khoury, Youssef; Dorlet, Pierre; Faller, Peter; Hellwig, Petra

    2011-12-15

    Alzheimer's disease is a neurodegenerative disorder in which the formation of amyloid-β (Aβ) aggregates plays a causative role. There is ample evidence that Cu(II) can bind to Aβ and modulate its aggregation. Moreover, Cu(II) bound to Aβ might be involved in the production of reactive oxygen species, a process supposed to be involved in the Alzheimer's disease. The native Aβ40 contains a high affinity binding site for Cu(II), which is comprised in the N-terminal portion. Thus, Aβ16 (amino acid 1-16 of Aβ) has often been used as a model for Cu(II)-binding to monomeric Aβ. The Cu(II)-binding to Aβ is pH dependent and at pH 7.4, two different type of Cu(II) coordinations exist in equilibrium. These two forms are predominant at pH 6.5 and pH 9.0. In either form, a variety of studies show that the N-terminal Asp and the three His play a key role in the coordination, although the exact binding of these amino acids has not been addressed. Therefore, we studied the coordination modes of Cu(II) at pH 6.5 and 9.0 with the help of Fourier transform infrared (FTIR) spectroscopy. Combined with isotopic labeling of the amino acids involved in the coordination sphere, the data points toward the coordination of Cu(II) via the carboxylate of Asp1 at both pH values in a pseudobridging monovalent fashion. At low pH, His6 binds copper via Nτ, while His13 and His14 are bound via Nπ. At high pH, direct evidence is given on the coordination of Cu(II) via the Nτ atom of His6. Additionally, this study clearly shows the effect of Cu(II) binding on the protonation state of the His residues where a proton displacement takes places on the nitrogen atoms of the imidazole ring.

  4. 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.

  5. Investigation of Membrane Peptides by Two-Dimensional Infrared Spectroscopy

    NASA Astrophysics Data System (ADS)

    Blanco, Emily Ann; Zanni, Martin T.

    2009-06-01

    Two-dimensional infrared spectroscopy (2D IR) is a useful tool for studying the structure of membrane peptides. Isotope labeling individual amino acids with 13C=18O decouples the isotope labeled amide I from the other amide I modes in the peptide. Work has been done on both the M2 ion channel and ovispirin antimicrobial peptide, studying the diagonal linewidths of the isotope labeled amide I. The diagonal linewidth of the isotope labeled amide I gives information about the local environment of that residue, which in turn gives structural information about the membrane peptide.

  6. 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.

  7. Food Labeling

    MedlinePlus

    ... in the U.S. have food labels. On every food label you will see Serving size, number of servings, and number of calories per serving Information on the amount of dietary fat, cholesterol, dietary fiber, dietary sodium, carbohydrates, dietary proteins, vitamins, ...

  8. Labeled Antimicrobial Peptides for Detection of Microorganisms

    DTIC Science & Technology

    2008-12-01

    sensitivity experiments. Bacterial spores ( Bacillus subtilis (QM 1611), B. stearothermophilus (ATCC 12980), B. megaterium (QMB 1551), B. atrophaeus (NRRL...Parham et aI., 2003), S. typhimurium (Yu and Bruno, 1996) and Bacillus stereothermophilus (Blake and Weimer, 1997). Although detection of single...Free Cy5 dye does not bind non-specifically to the cells. (B) Binding of 5 ug/ml purified Cy5 CPI and Cy5 SMAP to various Bacillus spp spores at 107 CFU

  9. 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.

  10. Food Labels

    MedlinePlus

    ... the food came from, whether the food is organic, and certain health claims. So who decides what ... make that claim. Foods that are labeled "USDA organic" are required to have at least 95% organic ...

  11. Improved collision-induced dissociation analysis of peptides by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry through 3-sulfobenzoic acid succinimidyl ester labeling.

    PubMed

    Alley, William R; Mechref, Yehia; Klouckova, Iveta; Novotny, Milos V

    2007-01-01

    The sulfonation reagent, a succinimidyl ester of 3-sulfobenzoic acid, has been synthesized for effective peptide sequencing. It is capable of incorporating an additional mobile proton into the peptide backbone, thus, facilitating efficient collision-induced dissociation. This reagent is easily and inexpensively prepared in short time. Tandem mass spectra of the guanidinated and reagent-sulfonated peptides consist mainly of the y-ion series with higher intensities than those observed for solely guanidinated peptides. These enhanced tandem MS attributes significantly improved MASCOT total-ion scores, thus, allowing more confident peptide sequencing. This derivatization was also very effective for the analysis of tryptic digest of human blood serum proteins separated by two-dimensional gel electrophoresis. When used in LC-MALDI/MS/MS format, this type of derivatization does not adversely affect chromatographic efficiencies.

  12. 4-[18F]Fluoro-N-methyl-N-(propyl-2-yn-1-yl)benzenesulfonamide ([18F]F-SA): a versatile building block for labeling of peptides, proteins and oligonucleotides with fluorine-18 via Cu(I)-mediated click chemistry.

    PubMed

    Ramenda, Theres; Steinbach, Jörg; Wuest, Frank

    2013-04-01

    Cu(I)-mediated [3+2]cycloaddition between azides and alkynes has evolved into a valuable bioconjugation tool in radiopharmaceutical chemistry. We have developed a simple, convenient and reliable radiosynthesis of 4-[18F]fluoro-N-methyl-N-(propyl-2-yn-1-yl)benzenesulfonamide ([18F]F-SA) as a novel aromatic sulfonamide-based click chemistry building block. [18F]F-SA could be prepared in a remotely controlled synthesis unit in 32 ± 5% decay-corrected radiochemical yield in a total synthesis time of 80 min. The determined lipophilicity of [18F]F-SA (logP = 1.7) allows handling of the radiotracer in aqueous solutions. The versatility of [18F]F-SA as click chemistry building block was demonstrated by the labeling of a model peptide (phosphopeptide), protein (HSA), and oligonucleotide (L-RNA). The obtained radiochemical yields were 77% (phosphopeptide), 55-60% (HSA), and 25% (L-RNA), respectively. Despite the recent emergence of a multitude of highly innovative novel bioconjugation methods for 18F labeling of biopolymers, Cu(I)-mediated click chemistry with [18F]F-SA represents a reliable, robust and efficient radiolabeling technique for peptides, proteins, and oligonucleotides with the short-lived positron emitter 18F.

  13. 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.

  14. 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...

  15. Protein quantification using a cleavable reporter peptide.

    PubMed

    Duriez, Elodie; Trevisiol, Stephane; Domon, Bruno

    2015-02-06

    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.

  16. Introduction to Pesticide Labels

    EPA Pesticide Factsheets

    Pesticide product labels provide critical information about how to safely and legally handle and use pesticide products. Unlike most other types of product labels, pesticide labels are legally enforceable. Learn about pesticide product labels.

  17. Poly-arginine conjugated triarylmethyl radical as intracellular spin label.

    PubMed

    Driesschaert, Benoit; Bobko, Andrey A; Eubank, Timothy D; Samouilov, Alexandre; Khramtsov, Valery V; Zweier, Jay L

    2016-04-01

    Stable triarylmethyl radicals are ideal spin labels used for biomedical electron paramagnetic resonance applications. Previously reported structures exhibit polar charged functions for water solubilization preventing them from crossing the cell membrane. We report the synthesis of a triarylmethyl radical conjugated to poly-arginine peptide allowing intracellular delivery of the paramagnetic label.

  18. 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.

  19. Förster Resonance Energy Transfer Mediated Photoluminescence Quenching in Stoichiometrically Assembled CdSe/ZnS Quantum Dot-Peptide Labeled Black Hole Quencher Conjugates for Matrix Metalloproteinase-2 Sensing.

    PubMed

    Pillai, Sreenadh Sasidharan; Yukawa, Hiroshi; Onoshima, Daisuke; Biju, Vasudevanpillai; Baba, Yoshinobu

    2017-01-01

    The steady state and time-resolved photoluminescence quenching of streptavidin modified CdSe/ZnS quantum dots (QDs) instigated by biotin-peptide-BHQ-1 (biotin-pep-BHQ-1) molecule was investigated. Here, we have achieved an efficient photoluminescence (PL) quenching of QDs with the conjugation of dark quencher (black hole quencher-BHQ) molecules intermediated with the GPLGVRGK peptide. The luminescence of streptavidin-QDs585 was decreased upon titration with a nano molar concentration of the biotin-GPLGVRGK-BHQ-1 molecule. It has been suggested that the decrease of QDs PL occurred through a Förster resonance energy transfer (FRET) mechanism from the analysis of steady state photoluminescence intensity measurements as well as time resolved lifetime measurements of streptavidin-QDs and QDs-(pep-BHQ-1)n conjugates. The sequence of intermediate peptide GPLG↓VRGK can act as a target material for matrix metalloproteinases-2 (MMP-2) produced by cancer cells at its Gly and Val region, shown by the down-headed arrow. Interestingly, here the reported self-assembled QDs-(pep-BHQ-1)n conjugates could detect the presence MMP-2 at a detection limit of 1 ng/mL with a clear luminescence recovery.

  20. Mechanisms of fragmentation of cationic peptide ions

    NASA Astrophysics Data System (ADS)

    Zhao, Hong; Adams, Jeanette

    1993-06-01

    Fragmentation mechanisms for formation of several commonly occurring product ions in high-energy collision-induced induced decomposition spectra of either (M + Cat2+ - H)+ ions of peptides cationized with alkaline earth metal ions, (M + Ca+)+ ions cationized with alkali metal ions, or (M + H)+ ions are evaluated by using deuterium-labelled peptides. The different sources of hydrogen transferred in the reactions are identified. Our study supports some previously proposed mechanisms but also provides evidence for others.

  1. Anionic phospholipids modulate peptide insertion into membranes.

    PubMed

    Liu, L P; Deber, C M

    1997-05-06

    While the insertion of a hydrophobic peptide or membrane protein segment into the bilayer can be spontaneous and driven mainly by the hydrophobic effect, anionic lipids, which comprise ca. 20% of biological membranes, provide a source of electrostatic attractions for binding of proteins/peptides into membranes. To unravel the interplay of hydrophobicity and electrostatics in the binding of peptides into membranes, we designed peptides de novo which possess the typical sequence Lys-Lys-Ala-Ala-Ala-X-Ala-Ala-Ala-Ala-Ala-X-Ala-Ala-Trp-Ala-Ala-X-Ala-Al a-Ala-Lys-Lys-Lys-Lys-amide, where X residues correspond to "guest" residues which encompass a range of hydrophobicity (Leu, Ile, Gly, and Ser). Circular dichroism spectra demonstrated that peptides were partially (40-90%) random in aqueous buffer but were promoted to form 100% alpha-helical structures by anionic lipid micelles. In neutral lipid micelles, only the relatively hydrophobic peptides (X = L and I) spontaneously adopted the alpha-helical conformation, but when 25% of negatively charged lipids were mixed in to mimic the content of anionic lipids in biomembranes, the less hydrophobic (X = S and G) peptides then formed alpha-helical conformations. Consistent with these findings, fluorescence quenching by the aqueous-phase quencher iodide indicated that in anionic (dimyristoylphosphatidylglycerol) vesicles, the peptide Trp residue was buried in the lipid vesicle hydrophobic core, while in neutral (dimyristoylphosphatidylcholine) vesicles, only hydrophobic (X = L and I) peptides were shielded from the aqueous solution. Trp emission spectra of peptides in the presence of phospholipids doxyl-labeled at the 5-, 7-, 10-, 12-, and 16-fatty acid positions implied not only a transbilayer orientation for inserted peptides but also that mixed peptide populations (transbilayer + surface-associated) may arise. Overall results suggest that for hydrophobic peptides with segmental threshold hydrophobicity below that which

  2. Determining bacteriophage endopeptidase activity using either fluorophore-quencher labeled peptides combined with liquid chromatography-mass spectrometry (LC-MS) or Förster resonance energy transfer (FRET) assays

    PubMed Central

    Molina, Henrik; Fischetti, Vincent A.

    2017-01-01

    The necessity of identifying novel methods to combat infections caused by antibiotic resistant bacteria is increasing each year. Recent advancements in the development of peptidoglycan hydrolases (e.g. lysins) from bacterial viruses (bacteriophages) have revealed the efficiency of this class of enzymes in treating serious bacterial infections. Though promising results have been obtained regarding the lethal action of lysin on bacterial pathogens both in vitro and in vivo, an often-overlooked factor in these studies is precisely identifying their peptidoglycan cleavage site. This knowledge would be useful for following the activity of the enzyme during development, without the need for whole-organism lytic assays. However, more importantly, it would enable the selection of lysins with different cleavage activities that would act synergistically for enhanced efficacy. Here, we have developed two new methods to accurately identify the cleavage site of lysins using liquid chromatography mass spectrometry (LC-MS) on peptidoglycan-like fluorophore-quencher modified synthetic peptides, as well as determining the enzymatic action and kinetics of the enzymes on modified peptides in a Förster resonance energy transfer (FRET) assay. These methods should facilitate progress within the lysin field, accelerating the development of therapeutic lysins to combat antibiotic resistant bacterial infections. PMID:28296948

  3. Combinatorial discovery of tumor targeting peptides using phage display.

    PubMed

    Landon, Linda A; Deutscher, Susan L

    2003-10-15

    Peptides possess appropriate pharmacokinetic properties to serve as cancer imaging or therapeutic targeting agents. Currently, only a small number of rationally-derived, labeled peptide analogues that target only a limited subset of antigens are available. Thus, finding new cancer targeting peptides is a central goal in the field of molecular targeting. Novel tumor-avid peptides can be efficiently identified via affinity selections using complex random peptide libraries containing millions of peptides that are displayed on bacteriophage. In vitro and in situ affinity selections may be used to identify peptides with high affinity for the target antigen in vitro. Unfortunately, it has been found that peptides selected in vitro or in situ may not effectively target tumors in vivo due to poor peptide stability and other problems. To improve in vivo targeting, methodological combinatorial chemistry innovations allow selections to be conducted in the environment of the whole animal. Thus, new targeting peptides with optimal in vivo properties can be selected in vivo in tumor-bearing animals. In vivo selections have been proven successful in identifying peptides that target the vasculature of specific organs. In addition, in vivo selections have identified peptides that bind specifically to the surface of or are internalized into tumor cells. In the future, direct selection of peptides for cancer imaging may be expedited using genetically engineered bacteriophage libraries that encode peptides with intrinsic radiometal-chelation or fluorescent sequences.

  4. 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 ...

  5. 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

  6. Affinity labeling of protein synthesis factors

    SciTech Connect

    Anthony, D.D.; Dever, T.E.; Abramson, R.D.; Lobur, M.; Merrick, W.C.

    1986-05-01

    The authors laboratory is interested in determining those eukaryotic protein synthesis factors which interact with nucleotides and mRNA. To study the binding the authors have used the nucleotides, their analogs, and mRNA analogs as listed below: (1) UV cross-linking with normal (/sup 32/P)XTP; (2) Oxidized GTP; (3) 3'p-azido benzoyl GDP (GTP); (4) 5'p-fluoro sulfonyl benzoyl guanosine; (5) 5'p-fluoro sulfonyl benzoyl adenosine; (6) oxidized mRNA. Currently, they are continuing their efforts to specifically label the proteins, and they are also trying to isolate a single labeled tryptic peptide from the proteins.

  7. 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

  8. Effects of Spin-Labels on Membrane Burial Depth of MARCKS-ED Residues.

    PubMed

    Qi, Yifei; Klauda, Jeffery B; Im, Wonpil

    2016-10-18

    Site-directed spin-labeling electron paramagnetic resonance spectroscopy is a useful tool to obtain information about the environment of specific residues. One of its applications is to investigate membrane protein topology based on the accessibility of the spin label, with the assumption that the position of the spin label in the membrane is close to that of the native residue. This assumption is valid in proteins with well-ordered structures, but could be problematic in small peptides because the labeling may cause a perturbation that is large enough to change local interactions between the peptide and the membrane. To quantitatively characterize such effects, we have simulated the association of a 25-amino-acid peptide, MARCKS-ED, to membranes with and without spin labels. Our simulations show that the depths of spin labels are ∼6-17 Å deeper than the unlabeled charged and polar residues in the wild-type. When the hydrophobic residue Phe is labeled, however, the spin-label depth is close to that of the native residue as well as the experimental value. Our study suggests that one should be cautious in interpretation of spin label data when charged and polar residues in small peptides are labeled.

  9. Narrow-range peptide isoelectric focusing as peptide prefractionation method prior to tandem mass spectrometry analysis.

    PubMed

    Pernemalm, Maria

    2013-01-01

    High sample complexity is one of the major challenges in mass spectrometry-based proteomics today. Despite massive improvement in instrumentation, sample prefractionation is still needed to reduce sample complexity and improve proteome coverage. Isoelectric focusing (IEF) has been traditionally used as a first-dimension protein separation technique in two-dimensional gel electrophoresis-based proteomics. Recently, peptide IEF has emerged as appealing alternative for anion exchange chromatography in multidimensional LC-MS/MS workflows. The rationale behind using narrow-range peptide isoelectric focusing as a prefractionation method prior to ms/ms is to reduce the complexity induced by tryptic digestion. This is done by selectively analyzing a sub-fraction of peptides with an acidic pI. The pI range is chosen as it has previously been shown that 96 % of human proteins have at least one tryptic peptide between pH 3.4 and 4.9. This ensures high proteome coverage while reducing the number of peptides with 2/3. In addition the focusing precision is optimal in this range. Therefore, by analyzing this sub-fraction of peptides the complexity of the sample can be reduced without significant loss of proteome coverage. As the theoretical pI of peptides can be calculated, the pI of the identified peptides can be used to validate the peptide sequence (identified peptides with pI outside the pH range 3.4-4.9 are more likely to be false positives). In addition, this approach is compatible with iTRAQ labelling as the different iTRAQ labels migrate similarly in IEF.

  10. C-Peptide Test

    MedlinePlus

    ... vital for the body to use its main energy source, glucose . Since C-peptide and insulin are produced ... these cases, C-peptide measurement is a useful alternative to testing for insulin. C-peptide measurements can ...

  11. Semiotic labelled deductive systems

    SciTech Connect

    Nossum, R.T.

    1996-12-31

    We review the class of Semiotic Models put forward by Pospelov, as well as the Labelled Deductive Systems developed by Gabbay, and construct an embedding of Semiotic Models into Labelled Deductive Systems.

  12. Soil Fumigant Labels

    EPA Pesticide Factsheets

    The 2012 updated pesticide labels include new safety requirements for buffer zones and related measures. Find labels for each different type of fumigant: chloropicrin, dazomet, dimethyl disulfide, metam sodium/potassium, and methyl bromide.

  13. Electronic Submission of Labels

    EPA Pesticide Factsheets

    Pesticide registrants can provide draft and final labels to EPA electronically for our review as part of the pesticide registration process. The electronic submission of labels by registrants is voluntary but strongly encouraged.

  14. Soil Fumigant Labels - Dazomet

    EPA Pesticide Factsheets

    Updated labels include new safety requirements for buffer zones and related measures. Find information from the Pesticide Product Labeling System (PPLS) for products such as Basamid G, manufactured by Amvac.

  15. 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)

  16. Pesticide Labeling Questions & Answers

    EPA Pesticide Factsheets

    Pesticide manufacturers, applicators, state regulatory agencies, and other stakeholders raise questions or issues about pesticide labels. The questions on this page are those that apply to multiple products or address inconsistencies among product labels.

  17. Soil Fumigant Labels - Chloropicrin

    EPA Pesticide Factsheets

    Search by EPA registration number, product name, or company name, and follow the link to the Pesticide Product Label System (PPLS) for details on each fumigant. Updated labels include new safety requirements for buffer zones and related measures.

  18. Structural determination of larger proteins using stable isotope labeling and NMR spectroscopy

    SciTech Connect

    Unkefer, C.; Hernandez, G.; Springer, P.; Trewhella, J.; Blumenthal, D.; Lidstrom, M.

    1996-04-01

    The project sought to employ stable isotope labeling and NMR spectroscopy to study protein structures and provide insight into important biochemical problems. A methylotrophic bacterial expression system has been developed for uniform deuterium and carbon-13 labeling of proteins for structural studies. These organisms grow using methanol as the sole source of carbon and energy. Because isotopically labeled methanol is relatively inexpensive, the methylotrophs are ideal for expressing proteins labeled uniformly with deuterium and/or carbon-13. This expression system has been employed to prepare deuterated troponin C. NMR spectroscopy measurements have been made on the inhibitory peptide from troponin I (residues 96--115), both as the free peptide and the peptide complexed with deuterated troponin C. Proton-NMR spectroscopy resonance-signal assignments have been made for the free peptide.

  19. Chemical visualization of an attractant peptide, LURE.

    PubMed

    Goto, Hiroaki; Okuda, Satohiro; Mizukami, Akane; Mori, Hitoshi; Sasaki, Narie; Kurihara, Daisuke; Higashiyama, Tetsuya

    2011-01-01

    The pollen tube attractant peptide LUREs of Torenia fournieri are diffusible peptides that attract pollen tubes in vitro. Here, we report a method enabling the direct visualization of a LURE peptide without inhibiting its attraction activity by conjugating it with the Alexa Fluor 488 fluorescent dye. After purifying and refolding the recombinant LURE2 with a polyhistidine tag, its amino groups were targeted for conjugation with the Alexa Fluor dye. Labeling of LURE2 was confirmed by its fluorescence and mass spectrometry. In our in vitro assay using gelatin beads, Alexa Fluor 488-labeled LURE2 appeared to have the same activity as unlabeled LURE2. Using the labeled LURE2, the relationship between the spatiotemporal change of distribution and activity of LURE2 was examined. LURE2 attracted pollen tubes when embedded in gelatin beads, but hardly at all when in agarose beads. Direct visualization suggested that the significant difference between these conditions was the retention of LURE2 in the gelatin bead, which might delay diffusion of LURE2 from the bead. Direct visualization of LURE peptide may open the way to studying the spatiotemporal dynamics of LURE in pollen tube attraction.

  20. Detection of Peptide-Based Nanoparticles in Blood Plasma by ELISA

    PubMed Central

    Bode, Gerard H.; Pickl, Karin E.; Sanchez-Purrà, Maria; Albaiges, Berta; Borrós, Salvador; Pötgens, Andy J. G.; Schmitz, Christoph; Sinner, Frank M.; Losen, Mario; Steinbusch, Harry W. M.; Frank, Hans-Georg; Martinez-Martinez, Pilar

    2015-01-01

    Aims The aim of the current study was to develop a method to detect peptide-linked nanoparticles in blood plasma. Materials & Methods A convenient enzyme linked immunosorbent assay (ELISA) was developed for the detection of peptides functionalized with biotin and fluorescein groups. As a proof of principle, polymerized pentafluorophenyl methacrylate nanoparticles linked to biotin-carboxyfluorescein labeled peptides were intravenously injected in Wistar rats. Serial blood plasma samples were analyzed by ELISA and by liquid chromatography mass spectrometry (LC/MS) technology. Results The ELISA based method for the detection of FITC labeled peptides had a detection limit of 1 ng/mL. We were able to accurately measure peptides bound to pentafluorophenyl methacrylate nanoparticles in blood plasma of rats, and similar results were obtained by LC/MS. Conclusions We detected FITC-labeled peptides on pentafluorophenyl methacrylate nanoparticles after injection in vivo. This method can be extended to detect nanoparticles with different chemical compositions. PMID:25996618

  1. Differential protein labeling based on electrochemically generated reactive intermediates.

    PubMed

    Büter, Lars; Faber, Helene; Wigger, Tina; Vogel, Martin; Karst, Uwe

    2015-10-06

    A specific labeling method for cysteine moieties in proteins was developed. Electrochemical oxidation of phenolic compounds such as phenol or acetaminophen leads to the generation of the reactive intermediates benzoquinone and N-acetyl-p-benzoquinone imine, which can subsequently react with nucleophilic thiol functions in peptides or proteins. Differential labeling of cysteine residues was successfully demonstrated with native as well as heavy-isotope labeled forms of the corresponding labeling compounds. The specific mass differences on the peptide level were successfully analyzed by mass spectrometry for the tripeptide glutathione. Free cysteines in various proteins such as β-lactoglobulin A, human serum albumin, hemoglobin, and human carbonic anhydrase I were successfully labeled. Tryptic digestion of differentially labeled carbonic anhydrase I and hemoglobin allowed the identification of the binding site in the proteins. The obtained mass difference allowed an easy identification of the cysteine containing peptides. With these experiments, it was successfully demonstrated that the developed method can serve as a tool for counting cysteine moieties in proteins and, thus, be used as an additional technique in protein identification experiments.

  2. 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

  3. Multifunctional Prenylated Peptides for Live Cell Analysis

    PubMed Central

    Wollack, James W.; Zeliadt, Nicholette A.; Mullen, Daniel G.; Amundson, Gregg; Geier, Suzanne; Falkum, Stacy; Wattenberg, Elizabeth V.; Barany, George; Distefano, Mark D.

    2009-01-01

    Protein prenylation is a common post-translational modification present in eukaryotic cells. Many key proteins involved in signal transduction pathways are prenylated and inhibition of prenylation can be useful as a therapeutic intervention. While significant progress has been made in understanding protein prenylation in vitro, we have been interested in studying this process in living cells, including the question of where prenylated molecules localize. Here, we describe the synthesis and live cell analysis of a series of fluorescently labeled multifunctional peptides, based on the C-terminus of the naturally prenylated protein CDC42. A synthetic route was developed that features a key Acm to Scm protecting group conversion. This strategy was compatible with acid-sensitive isoprenoid moieties, and allowed incorporation of an appropriate fluorophore as well as a cell-penetrating sequence (penetratin). These peptides are able to enter cells through different mechanisms, depending on the presence or absence of the penetratin vehicle and the nature of the prenyl group attached. Interestingly, prenylated peptides lacking penetratin are able to enter cells freely through an energy-independent process, and localize in a perinuclear fashion. This effect extends to a prenylated peptide that includes a full “CAAX box” sequence (specifically, CVLL). Hence, these peptides open the door for studies of protein prenylation in living cells, including enzymatic processing and intracellular peptide trafficking. Moreover, the synthetic strategy developed here should be useful for the assembly of other types of peptides that contain acid sensitive functionalities. PMID:19425596

  4. Label Review Training: Module 1: Label Basics, Page 22

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. Learn about what labels require review.

  5. Label Review Training: Module 1: Label Basics, Page 27

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. See examples of mandatory and advisory label statements.

  6. Label Review Training: Module 1: Label Basics, Page 21

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. Learn about types of labels.

  7. Label Review Training: Module 1: Label Basics, Page 24

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. This page is about which labels require review.

  8. Label Review Training: Module 1: Label Basics, Page 17

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. See an overview of the importance of labels.

  9. Label Review Training: Module 1: Label Basics, Page 23

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. Lists types of labels that do not require review.

  10. Label Review Training: Module 1: Label Basics, Page 16

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. Learn about the importance of labels and the role in enforcement.

  11. Label Review Training: Module 1: Label Basics, Page 15

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. Learn about the consequences of improper labeling.

  12. Low-energy collision-induced dissociation fragmentation analysis of cysteinyl-modified peptides.

    PubMed

    Borisov, Oleg V; Goshe, Michael B; Conrads, Thomas P; Rakov, V Sergey; Veenstra, Timothy D; Smith, Richard D

    2002-05-15

    The development of methods to chemically modify and isolate cysteinyl-residue-containing peptides (Cys-peptides) for LC-MS/MS analysis has generated considerable interest in the field of proteomics. Methods using isotope-coded affinity tags (ICAT) and (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine (iodoacetyl-PEO-biotin) employ similar Cys-modifying reagents that contain a thiolate-specific biotin group to modify and isolate Cys-containing peptides in conjunction with immobilized avidin. For these strategies to be effective on a proteome-wide level, the presence of the ICAT or acetyl-PEO-biotin tag should not interfere with the efficiency of induced dissociation in MS/MS experiments or with the identification of the modified Cys-peptides by automated database searching algorithms. We have compared the collision-induced dissociation (CID) fragmentation patterns of peptides labeled with iodoacetyl-PEO-biotin and the ICAT reagent to those of the unmodified peptides. CID of Cys-peptides modified with either reagent resulted in the formation of ions attributed to the modified Cys-peptides as well as those unique to the labeling reagent. As demonstrated by analyzing acetyl-PEO-biotin labeled peptides from ribonuclease A and the ICAT-labeled proteome of Deinococcus radiodurans, the presence of these label-specific product ions provides a useful identifier to discern whether a peptide has been modified with the Cys-specific reagent, especially when a number of peptides analyzed using these methods do not contain a modified Cys residue, and to differentiate identical Cys-peptides labeled with either ICAT-d0 or ICAT-d8.

  13. Low-Energy Collision-Induced Dissociation Fragmentation Analysis of Cysteinyl-Modified Peptides

    SciTech Connect

    Borisov, Oleg V.; Goshe, Michael B. ); Conrads, Thomas P. ); Rakov, Vsevolod S. ); Veenstra, Timothy D. ); Smith, Richard D. )

    2002-05-15

    The development of methods to chemically modify and isolate cysteinyl-residue containing peptides (Cys-peptides) for LC-MS/MS analysis has generated considerable interest in the field of proteomics. Methods using isotope-coded affinity tags (ICAT) and (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine (iodoacetyl-PEO-biotin) employ similar Cys-modifying reagents that contain a thiolate-specific biotin group to modify and isolate Cys-containing peptides in conjunction with immobilized avidin. For these strategies to be effective on a proteome-wide level, the presence of the ICAT or acetyl-PEO-biotin tag should not interfere with the efficiency of induced dissociation in MS/MS experiments or with the identification of the modified Cys-peptides by automated database searching algorithms. We have compared the collision-induced dissociation (CID) fragmentation patterns of peptides labeled with iodoacetyl-PEO-biotin and the ICAT reagent to those of the unmodified peptides. CID of Cys-peptides modified with either reagent resulted in the formation of ions attributed to the modified Cys-peptides as well as those unique to the labeling reagent. As demonstrated by analyzing acetyl-PEO-biotin labeled peptides from ribonuclease A and the ICAT-labeled proteome of D. radiodurans, the presence of these labeled-specific product ions provides a useful identifier to discern whether a peptide has been modified with the Cys-specific reagent, especially when a number of peptides analyzed using these methods do not contain a modified Cys-residue, and to differentiate identical Cys-peptides labeled with either ICAT-D0 or ICAT-D8.

  14. Sample Pesticide Label for Label Review Training

    EPA Pesticide Factsheets

    Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human health and the environment.

  15. Pesticide Product Label System

    EPA Pesticide Factsheets

    The Pesticide Product Label System (PPLS) provides a collection of pesticide product labels (Adobe PDF format) that have been approved by EPA under Section 3 of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA). New labels were added to PPLS on November 21, 2014. Pesticide product labels provide critical information about how to safely handle and use registered pesticide products. An approved pesticide product label represents the full content of EPAs registration decision regarding that product. Pesticide labels contain detailed information on the use, storage, and handling of a product. This information will be found on EPA stamped-approved labels and, in some cases, in subsequent related correspondence, which is also included in PPLS. You may need to review several PDF files for a single product to determine the complete current terms of registration.

  16. Selective purification of the thiol peptides of myosin

    PubMed Central

    Weeds, A. G.; Hartley, B. S.

    1968-01-01

    1. A method for selective purification of thiol peptides is described. Thiol groups in a protein are treated with radioactive cystine by disulphide–thiol interchange. The labelled cystine peptides in a digest can then be fractionated for peptide `maps'. Performic acid oxidation of paper strips containing the radioactive peptides followed by further ionophoresis yields the purified cysteic acid peptides. 2. The thiol peptides in a peptic digest of cystine-exchanged myosin were purified in this way, and their amino acid sequences were determined. 3. The conclusion that myosin contains at least 16, and probably between 20 and 22, unique thiol sequences indicates that the molecule consists of two chemically equivalent components. PMID:5660634

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

    DOE PAGES

    Wall, Jonathan S.; Williams, Angela; Wooliver, Craig; ...

    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.

  18. [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.

  19. 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

  20. Peptide nucleic acid probes with charged photocleavable mass markers

    PubMed Central

    Ball, Rachel J; Green, Philip S; Gale, Nittaya; Langley, G John

    2010-01-01

    Halogen-labelled peptide organic acid (HPOA) monomers have been synthesised and incorporated into sequence-specific peptide nucleic acid (PNA) probes. Three different types of probe have been prepared; the unmodified PNA probe, the PNA probe with a mass marker, and the PNA probe with photocleavable mass marker. All three types of probe have been used in model studies to develop a mass spectrometry-based hybridisation assay for detection of point mutations in DNA. PMID:21687524

  1. EBprot: Statistical analysis of labeling-based quantitative proteomics data.

    PubMed

    Koh, Hiromi W L; Swa, Hannah L F; Fermin, Damian; Ler, Siok Ghee; Gunaratne, Jayantha; Choi, Hyungwon

    2015-08-01

    Labeling-based proteomics is a powerful method for detection of differentially expressed proteins (DEPs). The current data analysis platform typically relies on protein-level ratios, which is obtained by summarizing peptide-level ratios for each protein. In shotgun proteomics, however, some proteins are quantified with more peptides than others, and this reproducibility information is not incorporated into the differential expression (DE) analysis. Here, we propose a novel probabilistic framework EBprot that directly models the peptide-protein hierarchy and rewards the proteins with reproducible evidence of DE over multiple peptides. To evaluate its performance with known DE states, we conducted a simulation study to show that the peptide-level analysis of EBprot provides better receiver-operating characteristic and more accurate estimation of the false discovery rates than the methods based on protein-level ratios. We also demonstrate superior classification performance of peptide-level EBprot analysis in a spike-in dataset. To illustrate the wide applicability of EBprot in different experimental designs, we applied EBprot to a dataset for lung cancer subtype analysis with biological replicates and another dataset for time course phosphoproteome analysis of EGF-stimulated HeLa cells with multiplexed labeling. Through these examples, we show that the peptide-level analysis of EBprot is a robust alternative to the existing statistical methods for the DE analysis of labeling-based quantitative datasets. The software suite is freely available on the Sourceforge website http://ebprot.sourceforge.net/. All MS data have been deposited in the ProteomeXchange with identifier PXD001426 (http://proteomecentral.proteomexchange.org/dataset/PXD001426/).

  2. Automated selected reaction monitoring software for accurate label-free protein quantification.

    PubMed

    Teleman, Johan; Karlsson, Christofer; Waldemarson, Sofia; Hansson, Karin; James, Peter; Malmström, Johan; Levander, Fredrik

    2012-07-06

    Selected reaction monitoring (SRM) is a mass spectrometry method with documented ability to quantify proteins accurately and reproducibly using labeled reference peptides. However, the use of labeled reference peptides becomes impractical if large numbers of peptides are targeted and when high flexibility is desired when selecting peptides. We have developed a label-free quantitative SRM workflow that relies on a new automated algorithm, Anubis, for accurate peak detection. Anubis efficiently removes interfering signals from contaminating peptides to estimate the true signal of the targeted peptides. We evaluated the algorithm on a published multisite data set and achieved results in line with manual data analysis. In complex peptide mixtures from whole proteome digests of Streptococcus pyogenes we achieved a technical variability across the entire proteome abundance range of 6.5-19.2%, which was considerably below the total variation across biological samples. Our results show that the label-free SRM workflow with automated data analysis is feasible for large-scale biological studies, opening up new possibilities for quantitative proteomics and systems biology.

  3. Labeling and Delinquency.

    ERIC Educational Resources Information Center

    Adams, Mike S.; Robertson, Craig T.; Gray-Ray, Phyllis; Ray, Melvin C.

    2003-01-01

    Index comprised of six contrasting descriptive adjectives was used to measure incarcerated youths' perceived negative labeling from the perspective of parents, teachers, and peers. Results provided partial support for hypothesis that juveniles who choose a greater number of negative labels will report more frequent delinquent involvement. Labeling…

  4. Government perspective: food labeling.

    PubMed

    Philipson, Tomas

    2005-07-01

    The Food and Drug Administration acknowledges the severity of the obesity epidemic. The Food and Drug Administration recognizes the importance of food labeling as a vehicle for dietary messages and, thus, enforces stringent guidelines to maintain the integrity of the food label. As food labels await another upgrade to make them more effective and easier to understand, the Food and Drug Administration considers what information will be most useful for consumers to make healthy choices. The causal relationship between food labels and subsequent diet choice is not well understood; more research in this area is needed. The Commissioner of the Food and Drug Administration has recently appointed an Obesity Working Group to develop proposals on pertinent topics of obesity, including the role of food labeling as a dietary guide.

  5. Mining Multi-label Data

    NASA Astrophysics Data System (ADS)

    Tsoumakas, Grigorios; Katakis, Ioannis; Vlahavas, Ioannis

    A large body of research in supervised learning deals with the analysis of single-label data, where training examples are associated with a single label λ from a set of disjoint labels L. However, training examples in several application domains are often associated with a set of labels Y ⊆ L. Such data are called multi-label.

  6. Label Review Training: Module 1: Label Basics, Page 29

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. This page is a quiz on Module 1.

  7. Comparative studies of adhesion peptides based on l- or d-amino acids.

    PubMed

    Nikitin, Sergey; Palmer, Daniel; Meldal, Morten; Diness, Frederik

    2016-10-01

    Detailed studies comparing solid-supported l- or d-amino acid adhesion peptides based on the sequence KLHRIRA were performed. Stability towards proteases and levels of cellular adhesion to the otherwise inert surface of PEGA resin were compared by using fluorescently labelled peptides. A clear difference in the peptide stability towards cleavage by subtilisin, trypsin, or papain was observed. However, all of the on-bead peptides provided an optimal surface for cell adhesion and proliferation. In long-term experiments, these properties were still found to be similar on the resins modified either with l- or with d-amino acids and unaffected by the nature of their fluorescence labelling at either terminus. These results support that the more accessible l-amino acids can be utilized for cell adhesion experiments and confirm the nonspecific interaction mechanism of cell binding to these peptides on the bead surface. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

  8. Peptide Antimicrobial Agents

    PubMed Central

    Jenssen, Håvard; Hamill, Pamela; Hancock, Robert E. W.

    2006-01-01

    Antimicrobial host defense peptides are produced by all complex organisms as well as some microbes and have diverse and complex antimicrobial activities. Collectively these peptides demonstrate a broad range of antiviral and antibacterial activities and modes of action, and it is important to distinguish between direct microbicidal and indirect activities against such pathogens. The structural requirements of peptides for antiviral and antibacterial activities are evaluated in light of the diverse set of primary and secondary structures described for host defense peptides. Peptides with antifungal and antiparasitic activities are discussed in less detail, although the broad-spectrum activities of such peptides indicate that they are important host defense molecules. Knowledge regarding the relationship between peptide structure and function as well as their mechanism of action is being applied in the design of antimicrobial peptide variants as potential novel therapeutic agents. PMID:16847082

  9. 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.

  10. 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

  11. PH dependent adhesive peptides

    SciTech Connect

    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.

  12. 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

  13. Off-Label Drug Use

    MedlinePlus

    ... their drugs for off-label uses. Off-label marketing is very different from off-label use. Why ... Employment Become a Supplier Report Fraud or Abuse Global Health ACS CAN Sign up for Email Policies ...

  14. Soil Fumigant Labels - Methyl Bromide

    EPA Pesticide Factsheets

    Search soil fumigant pesticide labels by EPA registration number, product name, or company, and follow the link to The Pesticide Product Label System (PPLS) for details. Updated labels include new safety requirements for buffer zones and related measures.

  15. Gelatin quantification by oxygen-18 labeling and liquid chromatography-high-resolution mass spectrometry.

    PubMed

    Sha, Xiao-Mei; Tu, Zong-Cai; Wang, Hui; Huang, Tao; Duan, Deng-Le; He, Na; Li, De-Jun; Xiao, Hui

    2014-12-10

    Combined with high-performance liquid chromatography (HPLC) and linear-ion trap/Orbitrap high-resolution mass spectrometry, trypsin-catalyzed (16)O-to-(18)O exchange was used to establish an accurate quantitative method for bovine or porcine gelatin. The sophisticated modifications for these two mammalian gelatins were unambiguously identified by accurate mass and tandem mass spectrometry. Eighteen marker peptides were successfully identified for the bovine and porcine gelatin, respectively. The gelatins were subjected to (18)O or (16)O labeling in the presence of trypsin and mixed together in various ratios for quantification. All of the (18)O-labeled peptides were also confirmed by accurate mass and tandem mass spectrometry. The 10 marker peptides with the strongest signals were chosen to calculate the average ratios of (18)O-labeled and (16)O-labeled gelatin. The measured ratios of (18)O-labeled and (16)O-labeled peptides were very close to the mixing ratios of 20:1, 5:1, 1:1, and 1:5 with low standard deviation values. The samples with a mixing ratio of 1:1 (18)O-labeled and (16)O-labeled peptides were determined to 1.00 and 0.99 with standard deviations of 0.02 and 0.04 for bovine and porcine gelatins, respectively, indicating the high accuracy of this method. Trypsin-catalyzed (18)O labeling was proved to be an excellent internal calibrant for gelatins. When combined with HPLC and high-resolution mass spectrometry, it is an accurate and sensitive quantitative method for gelatin in the food industry.

  16. 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.

  17. Capacitive label reader

    DOEpatents

    Arlowe, H.D.

    1985-11-12

    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. 5 figs.

  18. Capacitive label reader

    DOEpatents

    Arlowe, H. Duane

    1985-01-01

    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.

  19. Passive and active fragment ion mass defect labeling: distinct proteomics potential of iodine-based reagents.

    PubMed

    Shi, Yu; Bajrami, Bekim; Yao, Xudong

    2009-08-01

    The exact mass of a peptide differs characteristically from its nominal mass by a value called the mass defect. Limited by possible elemental compositions, the mass defect of peptides has a restricted range, resulting in an unoccupied mass spectral space in every mass-to-charge unit. The method of fragment ion mass defect labeling (FIMDL) places characteristic fragment ions of modified peptides as reporters into unused spectral space where no native peptide fragment ions exist. In this labeling method, peptides are chemically modified in solution and the modified peptides, upon gas-phase collision in a mass spectrometer, generate fragment ions with significantly shifted mass defects. In this work, the efficiency of iodine stable isotope-containing reagents for shifting mass defects of peptide fragment ions was systematically investigated, through derivatization of peptide N-termini with various reagents containing one or more chlorine, bromine, or iodine atoms. The observed efficiency for the iodine atom placing the labeled fragment ions into unoccupied spectral space agreed well with theoretical predictions from averagine-scaling analysis of ion masses. On the basis of the gas-phase stability of different labeling groups and their involvement in collisional dissociation of modified peptides, peptide modifications were classified into three categories: passive, type I active, and type II active. Each modification type has its unique potential in different proteome analyses. Possible proteomics applications of FIMDL are discussed and compared with proteome analyses currently being practiced in the field. Principles obtained from this survey study will provide a guideline in developing novel FIMDL reagents for advanced proteomics analysis.

  20. 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

  1. Postdiffusion of oligo-peptide within exponential growth multilayer films for localized peptide delivery.

    PubMed

    Wang, Xuefei; Ji, Jian

    2009-10-06

    The multilayers of poly(L-lysine) (PLL) and hyaluronic acid (HA) were constructed by alternating deposition of PLL at high pH and HA at low pH. The exponential growth of the multilayer was proved to be amplified by increasing the pH difference between the two deposition solutions. The exponential growth multilayers of PLL/HA assembled at different pH were utilized as reservoirs for loading a trans-activating transcriptional factor (TAT) peptide. The confocal laser scanning microscopy (CLSM) results indicated that the FITC-labeled TAT could diffuse throughout the exponentially growing PLL/HA film. The amount of peptide embedded within multilayer could be adjusted by both multilayer assembly pH and the TAT loading pH. Compared with (PLL/HA 6.5/6.5)5 multilayer (PLL/HA a/b means that the multilayer film was constructed by using PLL at pH a and HA at pH b), the (PLL/HA 9.5/2.9)5 film can be loaded with more TAT peptide at the same loading pH 6.5. The excess of positively charged TAT peptide within (PLL/HA 9.5/2.9)5 film could not only be ascribed to its extraordinary thickness but also be attributed to its uncompensated negative charge density enhanced by the pH difference between film buildup and peptide loading process. Increasing of the TAT loading pH from 6.5 to 9.5, which increases the pH difference between multilayer assembly and peptide loading process, enhances the uncompensated charge density within (PLL/HA 9.5/2.9)5 film and elevates the peptide density from 13.8 to 25.0 microg/cm2. Compared with direct layer-by-layer assembly of TAT and HA, the postdiffusion of TAT into (PLL/HA 9.5/2.9)5 film was loaded much more peptide. The postdiffusion of peptide into a rapid growth multilayer can be more favorable to load and sustainedly release functional oligo-peptide. The cell culture results indicated that the TAT embedded within the film maintained the ability to traverse across the Hep G2 cell membrane. The functionalized (PLL/HA 9.5/2.9)5 TAT 9.5 film was more

  2. Figuring Out Food Labels

    MedlinePlus

    ... milk dairy products also contribute to cholesterol level. Sodium Sodium, a component of salt, is listed on the Nutrition Facts label in milligrams. Small amounts of sodium are necessary for keeping proper body fluid balance, ...

  3. Label Review Training - Resources

    EPA Pesticide Factsheets

    Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human health and the environment.

  4. 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.

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

    SciTech Connect

    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.

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

    PubMed

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

    2016-03-01

    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.

  7. A switchable stapled peptide.

    PubMed

    Kalistratova, Aleksandra; Legrand, Baptiste; Verdié, Pascal; Naydenova, Emilia; Amblard, Muriel; Martinez, Jean; Subra, Gilles

    2016-03-01

    The O-N acyl transfer reaction has gained significant popularity in peptide and medicinal chemistry. This reaction has been successfully applied to the synthesis of difficult sequence-containing peptides, cyclic peptides, epimerization-free fragment coupling and more recently, to switchable peptide polymers. Herein, we describe a related strategy to facilitate the synthesis and purification of a hydrophobic stapled peptide. The staple consists of a serine linked through an amide bond formed from its carboxylic acid function and the side chain amino group of diaminopropionic acid and through an ester bond formed from its amino group and the side chain carboxylic acid function of aspartic acid. The α-amino group of serine was protonated during purification. Interestingly, when the peptide was placed at physiological pH, the free amino group initiated the O-N shift reducing the staple length by one atom, leading to a more hydrophobic stapled peptide.

  8. Matching isotopic distributions from metabolically labeled samples

    PubMed Central

    McIlwain, Sean; Page, David; Huttlin, Edward L.; Sussman, Michael R.

    2008-01-01

    Motivation: In recent years stable isotopic labeling has become a standard approach for quantitative proteomic analyses. Among the many available isotopic labeling strategies, metabolic labeling is attractive for the excellent internal control it provides. However, analysis of data from metabolic labeling experiments can be complicated because the spacing between labeled and unlabeled forms of each peptide depends on its sequence, and is thus variable from analyte to analyte. As a result, one generally needs to know the sequence of a peptide to identify its matching isotopic distributions in an automated fashion. In some experimental situations it would be necessary or desirable to match pairs of labeled and unlabeled peaks from peptides of unknown sequence. This article addresses this largely overlooked problem in the analysis of quantitative mass spectrometry data by presenting an algorithm that not only identifies isotopic distributions within a mass spectrum, but also annotates matches between natural abundance light isotopic distributions and their metabolically labeled counterparts. This algorithm is designed in two stages: first we annotate the isotopic peaks using a modified version of the IDM algorithm described last year; then we use a probabilistic classifier that is supplemented by dynamic programming to find the metabolically labeled matched isotopic pairs. Such a method is needed for high-throughput quantitative proteomic metabolomic experiments measured via mass spectrometry. Results: The primary result of this article is that the dynamic programming approach performs well given perfect isotopic distribution annotations. Our algorithm achieves a true positive rate of 99% and a false positive rate of 1% using perfect isotopic distribution annotations. When the isotopic distributions are annotated given ‘expert’ selected peaks, the same algorithm gets a true positive rate of 77% and a false positive rate of 1%. Finally, when annotating using

  9. UNiquant, a Program for Quantitative Proteomics Analysis Using Stable Isotope Labeling

    PubMed Central

    Huang, Xin; Tolmachev, Aleksey V.; Shen, Yulei; Liu, Miao; Huang, Lin; Zhang, Zhixin; Anderson, Gordon A.; Smith, Richard D.; Chan, Wing C.; Hinrichs, Steven H.; Fu, Kai; Ding, Shi-Jian

    2011-01-01

    Stable isotope labeling (SIL) methods coupled with nanoscale liquid chromatography and high resolution tandem mass spectrometry are increasingly useful for elucidation of the proteome-wide differences between multiple biological samples. Development of more effective programs for the sensitive identification of peptide pairs and accurate measurement of the relative peptide/protein abundance are essential for quantitative proteomic analysis. We developed and evaluated the performance of a new program, termed UNiquant, for analyzing quantitative proteomics data using stable isotope labeling. UNiquant was compared with two other programs, MaxQuant and Mascot Distiller, using SILAC-labeled complex proteome mixtures having either known or unknown heavy/light ratios. For the SILAC-labeled Jeko-1 cell proteome digests with known heavy/light ratios (H/L = 1:1, 1:5, and 1:10), UNiquant quantified a similar number of peptide pairs as MaxQuant for the H/L = 1:1 and 1:5 mixtures. In addition, UNiquant quantified significantly more peptides than MaxQuant and Mascot Distiller in the H/L = 1:10 mixtures. UNiquant accurately measured relative peptide/protein abundance without the need for post-measurement normalization of peptide ratios, which is required by the other programs. PMID:21158445

  10. Efficient sortase-mediated N-terminal labeling of TEV protease cleaved recombinant proteins.

    PubMed

    Sarpong, Kwabena; Bose, Ron

    2017-03-15

    A major challenge in attaching fluorophores or other handles to proteins is the availability of a site-specific labeling strategy that provides stoichiometric modification without compromising protein integrity. We developed a simple approach that combines TEV protease cleavage, sortase modification and affinity purification to N-terminally label proteins. To achieve stoichiometrically-labeled protein, we included a short affinity tag in the fluorophore-containing peptide for post-labeling purification of the modified protein. This strategy can be easily applied to any recombinant protein with a TEV site and we demonstrate this on Epidermal Growth Factor Receptor (EGFR) and Membrane Scaffold Protein (MSP) constructs.

  11. 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.

  12. Chromatographic behaviour of peptides following dimethylation with H2/D2-formaldehyde: implications for comparative proteomics.

    PubMed

    Boutilier, Joseph M; Warden, Hunter; Doucette, Alan A; Wentzell, Peter D

    2012-11-01

    The differential separation of deuterated and non-deuterated forms of isotopically substituted compounds in chromatography is a well-known but not well-understood phenomenon. This separation is relevant in comparative proteomics, where stable isotopes are used for differential labelling and the effect of isotope resolution on quantitation has been used to disqualify some deuterium labelling methods in favour of heavier isotopes. In this work, a detailed evaluation of the extent of isotopic separation and its impact on quantitation was performed for peptides labelled through dimethylation with H(2)/D(2) formaldehyde. The chromatographic behaviour of 71 labelled peptide pairs from quadruplicate tryptic digests of bovine serum albumin were analysed, focusing on differences in median retention times, resolution, and relative quantitation for each peptide. For 94% of peptides, the retention time difference (heavy-light) was less than 12s with a median value 3.4s. With the exception of a single anomalous pair, isotope resolution was below 0.6 with a median value 0.11. Quantitative assessment indicates that the bias in ratio calculation introduced by retention time shifts is only about 3%, substantially smaller than the variation in ratio measurements themselves. Computational studies on the dipole moments of deuterated labels indicate that these results are consistent with literature suggestions that retention time shifts are inversely related to the polarity of the label. This study suggests that the incorporation of deuterium isotopes through peptide dimethylation at amine residues is a viable route to proteome quantitation.

  13. 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.

  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. 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

  16. 16 CFR 305.17 - Television labeling.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... manufacturer may include the ENERGY STAR logo on the label as illustrated in Sample Labels 10, 11, and 12 in... labeled may add the ENERGY STAR logo to those labels. (g) Distribution of labels. For each...

  17. Mapping peptide thiol accessibility in membranes using a quaternary ammonium isotope-coded mass tag (ICMT)

    PubMed Central

    Su, Chiao-Yung; London, Erwin; Sampson, Nicole S.

    2013-01-01

    The plasma membrane contains a diverse array of proteins, including receptors, channels, and signaling complexes, that serve as decision-making centers. Investigation of membrane protein topology is important for understanding the function of these types of protein. Here, we report a method to determine protein topology in the membrane that utilizes labeling of cysteine with isotope-coded mass tags. The mass tags contain a thiol reactive moiety, linker, and a quaternary ammonium group to aid ionization in the mass spectrometer and were synthesizes as both light and heavy (deuterated) forms. The probes were found to be membrane impermeable when applied to lipid vesicles. To assess the utility of the probes for mapping peptide thiol topology, we employed a two-step labeling procedure. Vesicles containing α-helical transmembrane peptides were labeled with heavy (or light) probe, solubilized by detergent, and then labeled by an excess of the complementary probe. Peptide for which the cysteine was oriented in the center of the lipid bilayer was not labeled until the lipid vesicles were lysed with detergent, consistent with the membrane impermeability of the probes and reduced ionization of the thiol in the hydrophobic membrane. Peptide for which the cysteine was positioned in the head group zone of the lipid bilayer was labeled rapidly. Peptide for which the cysteine was positioned below the head group abutting the hydrocarbon region was labeled at a reduced rate compared to the fully accessible cysteine. Moreover, the effect of lipid bilayer structure on the kinetics of peptide and lipid flipping in the bilayer was readily measured with our two-step labeling method. The small sample size required, the ease and rapidity of sample preparation, and the amenability of MALDI-TOF mass spectral to analysis in the presence of lipids will enable future facile investigation of membrane proteins in a cellular context. PMID:23725486

  18. iTRAQ labeling is superior to mTRAQ for quantitative global proteomics and phosphoproteomics.

    PubMed

    Mertins, Philipp; Udeshi, Namrata D; Clauser, Karl R; Mani, D R; Patel, Jinal; Ong, Shao-en; Jaffe, Jacob D; Carr, Steven A

    2012-06-01

    Labeling of primary amines on peptides with reagents containing stable isotopes is a commonly used technique in quantitative mass spectrometry. Isobaric labeling techniques such as iTRAQ™ or TMT™ allow for relative quantification of peptides based on ratios of reporter ions in the low m/z region of spectra produced by precursor ion fragmentation. In contrast, nonisobaric labeling with mTRAQ™ yields precursors with different masses that can be directly quantified in MS1 spectra. In this study, we compare iTRAQ- and mTRAQ-based quantification of peptides and phosphopeptides derived from EGF-stimulated HeLa cells. Both labels have identical chemical structures, therefore precursor ion- and fragment ion-based quantification can be directly compared. Our results indicate that iTRAQ labeling has an additive effect on precursor intensities, whereas mTRAQ labeling leads to more redundant MS2 scanning events caused by triggering on the same peptide with different mTRAQ labels. We found that iTRAQ labeling quantified nearly threefold more phosphopeptides (12,129 versus 4,448) and nearly twofold more proteins (2,699 versus 1,597) than mTRAQ labeling. Although most key proteins in the EGFR signaling network were quantified with both techniques, iTRAQ labeling allowed quantification of twice as many kinases. Accuracy of reporter ion quantification by iTRAQ is adversely affected by peptides that are cofragmented in the same precursor isolation window, dampening observed ratios toward unity. However, because of tighter overall iTRAQ ratio distributions, the percentage of statistically significantly regulated phosphopeptides and proteins detected by iTRAQ and mTRAQ was similar. We observed a linear correlation of logarithmic iTRAQ to mTRAQ ratios over two orders of magnitude, indicating a possibility to correct iTRAQ ratios by an average compression factor. Spike-in experiments using peptides of defined ratios in a background of nonregulated peptides show that i

  19. 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.

  20. Assignment of disulfide-linked peptides using automatic a1 ion recognition.

    PubMed

    Huang, Sheng Yu; Wen, Chien Hsien; Li, Ding Tzai; Hsu, Jue Liang; Chen, Chinpan; Shi, Fong Ku; Lin, Yueh Yi

    2008-12-01

    We present a novel approach for the assignment of peptides containing disulfide linkages. Dimethyl labeling is introduced to generate labeled peptides which exhibit enhanced a1 ion signals during MS/MS fragmentation. For disulfide-linked peptides, multiple a1 ions can be observed due to multiple N-termini. This distinct feature allows sieving out the disulfide-linked peptides; meanwhile, the N-terminal amino acids can be identified. With such information, the number of possible peptide combinations involved in a disulfide bond dramatically narrows down. Furthermore, we developed a computational algorithm to perform target a1 ion screening followed by molecular weight matching of cysteine-containing peptides with specific amino acids at the N-termini. Once the protein sequence and the peak list from a LC-MS/MS survey scan of labeled peptides are imported, the identities of disulfide-linked peptides can be readily obtained. The presented approach is simple and straightforward, offering a valuable tool for protein structural characterization.

  1. Polycyclic peptide therapeutics.

    PubMed

    Baeriswyl, Vanessa; Heinis, Christian

    2013-03-01

    Owing to their excellent binding properties, high stability, and low off-target toxicity, polycyclic peptides are an attractive molecule format for the development of therapeutics. Currently, only a handful of polycyclic peptides are used in the clinic; examples include the antibiotic vancomycin, the anticancer drugs actinomycin D and romidepsin, and the analgesic agent ziconotide. All clinically used polycyclic peptide drugs are derived from natural sources, such as soil bacteria in the case of vancomycin, actinomycin D and romidepsin, or the venom of a fish-hunting coil snail in the case of ziconotide. Unfortunately, nature provides peptide macrocyclic ligands for only a small fraction of therapeutic targets. For the generation of ligands of targets of choice, researchers have inserted artificial binding sites into natural polycyclic peptide scaffolds, such as cystine knot proteins, using rational design or directed evolution approaches. More recently, large combinatorial libraries of genetically encoded bicyclic peptides have been generated de novo and screened by phage display. In this Minireview, the properties of existing polycyclic peptide drugs are discussed and related to their interesting molecular architectures. Furthermore, technologies that allow the development of unnatural polycyclic peptide ligands are discussed. Recent application of these technologies has generated promising results, suggesting that polycyclic peptide therapeutics could potentially be developed for a broad range of diseases.

  2. 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

  3. The natriuretic peptides.

    PubMed

    Baxter, Gary F

    2004-03-01

    The natriuretic peptides are a family of widely distributed, but evolutionarily conserved, polypeptide mediators that exert a range of actions throughout the body. In cardiovascular homeostasis, the endocrine roles of the cardiac-derived atrial and B-type natriuretic peptide (ANP and BNP) in regulating central fluid volume and blood pressure have been recognised for two decades. However, there is a growing realisation that natriuretic peptide actions go far beyond their volume regulating effects. These pleiotropic actions include local (autocrine/paracrine) regulatory actions of ANP and BNP within the heart, and of another natriuretic peptide, CNP, within the vessel wall. Effects on function and growth of the local tissue environment are likely to be of great importance, especially in disease states where tissue and circulating levels of ANP and BNP rise markedly. At present, the relevance of other natriuretic peptides (notably uroguanylin and DNP) to human physiology and pathology remain uncertain. Other articles in this issue of Basic Research in Cardiology review the molecular physiology of natriuretic peptide signalling, with a particular emphasis on the lessons from genetically targetted mice; the vascular activity of natriuretic peptides; the regulation and roles of natriuretic peptides in ischaemic myocardium; and the diagnostic, prognostic and therapeutic roles of natriuretic peptides in heart failure.

  4. Novel peptides functionally targeting in vivo human lung cancer discovered by in vivo peptide displayed phage screening.

    PubMed

    Lee, Kyoung Jin; Lee, Jae Hee; Chung, Hye Kyung; Choi, Jinhyang; Park, Jaesook; Park, Seok Soon; Ju, Eun Jin; Park, Jin; Shin, Seol Hwa; Park, Hye Ji; Ko, Eun Jung; Suh, Nayoung; Kim, InKi; Hwang, Jung Jin; Song, Si Yeol; Jeong, Seong-Yun; Choi, Eun Kyung

    2015-02-01

    Discovery of the cancer-specific peptidic ligands have been emphasized for active targeting drug delivery system and non-invasive imaging. For the discovery of useful and applicable peptidic ligands, in vivo peptide-displayed phage screening has been performed in this study using a xenograft mouse model as a mimic microenvironment to tumor. To seek human lung cancer-specific peptides, M13 phage library displaying 2.9 × 10(9) random peptides was intravenously injected into mouse model bearing A549-derived xenograft tumor through the tail vein. Then the phages emerged from a course of four rounds of biopanning in the xenograft tumor tissue. Novel peptides were categorized into four groups according to a sequence-homology phylogenicity, and in vivo tumor-targeting capacity of these peptides was validated by whole body imaging with Cy5.5-labeled phages in various cancer types. The result revealed that novel peptides accumulated only in adenocarcinoma lung cancer cell-derived xenograft tissue. For further confirmation of the specific targeting ability, in vitro cell-binding assay and immunohistochemistry in vivo tumor tissue were performed with a selected peptide. The peptide was found to bind intensely to lung cancer cells both in vitro and in vivo, which was efficiently compromised with unlabeled phages in an in vitro competition assay. In conclusion, the peptides specifically targeting human lung cancer were discovered in this study, which is warranted to provide substantive feasibilities for drug delivery and imaging in terms of a novel targeted therapeutics and diagnostics.

  5. Label-free quantitation, an extension to 2DB.

    PubMed

    Allmer, Jens

    2010-04-01

    Determining the differential expression of proteins under different conditions is of major importance in proteomics. Since mass spectrometry-based proteomics is often used to quantify proteins, several labelling strategies have been developed. While these are generally more precise than label-free quantitation approaches, they imply specifically designed experiments which also require knowledge about peptides that are expected to be measured and need to be modified. We recently designed the 2DB database which aids storage, analysis, and publication of data from mass spectrometric experiments to identify proteins. This database can aid identifying peptides which can be used for quantitation. Here an extension to the database application, named MSMAG, is presented which allows for more detailed analysis of the distribution of peptides and their associated proteins over the fractions of an experiment. Furthermore, given several biological samples in the database, label-free quantitation can be performed. Thus, interesting proteins, which may warrant further investigation, can be identified en passant while performing high-throughput proteomics studies.

  6. A Deceiving Label?

    ERIC Educational Resources Information Center

    Lum, Lydia

    2009-01-01

    The author reports on the growing debate among educators on whether the umbrella Asian Pacific Islander label conceals disparities among Asian American students or provides political power in numbers. Nationally, experts say that support services aimed at not only Southeast Asians, but all Asian Pacific Islander students, remain scarce in higher…

  7. 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

  8. Peptide bioregulators inhibit apoptosis.

    PubMed

    Khavinson, V K; Kvetnoii, I M

    2000-12-01

    The effects of peptide bioregulators epithalon and vilon on the dynamics of irradiation-induced apoptotic death of spleen lymphocytes in rats indicate that these agents inhibit physiologically programmed cell death. The antiapoptotic effect of vilon was more pronounced, which corroborates the concept on tissue-specific effect of peptide bioregulators.

  9. 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...

  10. Label Review Training: Module 1: Label Basics, Page 7

    EPA Pesticide Factsheets

    Page 7, Label Training, Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human he

  11. 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

  12. Issues and Applications in Label-Free Quantitative Mass Spectrometry

    PubMed Central

    Lai, Xianyin; Wang, Lianshui; Witzmann, Frank A.

    2013-01-01

    To address the challenges associated with differential expression proteomics, label-free mass spectrometric protein quantification methods have been developed as alternatives to array-based, gel-based, and stable isotope tag or label-based approaches. In this paper, we focus on the issues associated with label-free methods that rely on quantitation based on peptide ion peak area measurement. These issues include chromatographic alignment, peptide qualification for quantitation, and normalization. In addressing these issues, we present various approaches, assembled in a recently developed label-free quantitative mass spectrometry platform, that overcome these difficulties and enable comprehensive, accurate, and reproducible protein quantitation in highly complex protein mixtures from experiments with many sample groups. As examples of the utility of this approach, we present a variety of cases where the platform was applied successfully to assess differential protein expression or abundance in body fluids, in vitro nanotoxicology models, tissue proteomics in genetic knock-in mice, and cell membrane proteomics. PMID:23401775

  13. Quantitative analysis of bacterial and mammalian proteomes using a combination of cysteine affinity tags and 15N-metabolic labeling.

    PubMed

    Conrads, T P; Alving, K; Veenstra, T D; Belov, M E; Anderson, G A; Anderson, D J; Lipton, M S; Pasa-Tolić, L; Udseth, H R; Chrisler, W B; Thrall, B D; Smith, R D

    2001-05-01

    We describe the combined use of 15N-metabolic labeling and a cysteine-reactive biotin affinity tag to isolate and quantitate cysteine-containing polypeptides (Cys-polypeptides) from Deinococcus radiodurans as well as from mouse B16 melanoma cells. D. radiodurans were cultured in both natural isotopic abundance and 15N-enriched media. Equal numbers of cells from both cultures were combined and the soluble proteins extracted. This mixture of isotopically distinct proteins was derivatized using a commercially available cysteine-reactive reagent that contains a biotin group. Following trypsin digestion, the resulting modified peptides were isolated using immobilized avidin. The mixture was analyzed by capillary reversed-phase liquid chromatography (LC) online with ion trap mass spectrometry (MS) as well as Fourier transform ion cyclotron resonance (FTICR) MS. The resulting spectra contain numerous pairs of Cyspolypeptides whose mass difference corresponds to the number of nitrogen atoms present in each of the peptides. Designation of Cys-polypeptide pairs is also facilitated by the distinctive isotopic distribution of the 15N-labeled peptides versus their 14N-labeled counterparts. Studies with mouse B16 cells maintained in culture allowed the observation of hundreds of isotopically distinct pairs of peptides by LC-FTICR analysis. The ratios of the areas of the pairs of isotopically distinct peptides showed the expected 1:1 labeling of the 14N and 15N versions of each peptide. An additional benefit from the present strategy is that the 15N-labeled peptides do not display significant isotope-dependent chromatographic shifts from their 14N-labeled counterparts, therefore improving the precision for quantitating peptide abundances. The methodology presented offers an alternate, cost-effective strategy for conducting global, quantitative proteomic measurements.

  14. 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

  15. Rabies virus binding to an acetylcholine receptor alpha-subunit peptide.

    PubMed

    Lentz, T L

    1990-04-01

    The binding of 125I-labeled rabies virus to a synthetic peptide comprising residues 173-204 of the alpha 1-subunit of the nicotinic acetylcholine receptor was investigated. Binding of rabies virus to the receptor peptide was dependent on pH, could be competed with by unlabeled homologous virus particles, and was saturable. Synthetic peptides of snake venom, curaremimetic neurotoxins and of the structurally similar segment of the rabies virus glycoprotein, were effective in competing with labeled virus binding to the receptor peptide at micromolar concentrations. Similarly, synthetic peptides of the binding domain on the acetylcholine receptor competed for binding. These findings suggest that both rabies virus and neurotoxins bind to residues 173-204 of the alpha 1-subunit of the acetylcholine receptor. Competition studies with shorter alpha-subunit peptides within this region indicate that the highest affinity virus binding determinants are located within residues 179-192. A rat nerve alpha 3-subunit peptide, that does not bind alpha-bungarotoxin, inhibited binding of virus to the alpha 1 peptide, suggesting that rabies binds to neuronal nicotinic acetylcholine receptors. These studies indicate that synthetic peptides of the glycoprotein binding domain and of the receptor binding domain may represent useful antiviral agents by targeting the recognition event between the viral attachment protein and the host cell receptor, and inhibiting attachment of virus to the receptor.

  16. Fluorescence energy transfer as an indicator of Ca2+-ATPase interactions in sarcoplasmic reticulum.

    PubMed Central

    Papp, S; Pikula, S; Martonosi, A

    1987-01-01

    Ca2+-ATPase molecules were labeled in intact sarcoplasmic reticulum (SR) vesicles, sequentially with a donor fluorophore, fluorescein-5'-isothiocyanate (FITC), and with an acceptor fluorophore, eosin-5'-isothiocyanate (EITC), each at a mole ratio of 0.25-0.5 mol/mol of ATPase. The resonance energy transfer was determined from the effect of acceptor on the intensity and lifetime of donor fluorescence. Due to structural similarities, the two dyes compete for the same site(s) on the Ca2+-ATPase, and under optimal conditions each ATPase molecule is labeled either with donor or acceptor fluorophore, but not with both. There is only slight labeling of phospholipids and other proteins in SR, even at concentrations of FITC or EITC higher than those used in the reported experiments. Efficient energy transfer was observed from the covalently bound FITC to EITC that is assumed to reflect interaction between ATPase molecules. Protein denaturing agents (8 M urea and 4 M guanidine) or nonsolubilizing concentrations of detergents (C12E8 or lysolecithin) abolish the energy transfer. These results are consistent with earlier observations that a large portion of the Ca2+-ATPase is present in oligomeric form in the native membrane. The technique is suitable for kinetic analysis of the effect of various treatments on the monomer-oligomer equilibrium of Ca2+-ATPase. A drawback of the method is that the labeled ATPase, although it retains conformational responses, is enzymatically inactive. Images FIGURE 3 FIGURE 4 FIGURE 5 PMID:2950938

  17. 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 " ...

  18. Use the Nutrition Facts Label

    MedlinePlus

    ... Features Spokespeople News Archive eNewsletters Calendar Use the Nutrition Facts Label You can help your family eat ... to some of their favorite foods. Use the Nutrition Facts label found on food packages to make ...

  19. Biosynthesis of amidated joining peptide from pro-adrenocorticotropin-endorphin

    SciTech Connect

    Cullen, E.I.; Mains, R.E. )

    1987-09-01

    Joining peptide is the major alpha-amidated product of pro-ACTH/endorphin (PAE) in AtT-20 corticotropic tumor cells. To study intracellular joining peptide synthesis, affinity purified antibodies directed against gamma-MSH, joining peptide, and ACTH were used to immunoprecipitate extracts from biosynthetically labeled AtT-20 cells. Immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by tryptic peptide mapping on HPLC. In steady labeling experiments, radioactivity in amidated joining peptide (JP) increased roughly linearly with time, in the manner of a final product, whereas radioactivity associated with PAE (1-94)NH2 reached a constant value after 2-4 h, indicating that PAE(1-94)NH2 is an intermediate in the biosynthesis of JP. Radioactivity appeared in ACTH(1-39) well before JP, consistent with a cleavage order in which ACTH is cleaved from PAE(1-95) before JP sequences are cleaved from PAE(1-74). This conclusion was supported by tryptic peptide analyses of immunoprecipitates, which indicated that less than 5% of JP-related material is cleaved from PAE(1-74) before being cleaved from ACTH-related sequences. After a pulse label, radioactivity in PAE(1-94)NH2 reached a peak value after 1 h of chase and declined with a half-life of less than 1 h. Amidated JP increased to a constant level after 2 h of chase. Enough radiolabeled PAE(1-94)NH2 was detected to account for about half of the radioactivity found in amidated JP, indicating that about half of JP-related material is first cleaved from PAE(1-95) before being amidated. This result was corroborated using HPLC purification to determine both amidated and glycine-extended forms of JP.

  20. 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.

  1. Dynamic Proteomics: In Vivo Proteome-Wide Measurement of Protein Kinetics Using Metabolic Labeling.

    PubMed

    Holmes, W E; Angel, T E; Li, K W; Hellerstein, M K

    2015-01-01

    Control of biosynthetic and catabolic rates of polymers, including proteins, stands at the center of phenotype, physiologic adaptation, and disease pathogenesis. Advances in stable isotope-labeling concepts and mass spectrometric instrumentation now allow accurate in vivo measurement of protein synthesis and turnover rates, both for targeted proteins and for unbiased screening across the proteome. We describe here the underlying principles and operational protocols for measuring protein dynamics, focusing on metabolic labeling with (2)H2O (heavy water) combined with tandem mass spectrometric analysis of mass isotopomer abundances in trypsin-generated peptides. The core principles of combinatorial analysis (mass isotopomer distribution analysis or MIDA) are reviewed in detail, including practical advantages, limitations, and technical procedures to ensure optimal kinetic results. Technical factors include heavy water labeling protocols, optimal duration of labeling, clean up and simplification of sample matrices, accurate quantitation of mass isotopomer abundances in peptides, criteria for adequacy of mass spectrometric abundance measurements, and calculation algorithms. Some applications are described, including the noninvasive "virtual biopsy" strategy for measuring molecular flux rates in tissues through measurements in body fluids. In addition, application of heavy water labeling to measure flux lipidomics is noted. In summary, the combination of stable isotope labeling, particularly from (2)H2O, with tandem mass spectrometric analysis of mass isotopomer abundances in peptides, provides a powerful approach for characterizing the dynamics of proteins across the global proteome. Many applications in research and clinical medicine have been achieved and many others can be envisioned.

  2. Cyclic Opioid Peptides.

    PubMed

    Remesic, Michael; Lee, Yeon Sun; Hruby, Victor J

    2016-01-01

    For decades the opioid receptors have been an attractive therapeutic target for the treatment of pain. Since the first discovery of enkephalin, approximately a dozen endogenous opioid peptides have been known to produce opioid activity and analgesia, but their therapeutics have been limited mainly due to low blood brain barrier penetration and poor resistance to proteolytic degradation. One versatile approach to overcome these drawbacks is the cyclization of linear peptides to cyclic peptides with constrained topographical structure. Compared to their linear parents, cyclic analogs exhibit better metabolic stability, lower offtarget toxicity, and improved bioavailability. Extensive structure-activity relationship studies have uncovered promising compounds for the treatment of pain as well as further elucidate structural elements required for selective opioid receptor activity. The benefits that come with employing cyclization can be further enhanced through the generation of polycyclic derivatives. Opioid ligands generally have a short peptide chain and thus the realm of polycyclic peptides has yet to be explored. In this review, a brief history of designing ligands for the opioid receptors, including classic linear and cyclic ligands, is discussed along with recent approaches and successes of cyclic peptide ligands for the receptors. Various scaffolds and approaches to improve bioavailability are elaborated and concluded with a discourse towards polycyclic peptides.

  3. Search engine processor: Filtering and organizing peptide spectrum matches.

    PubMed

    Carvalho, Paulo C; Fischer, Juliana S G; Xu, Tao; Cociorva, Daniel; Balbuena, Tiago S; Valente, Richard H; Perales, Jonas; Yates, John R; Barbosa, Valmir C

    2012-04-01

    The search engine processor (SEPro) is a tool for filtering, organizing, sharing, and displaying peptide spectrum matches. It employs a novel three-tier Bayesian approach that uses layers of spectrum, peptide, and protein logic to lead the data to converge to a single list of reliable protein identifications. SEPro is integrated into the PatternLab for proteomics environment, where an arsenal of tools for analyzing shotgun proteomic data is provided. By using the semi-labeled decoy approach for benchmarking, we show that SEPro significantly outperforms a commercially available competitor.

  4. 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.

  5. Application of recombinant and non-recombinant peptides in the determination of tumor response to cancer therapy.

    PubMed

    Lopez-Barcons, Lluis A; Ali, Arif N; Diaz, Roberto

    2011-02-01

    An early and reliable assessment of therapeutic efficacy during the treatment of cancer is essential to achieve an optimal treatment regimen and patient outcome. The use of labeled peptides to monitor tumor response is associated with several advantages. For example, peptides are very stable, non-immunogenic, are easy to label for imaging, they undergo rapid clearance from the circulation, can penetrate tumor tissue, and are inexpensive to synthesize. In this review, studies using recombinant and non-recombinant peptides to monitor the response of glioblastoma multiforme, lung, breast, pancreas, colon, prostate, and skin carcinomas to radiation and/or chemotherapeutics such as camptothecin, doxorubicin, etoposide, 5-fluorouracil, paclitaxel, AG3340, sunitinib, and dasatinib, are presented. A consideration of the imaging techniques available to monitor peptide localization, including near-infrared (NIR) fluorescence, magnetic resonance imaging (MRI), positron emission tomography (PET), and ultrasonography, is also included. Peptides that have been successfully used to monitor various tumor types and therapies have been shown to target proteins that undergo changes in expression in response to treatment, endothelial cells that respond to radiation, or mediators of apoptosis. Peptides that are able to selectively bind responsive versus unresponsive tumors have also been identified. Therefore, the advantages associated with the use of peptides, combined with the capacity for selected peptides to assess tumor response as demonstrated in various studies, support the use of labeled peptides to evaluate the effectiveness of a given cancer therapy.

  6. Binding modes of thioflavin T molecules to prion peptide assemblies identified by using scanning tunneling microscopy.

    PubMed

    Mao, Xiaobo; Guo, Yuanyuan; Wang, Chenxuan; Zhang, Min; Ma, Xiaojing; Liu, Lei; Niu, Lin; Zeng, Qingdao; Yang, Yanlian; Wang, Chen

    2011-06-15

    The widely used method to monitor the aggregation process of amyloid peptide is thioflavin T (ThT) assay, while the detailed molecular mechanism is still not clear. In this work, we report here the direct identification of the binding modes of ThT molecules with the prion peptide GNNQQNY by using scanning tunneling microscopy (STM). The assembly structures of GNNQQNY were first observed by STM on a graphite surface, and the introduction of ThT molecules to the surface facilitated the STM observations of the adsorption conformations of ThT with peptide strands. ThT molecules are apt to adsorb on the peptide assembly with β-sheet structure and oriented parallel with the peptide strands adopting four different binding modes. This effort could benefit the understanding of the mechanisms of the interactions between labeling species or inhibitory ligands and amyloid peptides, which is keenly needed for developing diagnostic and therapeutic approaches.

  7. 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.

  8. Histidine-containing peptide catalysts developed by a facile library screening method.

    PubMed

    Akagawa, Kengo; Sakai, Nobutaka; Kudo, Kazuaki

    2015-02-02

    Although peptide catalysts have a high potential for the use as organocatalysts, the optimization of peptide sequences is laborious and time-consuming. To address this issue, a facile screening method for finding efficient aminocatalysts from a peptide library has been developed. In the screening for the Michael addition of a malonate to an enal, a dye-labeled product is immobilized on resin-bound peptides through reductive amination to visualize active catalysts. This procedure allows for the monitoring of the reactivity of entire peptides without modifying the resin beads beforehand. Peptides containing histidine at an appropriate position were identified by this method. A novel function of the histidyl residue, which enhances the binding of a substrate to the catalyst by capturing an iminium intermediate, was indicated.

  9. Identifying structural features of fibrillar islet amyloid polypeptide using site-directed spin labeling.

    PubMed

    Jayasinghe, Sajith A; Langen, Ralf

    2004-11-12

    Pancreatic amyloid deposits, composed primarily of the 37-residue islet amyloid polypeptide (IAPP), are a characteristic feature found in more than 90% of patients with type II diabetes. Although IAPP amyloid deposits are associated with areas of pancreatic islet beta-cell dysfunction and depletion and are thought to play a role in disease, their structure is unknown. We used electron paramagnetic resonance spectroscopy to analyze eight spin-labeled derivatives of IAPP in an effort to determine structural features of the peptide. In solution, all eight derivatives gave rise to electron paramagnetic resonance spectra with sharp lines indicative of rapid motion on the sub-nanosecond time scale. These spectra are consistent with a rapidly tumbling and highly dynamic peptide. In contrast, spectra for the fibrillar form exhibit reduced mobility and the presence of strong intermolecular spin-spin interactions. The latter implies that the peptide subunits are ordered and that the same residues from neighboring peptides are in close proximity to one another. Our data are consistent with a parallel arrangement of IAPP peptides within the amyloid fibril. Analysis of spin label mobility indicates a high degree of order throughout the peptide, although the N-terminal region is slightly less ordered. Possible similarities with respect to the domain organization and parallelism of Alzheimer's amyloid beta peptide fibrils are discussed.

  10. 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.

  11. Anti-antimicrobial Peptides

    PubMed Central

    Ryan, Lloyd; Lamarre, Baptiste; Diu, Ting; Ravi, Jascindra; Judge, Peter J.; Temple, Adam; Carr, Matthew; Cerasoli, Eleonora; Su, Bo; Jenkinson, Howard F.; Martyna, Glenn; Crain, Jason; Watts, Anthony; Ryadnov, Maxim G.

    2013-01-01

    Antimicrobial or host defense peptides are innate immune regulators found in all multicellular organisms. Many of them fold into membrane-bound α-helices and function by causing cell wall disruption in microorganisms. Herein we probe the possibility and functional implications of antimicrobial antagonism mediated by complementary coiled-coil interactions between antimicrobial peptides and de novo designed antagonists: anti-antimicrobial peptides. Using sequences from native helical families such as cathelicidins, cecropins, and magainins we demonstrate that designed antagonists can co-fold with antimicrobial peptides into functionally inert helical oligomers. The properties and function of the resulting assemblies were studied in solution, membrane environments, and in bacterial culture by a combination of chiroptical and solid-state NMR spectroscopies, microscopy, bioassays, and molecular dynamics simulations. The findings offer a molecular rationale for anti-antimicrobial responses with potential implications for antimicrobial resistance. PMID:23737519

  12. 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.

  13. Melanins from opioid peptides.

    PubMed

    Rosei, M A

    1996-12-01

    Opioid peptides and other Tyr-NH2-terminal peptides are substrates in vitro for mushroom and sepia tyrosine, giving rise to synthetic melanins retaining the peptide moiety (opiomelanins). The melanopeptides are characterized by a total solubility in hydrophylic solvents at neutral and basic pH. Opioid peptides (enkephalins, endorphins, and esorphins), if oxidized by tyrosinase in the presence of Dopa, are easily incorporated into Dopa-melanin, producing mixed-type pigments that can also be solubilized in hydrophylic solvents. Melanins derived from opioid peptides exhibit paramagnetism, as evidenced by an EPR spectrum identical to that of Dopa-melanin. However, the presence of the linked peptide chain is able to influence dramatically the electron transfer properties and the oxidizing behaviour of the melanopeptides, so that whereas Tyr-Gly-melanin appears to behave as Dopa-melanin, Enk-melanin does not exhibit any oxidizing activity. Opiomelanins are characterized by a peculiar UV-VIS spectrum; that is, by the presence of a distinct peak (330 nm) that disappears upon chemical treatment by acid hydrolysis. Opiomelanins are stable pigments at neutral and basic pH in the dark, whereas the addition of H2O2 leads to a 15% degradation. Under stimulated solar illumination, opiomelanins are more easily destroyed with respect to Dopa-melanin, with increasing degradation when exposed to increased hydrogen peroxide concentrations and more alkaline pH. Some speculations on the possible existence and role of opiomelanins have been outlined.

  14. Peptide Optical waveguides.

    PubMed

    Handelman, Amir; Apter, Boris; Shostak, Tamar; Rosenman, Gil

    2017-02-01

    Small-scale optical devices, designed and fabricated onto one dielectric substrate, create integrated optical chip like their microelectronic analogues. These photonic circuits, based on diverse physical phenomena such as light-matter interaction, propagation of electromagnetic waves in a thin dielectric material, nonlinear and electro-optical effects, allow transmission, distribution, modulation, and processing of optical signals in optical communication systems, chemical and biological sensors, and more. The key component of these optical circuits providing both optical processing and photonic interconnections is light waveguides. Optical confinement and transmitting of the optical waves inside the waveguide material are possible due to the higher refractive index of the waveguides in comparison with their surroundings. In this work, we propose a novel field of bionanophotonics based on a new concept of optical waveguiding in synthetic elongated peptide nanostructures composed of ordered peptide dipole biomolecules. New technology of controllable deposition of peptide optical waveguiding structures by nanofountain pen technique is developed. Experimental studies of refractive index, optical transparency, and linear and nonlinear waveguiding in out-of-plane and in-plane diphenylalanine peptide nanotubes have been conducted. Optical waveguiding phenomena in peptide structures are simulated by the finite difference time domain method. The advantages of this new class of bio-optical waveguides are high refractive index contrast, wide spectral range of optical transparency, large optical nonlinearity, and electro-optical effect, making them promising for new applications in integrated multifunctional photonic circuits. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

  15. 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

  16. Methods for studying transmembrane peptides in bicelles: consequences of hydrophobic mismatch and peptide sequence

    NASA Astrophysics Data System (ADS)

    Whiles, Jennifer A.; Glover, Kerney J.; Vold, Regitze R.; Komives, Elizabeth A.

    2002-09-01

    We have shown that bicelles prepared from dilauryl phosphatidylcholine (DLPC) and dipalmitoyl phosphatidylcholine (DPPC) align in a magnetic field under conditions similar to the more common dimyristoyl phosphatidylcholine (DMPC) bicelles. In addition, a model transmembrane peptide, P16, with a hydrophobic stretch of 24 Å, and specific alanine-d 3 labels, was incorporated into all of the different bicelles. The long-chain phospholipid (DLPC, DMPC, or DPPC) remained unperturbed upon incorporation of the peptide while the quadrupolar splitting of the short-chain phospholipid along the bicelle rim increased by varying degrees in the different bicelle systems. The change in quadrupolar splitting of the short-chain phospholipids was attributed to changes in either fluidity of the planar region of the bicelle or differences in overall lipid packing. When the hydrophobic stretch of the bilayer was 22.8 (DMPC) or 26.3 Å (DPPC), the peptide tilt was found to be transmembrane (33-35° with respect to the bicelle normal). When the hydrophobic stretch of the bilayer was 19.5 Å (DLPC), the peptide quadrupolar splittings suggested a loss of transmembrane orientation. When tryptophan was incorporated in the middle of the transmembrane region, the transmembrane orientation was also lost.

  17. Supplementing national menu labeling.

    PubMed

    Hodge, James G; White, Lexi C

    2012-12-01

    The US Food and Drug Administration's forthcoming national menu labeling regulations are designed to help curb the national obesity epidemic by requiring calorie counts on restaurants' menus. However, posted calories can be easily ignored or misunderstood by consumers and fail to accurately describe the healthiness of foods. We propose supplemental models that include nutritional information (e.g., fat, salt, sugar) or specific guidance (e.g., "heart-healthy" graphics). The goal is to empower restaurant patrons with better data to make healthier choices, and ultimately to reduce obesity prevalence.

  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. Selective labeling of polypeptides using protein farnesyltransferase via rapid oxime ligation.

    PubMed

    Rashidian, Mohammad; Dozier, Jonathan K; Lenevich, Stepan; Distefano, Mark D

    2010-12-21

    An aldehyde-containing alternative substrate for protein farnesyltransferase was prepared and shown to be enzymatically incorporated into a peptide and a protein. The protein was subsequently immobilized onto aminooxy-functionalized agarose beads or labeled with a fluorophore. This method for protein modification provides an alternative to the commonly employed Cu(I)-catalyzed click reaction.

  20. Selective Labeling of Polypeptides Using Protein Farnesyltransferase via Rapid Oxime Ligation

    PubMed Central

    Rashidian, Mohammad; Dozier, Jonathan K.; Lenevich, Stepan; Distefano, Mark D.

    2012-01-01

    An aldehyde-containing alternative substrate for protein farnesyltransferase was prepared and shown to be enzymatically incorporated into a peptide and a protein. The protein was subsequently immobilized onto aminooxy-functionalized agarose beads or labeled with a fluorophore. This method for protein modification provides an alternative to the commonly employed Cu(I)-catalyzed click reaction PMID:20967387

  1. Rapid peptide metabolism: A major component of soil nitrogen cycling?

    NASA Astrophysics Data System (ADS)

    Farrell, Mark; Hill, Paul W.; Wanniarachchi, Sudas D.; Farrar, John; Bardgett, Richard D.; Jones, Davey L.

    2011-09-01

    Proteinaceous and peptidic nitrogen is a potential direct nutrient source for both plants and microbes in the soil, without prior degradation to amino acids and mineralization. We used a series of five sites along an elevation gradient from 15 m a.s.l. to 710 m a.s.l. along which primary productivity decreases to investigate peptide utilization rates by soil microbes. Using 14C-labeled L-alanine, L-dialanine, and L-trialanine in a series of incubation experiments, we show that peptides are directly and rapidly assimilated by soil microbes, and that they are utilized for both biomass production and respiration. Alanine, dialanine, and trialanine were mineralized rapidly by soil microbes from the five sites along the gradient. Across all five sites, dialanine and trialanine were mineralized faster than alanine. In competition experiments, a 100-fold excess of alanine had no effect on the rate of trialanine mineralization in four of the five sites, and the same excess of trialanine had no effect on alanine mineralization. This is indicative of uptake of the intact peptide by the soil microbial community. Our findings have implications for understanding terrestrial nitrogen cycling because they point to a short-circuit whereby large peptides and proteins need only be extracellularly cleaved to short chain length peptides before direct assimilation by microbes.

  2. 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

  3. 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.

  4. Insulin biosynthesis: studies of Islet polyribosomes (nascent peptides-sucrose gradient analysis-gel filtration).

    PubMed

    Permutt, M A; Kipnis, D M

    1972-02-01

    A method is described for separation of polyribosomes from as few as 25 isolated Islets of Langerhans, representing about 250 mug of pancreatic tissue. Islets are labeled with [(3)H]leucine and polysomes are isolated with liver polyribosomes, which serve as carrier and inhibitor of ribonuclease activity. Islets incubated at 37 degrees C for 45 min in 15.5 mM glucose, then pulsed with [(3)H]leucine, incorporated about 2-3 times more label into nascent peptides on islet polysomes than islets incubated in 2.8 mM glucose. Sucrose gradient analysis of the labeled polysomes indicated that raising the glucose concentration preferentially stimulated synthesis of peptides on trisomes and larger polyribosomes. Islets incubated with [(3)H]leucine for 15 min incorporated two-thirds of the label into proteins on membrane-bound polysomes. At least 85% of the proinsulin synthesis during this time occurs on membrane-bound polysomes.

  5. 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.

  6. Optimizing connected component labeling algorithms

    NASA Astrophysics Data System (ADS)

    Wu, Kesheng; Otoo, Ekow; Shoshani, Arie

    2005-04-01

    This paper presents two new strategies that can be used to greatly improve the speed of connected component labeling algorithms. To assign a label to a new object, most connected component labeling algorithms use a scanning step that examines some of its neighbors. The first strategy exploits the dependencies among them to reduce the number of neighbors examined. When considering 8-connected components in a 2D image, this can reduce the number of neighbors examined from four to one in many cases. The second strategy uses an array to store the equivalence information among the labels. This replaces the pointer based rooted trees used to store the same equivalence information. It reduces the memory required and also produces consecutive final labels. Using an array instead of the pointer based rooted trees speeds up the connected component labeling algorithms by a factor of 5 ~ 100 in our tests on random binary images.

  7. 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.

  8. Label Ranking Algorithms: A Survey

    NASA Astrophysics Data System (ADS)

    Vembu, Shankar; Gärtner, Thomas

    Label ranking is a complex prediction task where the goal is to map instances to a total order over a finite set of predefined labels. An interesting aspect of this problem is that it subsumes several supervised learning problems, such as multiclass prediction, multilabel classification, and hierarchical classification. Unsurprisingly, there exists a plethora of label ranking algorithms in the literature due, in part, to this versatile nature of the problem. In this paper, we survey these algorithms.

  9. GEO label: The General Framework for Labeling and Certification

    NASA Astrophysics Data System (ADS)

    Bye, B. L.; McCallum, I.; Maso, J.

    2012-04-01

    The Group on Earth Observations (GEO) is coordinating efforts to build a Global Earth Observation System of Systems, or GEOSS. As part of a strategy to increase the involvement of the science and technology community in GEOSS, both as users and developers of GEOSS itself, GEO decided to develop a GEO label concept related to the scientific relevance, quality, acceptance and societal needs for services and data sets of GEOSS. The development of a GEO label is included in the GEO work plan and several projects address the challenges of developing a GEO label concept. Within the different projects developing the GEO label, various perspectives and approaches are being applied. In order to arrive at a generally accepted GEO label concept, a common understanding and basic knowledge of labeling is necessary. Assessment of quality of internationally standardized Earth observation data products implies possible certification. A general understanding of the framework for international standards and certification will also contribute to a more coherent discussion and more efficient development of a GEO label. We will describe the general labeling and certification framework emphasizing the relation to the three elements of the GEO label: quality, user acceptance and relevance. Based on a survey of international labels done by the EGIDA project, we have analyzed the legal framework and organization of labels and certification. We will discuss the frameworks for certification, user ratings, registration and analysis of user requirements. Quality assessment is a particular focus of the analysis and is based on the work done by the GeoViQua project. A GEO label will function both as a data distribution strategy and as a general management system for data. Through a label users can compare different data sets and get access to more information about the relevant data, including quality. A label will provide traceability of data both in the interest of users as well as data

  10. 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

  11. Cyclization in opioid peptides.

    PubMed

    Piekielna, Justyna; Perlikowska, Renata; Gach, Katarzyna; Janecka, Anna

    2013-06-01

    Endogenous opioid peptides have been studied extensively as potential therapeutics for the treatment of pain. The major problems of using natural opioid peptides as drug candidates are their poor receptor specificity, metabolic instability and inability to reach the brain after systemic administration. A lot of synthetic efforts have been made to opioid analogs with improved pharmacological properties. One important structural modification leading to such analogs is cyclization of linear sequences. Intramolecular cyclization has been shown to improve biological properties of various bioactive peptides. Cyclization reduces conformational freedom responsible for the simultaneous activation of two or more receptors, increases metabolic stability and lipophilicity which may result in a longer half-life and easier penetration across biological membranes. This review deals with various strategies that have been employed to synthesize cyclic analogs of opioid peptides. Discussed are such bridging bonds as amide and amine linkages, sulfur-containing bonds, including monosulfide, disulfide and dithioether bridges, bismethylene bonds, monosulfide bridges of lanthionine and, finally, carbonyl and guanidine linkages. Opioid affinities and activities of cyclic analogs are given and compared with linear opioid peptides. Analgesic activities of analogs evaluated in the in vivo pain tests are also discussed.

  12. Labeling conventions in isoelectronic sequences

    SciTech Connect

    Maniak, S.T.; Curtis, L.J. )

    1990-08-01

    The isoelectronic exposition of atomic structure properties involves labeling ambiguities when more than one level of the same total angular momentum and parity is present, and an energy ordered labeling of these levels can lead to apparent isoelectronic discontinuities. For example, in the recent oscillator strength calculations for S-like ions by Saloman and Kim (Phys. Rev. A 38, 577 (1988)), abrupt changes in the rates were sometimes observed between one isoelectronic element and the next. We suggest an alternative labeling scheme that removes these discontinuities and produces a smooth isoelectronic variation. This alternative labeling offers advantages for data exposition and for semiempirical interpolation and extrapolation.

  13. Measurement of the binding parameters of annexin derivative-erythrocyte membrane interactions.

    PubMed

    Yen, Tzu-Chen; Wey, Shiaw-Pyng; Liao, Chang-Hui; Yeh, Chi-Hsiao; Shen, Duan-Wen; Achilefu, Samuel; Wun, Tze-Chein

    2010-11-01

    Erythrocyte ghosts prepared from fresh blood expressed phosphatidylserine (PS) on the membrane surfaces in a rather stable fashion. The binding of fluorescein-5-isothiocyanate (FITC)-labeled annexin V (ANV) derivatives to these membranes was studied by titration with proteins and with calcium. Whereas the preaddition of ethylenediaminetetraacetic acid (EDTA) to reaction mixtures totally prevented membrane binding, Ca(2+)-dependent binding was only partially reversed by EDTA treatment, consistent with an initial Ca(2+)-dependent binding that became partially Ca(2+) independent. Data derived from saturation titration with ANV derivatives poorly fit the simple protein-membrane equilibrium binding equation and showed negative cooperativity of binding with increasing membrane occupancy. In contrast, calcium titration at low binding site occupancy resulted in excellent fit into the protein-Ca(2+)-membrane equilibrium binding equation. Calcium titrations of FITC-labeled ANV and ANV-6L15 (a novel ANV-Kunitz protease inhibitor fusion protein) yielded a Hill coefficient of approximately 4 in both cases. The apparent dissociation constant for ANV-6L15 was approximately 4-fold lower than that of ANV at 1.2-2.5mM Ca(2+). We propose that ANV-6L15 may provide improved detection of PS exposed on the membrane surfaces of pathological cells in vitro and in vivo.

  14. Labeled Cocaine Analogs

    DOEpatents

    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.

  15. 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.

  16. 78 FR 66826 - Prior Label Approval System: Generic Label Approval

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-07

    ... poultry products inspection regulations to expand the circumstances in which FSIS will generically approve the labels of meat and poultry products. The Agency also is consolidating the regulations that provide for the approval of labels for meat products and poultry products into a new Code of...

  17. 76 FR 75809 - Prior Label Approval System: Generic Label Approval

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-05

    ... amend the meat and poultry products inspection regulations to expand the circumstances in which FSIS will generically approve the labels of meat and poultry products. The Agency also is proposing to combine the regulations that provide for the approval of labels for meat products and poultry...

  18. 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...

  19. 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...

  20. Peptides in oral diseases.

    PubMed

    Lucchese, Alberta; Guida, Agostino; Petruzzi, Massimo; Capone, Giovanni; Laino, Luigi; Serpico, Rosario

    2012-01-01

    The oral cavity is home to numerous viruses and micro-organisms recognized as having a role in various oral diseases as well as in infections in other parts of the body. Indeed, in general a microbial infection underlies or is believed to underlie the ample spectrum of oral diseases, from tooth enamel decay to periodontal lesions, from candidiasis to virus-induced oral squamous cell carcinomas, and bullous autoimmune oral disorders. This clinico-pathological context stresses the need of targeted therapies to specifically kill infectious agents in a complex environment such as the oral cavity, and explains the current interest in exploring peptide-based therapeutic approaches in oral and dental research. Here, we review the therapeutic potential of antimicrobial peptides such as LL-37, beta defensins, adrenomedullin, histatins, and of various peptides modulating gene expression and immuno-biological interaction(s) in oral diseases.

  1. Molecular modeling of peptides.

    PubMed

    Kuczera, Krzysztof

    2015-01-01

    This article presents a review of the field of molecular modeling of peptides. The main focus is on atomistic modeling with molecular mechanics potentials. The description of peptide conformations and solvation through potentials is discussed. Several important computer simulation methods are briefly introduced, including molecular dynamics, accelerated sampling approaches such as replica-exchange and metadynamics, free energy simulations and kinetic network models like Milestoning. Examples of recent applications for predictions of structure, kinetics, and interactions of peptides with complex environments are described. The reliability of current simulation methods is analyzed by comparison of computational predictions obtained using different models with each other and with experimental data. A brief discussion of coarse-grained modeling and future directions is also presented.

  2. 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.

  3. Epitope peptides and immunotherapy.

    PubMed

    Tanabe, Soichi

    2007-02-01

    Allergic diseases affect atopic individuals, who synthesize specific Immunoglobulins E (IgE) to environmental allergens, usually proteins or glycoproteins. These allergens include grass and tree pollens, indoor allergens such as house dust mites and animal dander, and various foods. Because allergen-specific IgE antibodies are the main effector molecules in the immune response to allergens, many studies have focused on the identification of IgE-binding epitopes (called B cell epitopes), specific and minimum regions of allergen molecules that binds to IgE. Our initial studies have provided evidence that only four to five amino acid residues are enough to comprise an epitope, since pentapeptide QQQPP in wheat glutenin is minimally required for IgE binding. Afterwards, various kinds of B cell epitope structures have been clarified. Such information contributes greatly not only to the elucidation of the etiology of allergy, but also to the development of strategies for the treatment and prevention of allergy. Allergen-specific T cells also play an important role in allergy and are obvious targets for intervention in the disease. Currently, the principle approach is to modify B cell epitopes to prevent IgE binding while preserving T cell epitopes to retain the capacity for immunotherapy. There is mounting evidence that the administration of peptide(s) containing immunodominant T cell epitopes from an allergen can induce T cell nonresponsiveness (immunotherapy). There have been clinical studies of peptide immunotherapy performed, the most promising being for bee venom sensitivity. Clinical trials of immunotherapy for cat allergen peptide have also received attention. An alternative strategy for the generation of an effective but hypoallergenic preparation for immunotherapy is to modify T cell epitope peptides by, for example, single amino acid substitution. In this article, I will present an overview of epitopes related to allergic disease, particularly stress on

  4. 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.

  5. Efficient Covalent Bond Formation in Gas-Phase Peptide-Peptide Ion Complexes with the Photoleucine Stapler.

    PubMed

    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 (13)C 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. Graphical Abstract ᅟ.

  6. 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

  7. 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.

  8. 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…

  9. Design of cell-permeable stapled peptides as HIV-1 integrase inhibitors.

    PubMed

    Long, Ya-Qiu; Huang, Shao-Xu; Zawahir, Zahrah; Xu, Zhong-Liang; Li, Huiyuan; Sanchez, Tino W; Zhi, Ying; De Houwer, Stephanie; Christ, Frauke; Debyser, Zeger; Neamati, Nouri

    2013-07-11

    HIV-1 integrase (IN) catalyzes the integration of viral DNA into the host genome, involving several interactions with the viral and cellular proteins. We have previously identified peptide IN inhibitors derived from the α-helical regions along the dimeric interface of HIV-1 IN. Herein, we show that appropriate hydrocarbon stapling of these peptides to stabilize their helical structure remarkably improves the cell permeability, thus allowing inhibition of the HIV-1 replication in cell culture. Furthermore, the stabilized peptides inhibit the interaction of IN with the cellular cofactor LEDGF/p75. Cellular uptake of the stapled peptide was confirmed in four different cell lines using a fluorescein-labeled analogue. Given their enhanced potency and cell permeability, these stapled peptides can serve as not only lead IN inhibitors but also prototypical biochemical probes or "nanoneedles" for the elucidation of HIV-1 IN dimerization and host cofactor interactions within their native cellular environment.

  10. Label-free detection of immune complexes with myeloid cells.

    PubMed

    Szittner, Z; Bentlage, A E H; Rovero, P; Migliorini, P; Lóránd, V; Prechl, J; Vidarsson, G

    2016-07-01

    The aim of this study was to provide proof-of-concept for quantitative and qualitative label-free detection of immune complexes through myeloid cells with imaging surface plasmon resonance. Surface plasmon resonance imaging was first applied to monitor the binding of human sera from healthy and rheumatoid arthritis (RA) patients to immobilized citrullinated RA-specific peptide antigens, histone citrullinated peptide 2 (HCP2) and viral citrullinated peptide 2 (VCP2). Next, the binding of monocytoid cell line U937 to the resulting immune complexes on the sensor surface was monitored. As control, binding of U937 was monitored to immunoglobulin (Ig)G subclasses simultaneously. Cell response results were compared to results of cyclic citrullinated peptide 2 (CCP2) enzyme-linked immunosorbent assay (ELISA), clinical RA diagnosis and antigen-specific antibody distribution of the samples. Human IgG3 triggered the most pronounced response, followed by IgG1 and IgG4, while IgG2 did not result in U937 cell binding. Serum samples obtained from RA patients resulted in a significantly increased cell response to VCP2 compared to healthy controls. The strength of cell response towards VCP2 immune complexes showed significant correlation with levels of antigen-specific IgA, IgG and IgG3. Cellular responses on VCP2 immune complexes showed significant association with both CCP2-based serological positivity and European League Against Rheumatism (EULAR) criteria-based clinical RA diagnosis. Immunoglobulin-triggered binding of monocytoid cells can be monitored using a label-free multiplex technology. Because these binding events are presumably initiated by Fc receptors, the system provides a tool for biological detection of autoantibodies with diagnostic value, here exemplified by anti-citrullinated antibodies. This provides added information to antibody levels, as interaction with Fc-receptor-expressing cells is also affected by post-translational modification of the immunoglobulins.

  11. Related impurities in peptide medicines.

    PubMed

    D'Hondt, Matthias; Bracke, Nathalie; Taevernier, Lien; Gevaert, Bert; Verbeke, Frederick; Wynendaele, Evelien; De Spiegeleer, Bart

    2014-12-01

    Peptides are an increasingly important group of pharmaceuticals, positioned between classic small organic molecules and larger bio-molecules such as proteins. Currently, the peptide drug market is growing twice as fast as other drug markets, illustrating the increasing clinical as well as economical impact of this medicine group. Most peptides today are manufactured by solid-phase peptide synthesis (SPPS). This review will provide a structured overview of the most commonly observed peptide-related impurities in peptide medicines, encompassing the active pharmaceutical ingredients (API or drug substance) as well as the finished drug products. Not only is control of these peptide-related impurities and degradants critical for the already approved and clinically used peptide-drugs, these impurities also possess the capability of greatly influencing initial functionality studies during early drug discovery phases, possibly resulting in erroneous conclusions. The first group of peptide-related impurities is SPPS-related: deletion and insertion of amino acids are related to inefficient Fmoc-deprotection and excess use of amino acid reagents, respectively. Fmoc-deprotection can cause racemization of amino acid residues and thus diastereomeric impurities. Inefficient deprotection of amino acid side chains results into peptide-protection adducts. Furthermore, unprotected side chains can react with a variety of reagents used in the synthesis. Oxidation of amino acid side chains and dimeric-to-oligomeric impurities were also observed. Unwanted peptide counter ions such as trifluoroacetate, originating from the SPPS itself or from additional purification treatments, may also be present in the final peptide product. Contamination of the desired peptide product by other unrelated peptides was also seen, pointing out the lack of appropriate GMP. The second impurity group results from typical peptide degradation mechanisms such as β-elimination, diketopiperazine, pyroglutamate

  12. Metabolism of 125I-labeled lipoproteins by the isolated rat lung

    PubMed Central

    1976-01-01

    The capacity of the isolated perfused rat lung to metabolize the protein moieties of serum lipoproteins was assessed using homologous (rat) and heterologous (human) plasma lipoproteins. The protein and lipid moieties of the plasma lipoproteins were labeled in vivo with Na[125I]. In selected cases the lipoprotein peptides were labeled in vivo with 14C- or 3H-labeled amino acids. Uptake of lipoprotein label during perfusion was monitored by measure of losses in perfusate label and by rises in pulmonary tissue labeling as shown by radioassay and by light and electron microscope radioautography. Lipoprotein degradation was assessed by fractionation of perfusate and lung tissue radioactive material into trichloroacetic acid (TCA)-isoluble, TCA-soluble, and ether-ethanol-soluble fractions. When heparin was included in the perfusion medium, there was selective degradation of the protein portion of very low density lipoprotein (VLDL) in the perfusate and concomitant uptake of radioactive label by the lungs. Low density lipoprotein (LDL)) was neither taken up nor catabolized by the isolated rat lung in the absence or presence of heparin. By light and electron microscopy, the label was localized over the interalveolar septa, predominantly the capillary endothelium. Disappearance of TCA-insoluble radioactivity from the perfusate was associated with the generation of both TCA-soluble iodide and noniodide radioactivity. Greater than 50% of the radioactive label taken up by the lungs was found in the delipidated TCA-insoluble fraction. This study provides in vitro evidence for pulmonary catabolism of VLDL apolipoproteins and uptake of peptide catabolic products of VLDL by the lung. PMID:180034

  13. Peptide -- Silica Hybrid Networks

    NASA Astrophysics Data System (ADS)

    Altunbas, Aysegul; Sharma, Nikhil; Nagarkar, Radhika; Schneider, Joel; Pochan, Darrin

    2010-03-01

    In this study, a bio-inspired route was used to fabricate scaffolds that display hierarchical organization of an inorganic layer around an organic self-assembled peptide fibril template. The 20 amino acid peptide used in this study intramolecular folds into a beta-hairpin conformation on addition of a desired solution stimulus. This intramolecular folding is followed by intermolecular self-assembly of the peptides into a three dimensional network of entangled fibrils rich in beta-sheet with a high density of lysine groups exposed on the fibril-surfaces. The lysine-rich surface chemistry was utilized to create a silica shell around the fibrils. The mineralization process of the fibrils results in a rigid, porous silica network that retains the microscale and nanoscale structure of the peptide fibril network. Structural characterization via Transmission Electron Microscopy, cryogenic-Scanning Electron Microscopy, mechanical characterization via oscillatory rheology, Small Angle X-ray and Neutron Scattering of the silicified hydrogels will be presented.

  14. 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)

  15. Peptide Nanofilament Engineering

    DTIC Science & Technology

    2006-05-31

    This laboratory studied four systems involving molecular self-assembly during this project period. Each system will open a new avenue of research in developing novel applications for use in biomedical engineering and materials science. These systems include self-assembling oligopeptides that form stable beta sheets in water, peptides that form inter- and

  16. Bioinformatic identification of plant peptides.

    PubMed

    Lease, Kevin A; Walker, John C

    2010-01-01

    Plant peptides play a number of important roles in defence, development and many other aspects of plant physiology. Identifying additional peptide sequences provides the starting point to investigate their function using molecular, genetic or biochemical techniques. Due to their small size, identifying peptide sequences may not succeed using the default bioinformatic approaches that work well for average-sized proteins. There are two general scenarios related to bioinformatic identification of peptides to be discussed in this paper. In the first scenario, one already has the sequence of a plant peptide and is trying to find more plant peptides with some sequence similarity to the starting peptide. To do this, the Basic Local Alignment Search Tool (BLAST) is employed, with the parameters adjusted to be more favourable for identifying potential peptide matches. A second scenario involves trying to identify plant peptides without using sequence similarity searches to known plant peptides. In this approach, features such as protein size and the presence of a cleavable amino-terminal signal peptide are used to screen annotated proteins. A variation of this method can be used to screen for unannotated peptides from genomic sequences. Bioinformatic resources related to Arabidopsis thaliana will be used to illustrate these approaches.

  17. 16 CFR 1633.12 - Labeling.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... the Standard shall bear a permanent, conspicuous, and legible label(s) containing the following... with black text. The label text shall comply with the following format requirements: (1) All... as needed for varying information. The label must be white with black text. The label shall...

  18. 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...

  19. 21 CFR 201.72 - Potassium labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 4 2011-04-01 2011-04-01 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...

  20. 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...

  1. 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...

  2. 30 CFR 47.42 - Label contents.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Label contents. 47.42 Section 47.42 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING HAZARD COMMUNICATION (HazCom) Container Labels and Other Forms of Warning § 47.42 Label contents. When an operator must make a label, the label must— (a)...

  3. 30 CFR 47.42 - Label contents.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Label contents. 47.42 Section 47.42 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING HAZARD COMMUNICATION (HazCom) Container Labels and Other Forms of Warning § 47.42 Label contents. When an operator must make a label, the label must— (a)...

  4. 30 CFR 47.42 - Label contents.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Label contents. 47.42 Section 47.42 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING HAZARD COMMUNICATION (HazCom) Container Labels and Other Forms of Warning § 47.42 Label contents. When an operator must make a label, the label must— (a)...

  5. 30 CFR 47.42 - Label contents.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 30 Mineral Resources 1 2014-07-01 2014-07-01 false Label contents. 47.42 Section 47.42 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING HAZARD COMMUNICATION (HazCom) Container Labels and Other Forms of Warning § 47.42 Label contents. When an operator must make a label, the label must— (a)...

  6. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 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 of over-the-counter (OTC) drug products intended for oral ingestion shall contain the sodium content...

  7. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 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 of over-the-counter (OTC) drug products intended for oral ingestion shall contain the sodium content...

  8. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 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 of over-the-counter (OTC) drug products intended for oral ingestion shall contain the sodium content...

  9. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 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 of over-the-counter (OTC) drug products intended for oral ingestion shall contain the sodium content...

  10. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 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 of over-the-counter (OTC) drug products intended for oral ingestion shall contain the sodium content...

  11. 30 CFR 47.42 - Label contents.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Label contents. 47.42 Section 47.42 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING HAZARD COMMUNICATION (HazCom) Container Labels and Other Forms of Warning § 47.42 Label contents. When an operator must make a label, the label must— (a)...

  12. 21 CFR 201.71 - Magnesium labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 4 2011-04-01 2011-04-01 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...

  13. 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...

  14. 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...

  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. Cell Labeling via Membrane-Anchored Lipophilic MR Contrast Agents

    PubMed Central

    2015-01-01

    Cell tracking in vivo with MR imaging requires the development of contrast agents with increased sensitivity that effectively label and are retained by cells. Most clinically approved Gd(III)-based contrast agents require high incubation concentrations and prolonged incubation times for cellular internalization. Strategies to increase contrast agent permeability have included conjugating Gd(III) complexes to cell penetrating peptides, nanoparticles, and small molecules which have greatly improved cell labeling but have not resulted in improved cellular retention. To overcome these challenges, we have synthesized a series of lipophilic Gd(III)-based MR contrast agents that label cell membranes in vitro. Two of the agents were synthesized with a multiplexing strategy to contain three Gd(III) chelates (1 and 2) while the third contains a single Gd(III) chelate (3). These new agents exhibit significantly enhanced labeling and retention in HeLa and MDA-MB-231-mcherry cells compared to agents that are internalized by cells (4 and Prohance). PMID:24787689

  17. Photoaffinity labeling of uncoupler binding sites on mitochondrial membrane.

    PubMed

    Kurup, C K; Sanadi, D R

    1977-02-01

    3H 2-azido-4-nitrophenol, a photoactive uncoupler, has been synthesized, and its uncoupling action on oxidative phosphorylation and its binding to the mitochondrial membrane have been studied. The uncoupler bound covalently to the mitochondrial membrane on photoirradiation was 3-4 times that bound reversibly in the absence of light. When irradiation was carried out in the presence of serum albumin, covalent binding was significantly depressed. The pattern of loss of ATP-Pi exchange activity with increasing amounts of the uncoupler suggests that serum albumin prevents the binding of the uncoupler to the functional sites as well. Polyacrylamide gel electrophoresis of photoaffinity labeled submitochondrial particles in the presence of sodium dodecyl sulfate revealed that a 9000 dalton peptide bound high levels of uncoupler. Other proteins in the molecular weight range of 20,000-40,000 and 55,000 were also labeled. Photolysis in the presence of serum albumin or ATP decreased the covalent binding of the uncoupler to all the proteins, but particularly to the 20,000 dalton component. Soluble ATPase and the mitochondrial proteolipid purified from labeled mitochondria showed the presence of label.

  18. 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.

  19. 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.

  20. Labeled Cocaine Analogs

    DOEpatents

    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.

  1. 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.

  2. 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.

  3. 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

  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. 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.

  6. The vectorial release of nascent immunoglobulin peptides

    PubMed Central

    Bevan, Michael J.

    1971-01-01

    A microsomal preparation from a mouse plasmacytoma, MOPC 47A, that secretes immunoglobulin A was used to study the release of nascent immunoglobulin peptides in vitro. Nascent chains were released with puromycin and characterized with specific antiserum against the immunoglobulin product of the tumour. When the tissue had been prelabelled with [3H]leucine the experiments were complicated by the large background of completed radioactive polypeptides in the microsomal preparation. Up to one-third of the released radioactivity in the microsomal preparation could be recognized as immunoglobulin. With [3H]-puromycin as the radioactive label, however, the results are much easier to interpret, although the proportion of released radioactivity that can be identified as immunoglobulin is lower (up to one-tenth). Both types of experiment demonstrate that all of the recognizable nascent immunoglobulin chains remain in association with the microsomal vesicles after release from the ribosomes. PMID:5124814

  7. Invention of stimulus-responsive peptide-bond-cleaving residue (Spr) and its application to chemical biology tools.

    PubMed

    Shigenaga, Akira; Yamamoto, Jun; Kohiki, Taiki; Inokuma, Tsubasa; Otaka, Akira

    2017-01-19

    Elucidation of biological functions of peptides and proteins is essential for understanding peptide/protein-related biological events and developing drugs. Caged peptides and proteins that release a parent active peptide/protein by photo-irradiation have successfully been employed to elucidate the functions. Whereas the usual caged peptide/protein enables conversion of an inactive form to an active form (OFF-to-ON conversion) by photo-induced deprotection, photo-triggered main chain cleavage is reported to be applicable to ON-to-OFF conversion. These peptides and proteins are photo-responsive; however, if peptides and proteins could respond to other stimuli such as disease-related environment or enzymes, their range of application should be widened. To convert the photo-responsive peptide/protein into other stimulus-responsive peptide/protein, quite laborious de novo design and synthesis of the stimulus-responsive unit are required. In this context, we designed a stimulus-responsive peptide-bond-cleaving residue (Spr) in which the stimuli available for the main chain cleavage vary according to the choice of protecting groups on the residue. In this review, design and synthesis of Spr are introduced, and challenges to apply Spr to other fields to enable, for example, functional control, localization control, delivery of cargos, labeling of a protein of interest in living cells, and identification of target proteins of bioactive ligands are discussed. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

  8. Algorithms for Labeling Focus Regions.

    PubMed

    Fink, M; Haunert, Jan-Henrik; Schulz, A; Spoerhase, J; Wolff, A

    2012-12-01

    In this paper, we investigate the problem of labeling point sites in focus regions of maps or diagrams. This problem occurs, for example, when the user of a mapping service wants to see the names of restaurants or other POIs in a crowded downtown area but keep the overview over a larger area. Our approach is to place the labels at the boundary of the focus region and connect each site with its label by a linear connection, which is called a leader. In this way, we move labels from the focus region to the less valuable context region surrounding it. In order to make the leader layout well readable, we present algorithms that rule out crossings between leaders and optimize other characteristics such as total leader length and distance between labels. This yields a new variant of the boundary labeling problem, which has been studied in the literature. Other than in traditional boundary labeling, where leaders are usually schematized polylines, we focus on leaders that are either straight-line segments or Bezier curves. Further, we present algorithms that, given the sites, find a position of the focus region that optimizes the above characteristics. We also consider a variant of the problem where we have more sites than space for labels. In this situation, we assume that the sites are prioritized by the user. Alternatively, we take a new facility-location perspective which yields a clustering of the sites. We label one representative of each cluster. If the user wishes, we apply our approach to the sites within a cluster, giving details on demand.

  9. Label Review Training: Module 1: Label Basics, Page 6

    EPA Pesticide Factsheets

    Page 6, Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human health and the environment

  10. Label Review Training: Module 1: Label Basics, Page 5

    EPA Pesticide Factsheets

    Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human health and the environment.

  11. Label Review Training: Module 1: Label Basics, Page 8

    EPA Pesticide Factsheets

    Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human he

  12. Label Review Training: Module 1: Label Basics, Page 9

    EPA Pesticide Factsheets

    Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human health and the environment.

  13. Label Review Training: Module 1: Label Basics, Page 2

    EPA Pesticide Factsheets

    Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human health and the environment.

  14. Label Review Training: Module 1: Label Basics, Page 4

    EPA Pesticide Factsheets

    Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human health and the environment.

  15. Label Review Training: Module 1: Label Basics, Page 3

    EPA Pesticide Factsheets

    Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human health and the environment.

  16. 49 CFR 172.442 - CORROSIVE label.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION HAZARDOUS MATERIALS REGULATIONS HAZARDOUS MATERIALS TABLE, SPECIAL... SECURITY PLANS Labeling § 172.442 CORROSIVE label. (a) Except for size and color, the CORROSIVE label...

  17. 49 CFR 172.442 - CORROSIVE label.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION HAZARDOUS MATERIALS REGULATIONS HAZARDOUS MATERIALS TABLE, SPECIAL... SECURITY PLANS Labeling § 172.442 CORROSIVE label. (a) Except for size and color, the CORROSIVE label...

  18. 49 CFR 172.442 - CORROSIVE label.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION HAZARDOUS MATERIALS REGULATIONS HAZARDOUS MATERIALS TABLE, SPECIAL... SECURITY PLANS Labeling § 172.442 CORROSIVE label. (a) Except for size and color, the CORROSIVE label...

  19. 40 CFR 94.212 - Labeling.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... shall be of a color that contrasts with the background of the label: (1) The label heading: Marine...) to be designated as Blue Sky Series engines must contain the statement on the label: “Blue Sky...

  20. 40 CFR 94.212 - Labeling.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... shall be of a color that contrasts with the background of the label: (1) The label heading: Marine...) to be designated as Blue Sky Series engines must contain the statement on the label: “Blue Sky...

  1. 40 CFR 94.212 - Labeling.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... shall be of a color that contrasts with the background of the label: (1) The label heading: Marine...) to be designated as Blue Sky Series engines must contain the statement on the label: “Blue Sky...

  2. 40 CFR 94.212 - Labeling.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... shall be of a color that contrasts with the background of the label: (1) The label heading: Marine...) to be designated as Blue Sky Series engines must contain the statement on the label: “Blue Sky...

  3. 16 CFR 305.17 - Television labeling.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... STAR logo on the label as illustrated in Sample Labels in appendix L. The logo must be 0.375″ wide... Environmental Protection Agency covering the televisions to be labeled may add the ENERGY STAR logo to...

  4. 21 CFR 331.80 - Professional labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... HUMAN USE ANTACID PRODUCTS FOR OVER-THE-COUNTER (OTC) HUMAN USE Labeling § 331.80 Professional labeling..., muscle weakness, and osteomalacia. (b) Professional labeling for an antacid-antiflatulent combination...

  5. 21 CFR 331.80 - Professional labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... HUMAN USE ANTACID PRODUCTS FOR OVER-THE-COUNTER (OTC) HUMAN USE Labeling § 331.80 Professional labeling..., muscle weakness, and osteomalacia. (b) Professional labeling for an antacid-antiflatulent combination...

  6. 21 CFR 331.80 - Professional labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... HUMAN USE ANTACID PRODUCTS FOR OVER-THE-COUNTER (OTC) HUMAN USE Labeling § 331.80 Professional labeling..., muscle weakness, and osteomalacia. (b) Professional labeling for an antacid-antiflatulent combination...

  7. Soil Fumigant Labels - Dimethyl Disulfide (DMDS)

    EPA Pesticide Factsheets

    Search by EPA registration number, product name, or company and follow the link to the Pesticide Product Labeling System (PPLS) for label details. Updated labels include new safety requirements for buffer zones and related measures.

  8. Mobile Application for Pesticide Label Matching

    EPA Pesticide Factsheets

    The label matching application will give inspectors the ability to instantly compare pesticide product labels against state and federal label databases via their cell phone, tablet or other mobile device.

  9. 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.

  10. 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.

  11. D-[Ala1]-peptide T-amide is transported from blood to brain by a saturable system.

    PubMed

    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 125I-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 125I-Peptide T analog indicate that saturable systems can transport peptides from the blood into the central nervous system.

  12. 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.

  13. Multi-frequency, multi-technique pulsed EPR investigation of the copper binding site of murine amyloid β peptide.

    PubMed

    Kim, Donghun; Bang, Jeong Kyu; Kim, Sun Hee

    2015-01-26

    Copper-amyloid peptides are proposed to be the cause of Alzheimer's disease, presumably by oxidative stress. However, mice do not produce amyloid plaques and thus do not suffer from Alzheimer's disease. Although much effort has been focused on the structural characterization of the copper- human amyloid peptides, little is known regarding the copper-binding mode in murine amyloid peptides. Thus, we investigated the structure of copper-murine amyloid peptides through multi-frequency, multi-technique pulsed EPR spectroscopy in conjunction with specific isotope labeling. Based on our pulsed EPR results, we found that Ala2, Glu3, His6, and His14 are directly coordinated with the copper ion in murine amyloid β peptides at pH 8.5. This is the first detailed structural characterization of the copper-binding mode in murine amyloid β peptides. This work may advance the knowledge required for developing inhibitors of Alzheimer's disease.

  14. VGF Changes during the Estrous Cycle: A Novel Endocrine Role for TLQP Peptides?

    PubMed Central

    Noli, Barbara; Brancia, Carla; D’Amato, Filomena; Ferri, Gian-Luca; Cocco, Cristina

    2014-01-01

    Although the VGF derived peptide TLQP-21 stimulates gonadotropin-releasing hormone (GnRH) and gonadotropin secretion, available data on VGF peptides and reproduction are limited. We used antibodies specific for the two ends of the VGF precursor, and for two VGF derived peptides namely TLQP and PGH, to be used in immunohistochemistry and enzyme-linked immunosorbent assay complemented with gel chromatography. In cycling female rats, VGF C-/N-terminus and PGH peptide antibodies selectively labelled neurones containing either GnRH, or kisspeptin (VGF N-terminus only), pituitary gonadotrophs and lactotrophs, or oocytes (PGH peptides only). Conversely, TLQP peptides were restricted to somatostatin neurones, gonadotrophs, and ovarian granulosa, interstitial and theca cells. TLQP levels were highest, especially in plasma and ovary, with several molecular forms shown in chromatography including one compatible with TLQP-21. Among the cycle phases, TLQP levels were higher during metestrus-diestrus in median eminence and pituitary, while increased in the ovary and decreased in plasma during proestrus. VGF N- and C-terminus peptides also showed modulations over the estrous cycle, in median eminence, pituitary and plasma, while PGH peptides did not. In ovariectomised rats, plasmatic TLQP peptide levels showed distinct reduction suggestive of a major origin from the ovary, while the estrogen-progesterone treatment modulated VGF C-terminus and TLQP peptides in the hypothalamus-pituitary complex. In in vitro hypothalamus, TLQP-21 stimulated release of growth hormone releasing hormone but not of somatostatin. In conclusion, various VGF peptides may regulate the hypothalamus-pituitary complex via specific neuroendocrine mechanisms while TLQP peptides may act at further, multiple levels via endocrine mechanisms involving the ovary. PMID:25280008

  15. Location of the epidermal growth factor binding site on the EGF receptor. A resonance energy transfer study.

    PubMed

    Carraway, K L; Koland, J G; Cerione, R A

    1990-09-18

    As a first step toward developing a structural map of key sites on the epidermal growth factor (EGF) receptor, we have used resonance energy transfer to measure the distance of closest approach between the receptor-bound growth factor molecule and lipid molecules at the surface of the plasma membrane. EGF, specifically labeled at its amino terminus with fluorescein 5-isothiocyanate, was used as an energy donor in these experiments, while either octadecylrhodamine B or octadecylrhodamine 101, inserted into plasma membranes isolated from human epidermoid carcinoma (A431) cells, served as the energy acceptors. The energy transfer measurements indicate that the amino terminus of the bound growth factor is about 67 A away from the plasma membrane. On the basis of the dimensions of the EGF molecule, this suggests that EGF binds to a site on its receptor that is a considerable distance (52-82 A) from the surface of these cells. Identical results were obtained under conditions where the receptor functions as an active tyrosine kinase, suggesting that the relative juxtaposition of the EGF binding domain to the membrane surface does not change with receptor autophosphorylation or with the activation of the receptor tyrosine kinase activity.

  16. Human milk mucin 1 and mucin 4 inhibit Salmonella enterica serovar Typhimurium invasion of human intestinal epithelial cells in vitro.

    PubMed

    Liu, Bo; Yu, Zhuoteng; Chen, Ceng; Kling, David E; Newburg, David S

    2012-08-01

    Many human milk glycans inhibit pathogen binding to host receptors and their consumption by infants is associated with reduced risk of disease. Salmonella infection is more frequent among infants than among the general population, but the incidence is lower in breast-fed babies, suggesting that human milk could contain components that inhibit Salmonella. This study aimed to test whether human milk per se inhibits Salmonella invasion of human intestinal epithelial cells in vitro and, if so, to identify the milk components responsible for inhibition. Salmonella enterica serovar Typhimurium SL1344 (SL1344) invasion of FHs 74 Int and Caco-2 cells were the models of human intestinal epithelium infection. Internalization of fluorescein-5-isothiocyanate-labeled SL1344 into intestinal cells was measured by flow cytometry to quantify infection. Human milk and its fractions inhibited infection; the inhibitory activity localized to the high molecular weight glycans. Mucin 1 and mucin 4 were isolated to homogeneity. At 150 μg/L, a typical concentration in milk, human milk mucin 1 and mucin 4 inhibited SL1344 invasion of both target cell types. These mucins inhibited SL1344 invasion of epithelial cells in a dose-dependent manner. Thus, mucins may prove useful as a basis for developing novel oral prophylactic and therapeutic agents that inhibit infant diseases caused by Salmonella and related pathogens.

  17. Concepts for Biologically Active Peptides

    PubMed Central

    Kastin, Abba J.; Pan, Weihong

    2012-01-01

    Here we review a unique aspect of CNS research on biologically active peptides that started against a background of prevalent dogmas but ended by exerting considerable influence on the field. During the course of refuting some doctrines, we introduced several concepts that were unconventional and paradigm-shifting at the time. We showed that (1) hypothalamic peptides can act ‘up’ on the brain as well as ‘down’ on the pituitary, (2) peripheral peptides can affect the brain, (3) peptides can cross the blood-brain barrier, (4) the actions of peptides can persist longer than their half-lives in blood, (5) perinatal administration of peptides can exert actions persisting into adulthood, (6) a single peptide can have more than one action, (7) dose-response relationships of peptides need not be linear, (8) the brain produces antiopiate as well as opiate peptides, (9) there is a selective high affinity endogenous peptide ligand for the mu-opiate receptor, (10) a peptide’s name does not restrict its effects, and (11) astrocytes assume an active role in response to metabolic disturbance and hyperleptinemia. The evolving questions in our laboratories reflect the diligent effort of the neuropeptide community to identify the roles of peptides in the CNS. The next decade is expected to see greater progress in the following areas: (a) interactions of peptides with other molecules in the CNS; (b) peptide involvement in cell-cell interactions; and (c) peptides in neuropsychiatric, autoimmune, and neurodegenerative diseases. The development of peptidomics and gene silencing approaches will expedite the formation of many new concepts in a new era. PMID:20726835

  18. Approximation Algorithms for Free-Label Maximization

    NASA Astrophysics Data System (ADS)

    de Berg, Mark; Gerrits, Dirk H. P.

    Inspired by air traffic control and other applications where moving objects have to be labeled, we consider the following (static) point labeling problem: given a set P of n points in the plane and labels that are unit squares, place a label with each point in P in such a way that the number of free labels (labels not intersecting any other label) is maximized. We develop efficient constant-factor approximation algorithms for this problem, as well as PTASs, for various label-placement models.

  19. 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.

  20. Identification of oligomerizing peptides.

    PubMed

    Dhiman, A; Rodgers, M E; Schleif, R

    2001-06-08

    The AraC DNA binding domain is inactive in a monomeric form but can activate transcription from the arabinose operon promoters upon its dimerization. We used this property to identify plasmids encoding peptide additions to the AraC DNA binding domain that could dimerize the domain. We generated a high diversity library of plasmids by inserting 90-base oligonucleotides of random sequence ahead of DNA coding for the AraC DNA binding domain in an expression vector, transforming, and selecting colonies containing functional oligomeric peptide-AraC DNA binding domain chimeric proteins by their growth on minimal arabinose medium. Six of seven Ara(+) candidates were partially characterized, and one was purified. Equilibrium analytical centrifugation experiments showed that it dimerizes with a dissociation constant of approximately 2 micrometer.

  1. Avian host defense peptides.

    PubMed

    Cuperus, Tryntsje; Coorens, Maarten; van Dijk, Albert; Haagsman, Henk P

    2013-11-01

    Host defense peptides (HDPs) are important effector molecules of the innate immune system of vertebrates. These antimicrobial peptides are also present in invertebrates, plants and fungi. HDPs display broad-spectrum antimicrobial activities and fulfill an important role in the first line of defense of many organisms. It is becoming increasingly clear that in the animal kingdom the functions of HDPs are not confined to direct antimicrobial actions. Research in mammals has indicated that HDPs have many immunomodulatory functions and are also involved in other physiological processes ranging from development to wound healing. During the past five years our knowledge about avian HDPs has increased considerably. This review addresses our current knowledge on the evolution, regulation and biological functions of HDPs of birds.

  2. New Labeling for Neonicotinoid Pesticides

    EPA Pesticide Factsheets

    These documents, a graphic of the bee advisory box and letters to pesticide registrants, describe steps by EPA to change pesticide labels to better protect pollinators by being clearer and more precise in their directions for pesticide application.

  3. Relaxation labeling using modular operators

    SciTech Connect

    Duncan, J.S.; Frei, W.

    1983-01-01

    Probabilistic relaxation labeling has been shown to be useful in image processing, pattern recognition, and artificial intelligence. The approaches taken to date have been encumbered with computationally extensive summations which generally prevent real-time operation and/or easy hardware implementation. The authors present a new and unique approach to the relaxation labeling problem using modular, VLSI-oriented hierarchical complex operators. One of the fundamental concepts of this work is the representation of the probability distribution of the possible labels for a given object (pixel) as an ellipse, which may be summed with neighboring object's distribution ellipses, resulting in a new, relaxed label space. The mathematical development of the elliptical approach will be presented and compared to more classical approaches, and a hardware block diagram that shows the implementation of the relaxation scheme using vlsi chips will be presented. Finally, results will be shown which illustrate applications of the modular scheme, iteratively, to both edges and lines. 13 references.

  4. "Off-Label" Drug Use

    MedlinePlus

    ... the Connecticut Attorney General for possible promotion and marketing of the off-label uses of the drug. ... cited improved energy and quality of life. The marketing of these three drugs and the doses used ...

  5. Meat and Poultry Labeling Terms

    MedlinePlus

    ... Food Safety and Inspection Service and the Agriculture Marketing Service have officially evaluated a meat product for ... refer to these factsheets from the USDA Agricultural Marketing Service: Organic Food Standards and Labels: The Facts ...

  6. How to Read Drug Labels

    MedlinePlus

    ... Home > Healthy Aging > Drugs and alternative medicine Healthy Aging How to read drug labels Printer-friendly version ... html Connect with other organizations National Institute on Aging, NIH, HHS http://www.nia.nih.gov/ U.S. ...

  7. Locating the Vehicle Emissions Label

    EPA Pesticide Factsheets

    The EPA vehicle emissions label is entitled Vehicle Emission Control Information and contains the name and trademark of the manufacturer and an unconditional statement of compliance with EPA emission regulations.

  8. Evaluation of Phosphatidylserine-Binding Peptides Radiolabeled with Fluorine 18 for in vivo Imaging of Apoptosis

    NASA Astrophysics Data System (ADS)

    Kapty, Janice Sarah

    We currently do not have a clinical method to directly assess apoptosis induced by cancer therapies. Phosphatidylserine (PS) is an attractive target for imaging apoptosis since it is on the exterior of the apoptotic cells and PS externalization is an early marker of apoptosis. PS-binding peptides are an attractive option for developing an imaging probe to detect apoptosis using positron emission tomography. In this study we evaluated binding characteristics of PS-binding peptides for ability to bind to PS, radiolabeled PS-binding peptides with fluorine-18, and performed in vitro and in vivo analysis of 18F radiolabeled PS-binding peptides including biodistribution analysis and dynamic PET imaging in a murine tumor model of apoptosis. Four peptides were evaluated for PS binding characteristics using a plate based assay system, a liposome mimic of cell membrane PS presentation, and a cell assay of apoptosis. The results indicate that all four peptides bind to PS and are specific to apoptotic cells. The widely used 18 F prosthetic group N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) and the recently developed N-[6-(4-[ 18F]fluorobenzylidene) aminooxyhexyl]maleimide ([18F]FBAM) were investigated for radiolabeling of two representative phosphatidylserine-binding peptides. The prosthetic groups were compared with respect to required reaction conditions for optimum labeling, radiolabeling yield and chemoselectivity. The N-terminus labeled product produced by reaction of [18F]SFB with binding peptide LIKKPF was produced in 18% radiochemical yield while no N-terminus labeled product could be isolated following [18F]SFB reaction with PDGLSR. When the peptides were modified by addition of a cysteine residue at the N-terminus they provided almost quantitative radiochemical yields with [18F]FBAM. Results indicate that for the peptides in this study, [18F]FBAM is a more useful prosthetic group compared to [18F]SFB due to its excellent chemo-selectivity and high radiochemical

  9. Antimicrobial Peptides and Colitis

    PubMed Central

    Ho, Samantha; Pothoulakis, Charalabos; Koon, Hon Wai

    2013-01-01

    Antimicrobial peptides (AMPs) are important components of innate immunity. They are often expressed in response to colonic inflammation and infection. Over the last several years, the roles of several antimicrobial peptides have been explored. Gene expression of many AMPs (beta defensin HBD2-4 and cathelicidin) is induced in response to invasion of gut microbes into the mucosal barrier. Some AMPs are expressed in a constitutive manner (alpha defensin HD 5-6 and beta defensin HBD1), while others (defensin and bactericidal/permeability increasing protein BPI) are particularly associated with Inflammatory Bowel Disease (IBD) due to altered defensin expression or development of autoantibodies against Bactericidal/permeability increasing protein (BPI). Various AMPs have different spectrum and strength of antimicrobial effects. Some may play important roles in modulating the colitis (cathelicidin) while others (lactoferrin, hepcidin) may represent biomarkers of disease activity. The use of AMPs for therapeutic purposes is still at an early stage of development. A few natural AMPs were shown to be able to modulate colitis when delivered intravenously or intracolonically (cathelicidin, elafin and SLPI) in mouse colitis models. New AMPs (synthetic or artificial non-human peptides) are being developed and may represent new therapeutic approaches against colitis. This review discusses the latest research developments in the AMP field with emphasis in innate immunity and pathophysiology of colitis. PMID:22950497

  10. 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.

  11. Decision peptide-driven: a free software tool for accurate protein quantification using gel electrophoresis and matrix assisted laser desorption ionization time of flight mass spectrometry.

    PubMed

    Santos, Hugo M; Reboiro-Jato, Miguel; Glez-Peña, Daniel; Nunes-Miranda, J D; Fdez-Riverola, Florentino; Carvallo, R; Capelo, J L

    2010-09-15

    The decision peptide-driven tool implements a software application for assisting the user in a protocol for accurate protein quantification based on the following steps: (1) protein separation through gel electrophoresis; (2) in-gel protein digestion; (3) direct and inverse (18)O-labeling and (4) matrix assisted laser desorption ionization time of flight mass spectrometry, MALDI analysis. The DPD software compares the MALDI results of the direct and inverse (18)O-labeling experiments and quickly identifies those peptides with paralleled loses in different sets of a typical proteomic workflow. Those peptides are used for subsequent accurate protein quantification. The interpretation of the MALDI data from direct and inverse labeling experiments is time-consuming requiring a significant amount of time to do all comparisons manually. The DPD software shortens and simplifies the searching of the peptides that must be used for quantification from a week to just some minutes. To do so, it takes as input several MALDI spectra and aids the researcher in an automatic mode (i) to compare data from direct and inverse (18)O-labeling experiments, calculating the corresponding ratios to determine those peptides with paralleled losses throughout different sets of experiments; and (ii) allow to use those peptides as internal standards for subsequent accurate protein quantification using (18)O-labeling. In this work the DPD software is presented and explained with the quantification of protein carbonic anhydrase.

  12. Solution structure of nickel-peptide deformylase.

    PubMed

    Dardel, F; Ragusa, S; Lazennec, C; Blanquet, S; Meinnel, T

    1998-07-17

    In the accompanying paper, we report that zinc is unlikely to be the co-factor supporting peptide deformylase activity in vivo. In contrast, nickel binding promotes full enzyme activity. The three-dimensional structure of the resulting nickel-containing peptide deformylase (catalytic domain, residues 1 to 147) was solved by NMR using a 13C-15N-doubly labelled protein sample. A set of 2261 restraints could be collected, with an average of 15.4 per amino acid. The resolution, which shows a good definition for the position of most side-chains, is greatly improved compared to that previously reported for the zinc-containing, inactive form. A comparison of the two stuctures indicates however that both share the same 3D organization. This shows that the nature of the bound metal is the primary determinant of the hydrolytic activity of this enzyme. Site-directed mutagenesis enabled us to determine the conserved residues of PDF involved in the structure of the active site. In particular, a buried arginine appears to be critical for the positioning of Cys90, one of the metal ligands. Furthermore, the 3D structure of peptide deformylase was compared to thermolysin and metzincins. Although the structural folds are very different, they all display a common structural motif involving an alpha-helix and a three-stranded beta-sheet. These conserved structural elements build a common scaffold which includes the active site, suggesting a common hydrolytic mechanism for these proteases. Finally, an invariant glycine shared by both PDF and metzincins enables us to extend the conserved motif from HEXXH to HEXXHXXG.

  13. Quantitative label-free phosphoproteomics strategy for multifaceted experimental designs.

    PubMed

    Soderblom, Erik J; Philipp, Melanie; Thompson, J Will; Caron, Marc G; Moseley, M Arthur

    2011-05-15

    Protein phosphorylation is a critical regulator of signaling in nearly all eukaryotic cellular pathways and dysregulated phosphorylation has been implicated in an array of diseases. The majority of MS-based quantitative phosphorylation studies are currently performed from transformed cell lines because of the ability to generate large amounts of starting material with incorporated isotopically labeled amino acids during cell culture. Here we describe a general label-free quantitative phosphoproteomic strategy capable of directly analyzing relatively small amounts of virtually any biological matrix, including human tissue and biological fluids. The strategy utilizes a TiO(2) enrichment protocol in which the selectivity and recovery of phosphopeptides were optimized by assessing a twenty-point condition matrix of binding modifier concentrations and peptide-to-resin capacity ratios. The quantitative reproducibility of the TiO(2) enrichment was determined to be 16% RSD through replicate enrichments of a wild-type Danio rerio (zebrafish) lysate. Measured phosphopeptide fold-changes from alpha-casein spiked into wild-type zebrafish lysate backgrounds were within 5% of the theoretical value. Application to a morpholino induced knock-down of G protein-coupled receptor kinase 5 (GRK5) in zebrafish embryos resulted in the quantitation of 719 phosphorylated peptides corresponding to 449 phosphorylated proteins from 200 μg of zebrafish embryo lysates.

  14. Photoaffinity labeling of cytochrome P4501A1 with azidocumene: identification of cumene hydroperoxide binding region.

    PubMed

    Cvrk, T; Strobel, H W

    1998-01-01

    Cumene hydroperoxide can support cytochrome P450-catalyzed reactions in the absence of molecular oxygen, NADPH, and cytochrome P450-NADPH oxidoreductase. Its binding at the cytochrome P450 active site is governed by the structure of the cumene hydroperoxide binding region. In order to define the region of cytochrome P4501A1 at which cumene hydroperoxide binds, we prepared an analog of cumene hydroperoxide for use as a photoaffinity label. p-Azido-isopro-pylbenzene (azidocumene) and its tritiated derivative were photolyzed in water solution by uv light with a half-life of 29 s. The 7-ethoxycoumarin deethylatation catalyzed by P450 using the cumene hydroperoxide-supported system was strongly inhibited by the presence of the label. Covalent binding to the protein after photoactivation was blocked by 50% in the presence of cumene hydroperoxide. HPLC analysis after trypsin digestion of the labeled protein showed that [3H]-azidocumene was attached covalently to the peptide VDMTPAYGLTLK corresponding to residues 492-503 in the 1A1 sequence. The radioactivity level of this fraction was reduced by 50% when the labeling was carried out in the presence of cumene hydroperoxide. To confirm the identified region the labeled protein was cleaved by cyanogen bromide. HPLC separation of the CNBr digest showed two peaks with a high level of radioactivity. The SDS/Tricine PAGE analysis of the radioactive fraction with an elution time of 43 min revealed a 2.4-kDa peptide carrying a high level of covalently bound radioactivity. The N-terminal sequence identified the labeled peptide to be a fragment generated by CNBr corresponding to residues 494-512. The N-terminal sequence of the labeled peptide with elution time of 27 min, TLKH, matches amino acid residues 501-504 in the P4501A1 sequence. We can conclude that in the overlapping region of all three identified peptides, T501-L502-K503, is the site where azidocumene covalently binds to P4501A1. The sequence alignment of cytochrome P4501A

  15. A peptide-retrieval strategy enables significant improvement of quantitative performance without compromising confidence of identification.

    PubMed

    Tu, Chengjian; Shen, Shichen; Sheng, Quanhu; Shyr, Yu; Qu, Jun

    2017-01-30

    Reliable quantification of low-abundance proteins in complex proteomes is challenging largely owing to the limited number of spectra/peptides identified. In this study we developed a straightforward method to improve the quantitative accuracy and precision of proteins by strategically retrieving the less confident peptides that were previously filtered out using the standard target-decoy search strategy. The filtered-out MS/MS spectra matched to confidently-identified proteins were recovered, and the peptide-spectrum-match FDR were re-calculated and controlled at a confident level of FDR≤1%, while protein FDR maintained at ~1%. We evaluated the performance of this strategy in both spectral count- and ion current-based methods. >60% increase of total quantified spectra/peptides was respectively achieved for analyzing a spike-in sample set and a public dataset from CPTAC. Incorporating the peptide retrieval strategy significantly improved the quantitative accuracy and precision, especially for low-abundance proteins (e.g. one-hit proteins). Moreover, the capacity of confidently discovering significantly-altered proteins was also enhanced substantially, as demonstrated with two spike-in datasets. In summary, improved quantitative performance was achieved by this peptide recovery strategy without compromising confidence of protein identification, which can be readily implemented in a broad range of quantitative proteomics techniques including label-free or labeling approaches.

  16. Characterization of the triphenylphosphonium derivative of peptides by fast atom bombardment-tandem mass spectrometry, and investigations of the mechanisms of fragmentation of peptides

    SciTech Connect

    Wagner, D.S.

    1992-01-01

    Fast atom bombardment collisionally activated dissociation tandem mass spectrometry is a powerful technique for the determination of the primary structure of peptides. However, there are factors that frequently prevent successful sequence analysis by mass spectrometry. Two such factors are the poor ionization efficiency of some hydrophilic peptides and, for many peptides, ambiguities in interpretation of the spectra when key sequence ions are weak or absent. Novel and simple procedures for preparing ethyl-triphenylphosphonium derivatives of peptides are described. These procedures allow an ethyl-triphenylphosphonium moiety to be selectively attached to either the N- or C-terminus. Modification of peptides by these chemical methods significantly enhances the efficiency of fast atom bombardment ionization. Moreover, upon collisionally activated dissociation, the derivatized peptides generate a predictable series of sequence ions from either the C-terminus or the N-terminus, depending on the location of the ethyl-triphenylphosphonium moiety. The potential utility of the ethyl-triphenylphosphonium derivative in structure elucidation is illustrated by a comparison of the mass spectra of underivatized and derivatized peptides containing up to 20 amino acid residues, or contain an N-terminal blocking group, or contain a phosphate group, or contain a disulfide bond, or contain a backbone modification. When protonated peptide molecules and cationized peptide molecules are subjected to high-energy collisionally activated dissociation, skeletal bonds cleave generating sequence-specific fragment ions. These bond cleavages usually involve H-shifts. The utility of selective deuterium labeling was applied here to elucidate fragmentation mechanisms. Skeletal bond cleavages in the ionized peptide H-VGVAPG-OH were investigated, in which the molecule was analyzed in the protonated form, cationized form, or as the charge-localized ethyl-triphenylphosphonium derivative.

  17. Spontaneous intermolecular amide bond formation between side chains for irreversible peptide targeting.

    PubMed

    Zakeri, Bijan; Howarth, Mark

    2010-04-07

    Peptides and synthetic peptide-like molecules are powerful tools for analysis and control of biological function. One major limitation of peptides is the instability of their interactions with biomolecules, because of the limited accessible surface area for noncovalent interactions and the intrinsic flexibility of peptides. Peptide tags are nonetheless fundamental for protein detection and purification, because their small size minimizes the perturbation to protein function. Here we have designed a 16 amino acid peptide that spontaneously forms an amide bond to a protein partner, via reaction between lysine and asparagine side chains. This depended upon splitting a pilin subunit from a human pathogen, Streptococcus pyogenes, which usually undergoes intramolecular amide bond formation to impart mechanical and proteolytic stability to pili. Reaction of the protein partner was able to proceed to 98% conversion. The amide bond formation was independent of redox state and occurred at pH 5-8. The reaction was efficient in phosphate buffered saline and a wide range of biological buffers. Surprisingly, amide bond formation occurred at a similar rate at 4 and 37 degrees C. Both peptide and protein partners are composed of the regular 20 amino acids and reconstituted efficiently inside living E. coli. Labeling also showed high specificity on the surface of mammalian cells. Irreversible targeting of a peptide tag may have application in bioassembly, in cellular imaging, and to lock together proteins subject to high biological forces.

  18. 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

    2016-08-24

    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.

  19. Antimicrobial peptides temporins B and L induce formation of tubular lipid protrusions from supported phospholipid bilayers.

    PubMed

    Domanov, Yegor A; Kinnunen, Paavo K J

    2006-12-15

    The binding of the antimicrobial peptides temporins B and L to supported lipid bilayer (SLB) model membranes composed of phosphatidylcholine and phosphatidylglycerol (4:1, mol/mol) caused the formation of fibrillar protrusions, visible by fluorescent microscopy of both a fluorescent lipid analog and a labeled peptide. Multicolor imaging at low peptide-to-lipid ratios (P/L < approximately 1:5) revealed an initial in-plane segregation of membrane-bound peptide and partial exclusion of lipid from the peptide-enriched areas. Subsequently, at higher P/L numerous flexible lipid fibrils were seen growing from the areas enriched in lipid. The fibrils have diameters <250 nm and lengths of up to approximately 1 mm. Fibril formation reduces the in-plane heterogeneity and results in a relatively even redistribution of bound peptide over the planar bilayer and the fibrils. Physical properties of the lipid fibrils suggest that they have a tubular structure. Our data demonstrate that the peptide-lipid interactions alone can provide a driving force for the spontaneous membrane shape transformations leading to tubule outgrowth and elongation. Further experiments revealed the importance of positive curvature strain in the tubulation process as well as the sufficient positive charge on the peptide (>/=+2). The observed membrane transformations could provide a simplified in vitro model for morphogenesis of intracellular tubular structures and intercellular connections.

  20. Antimicrobial Peptides Temporins B and L Induce Formation of Tubular Lipid Protrusions from Supported Phospholipid Bilayers

    PubMed Central

    Domanov, Yegor A.; Kinnunen, Paavo K. J.

    2006-01-01

    The binding of the antimicrobial peptides temporins B and L to supported lipid bilayer (SLB) model membranes composed of phosphatidylcholine and phosphatidylglycerol (4:1, mol/mol) caused the formation of fibrillar protrusions, visible by fluorescent microscopy of both a fluorescent lipid analog and a labeled peptide. Multicolor imaging at low peptide-to-lipid ratios (P/L < ∼1:5) revealed an initial in-plane segregation of membrane-bound peptide and partial exclusion of lipid from the peptide-enriched areas. Subsequently, at higher P/L numerous flexible lipid fibrils were seen growing from the areas enriched in lipid. The fibrils have diameters <250 nm and lengths of up to ∼1 mm. Fibril formation reduces the in-plane heterogeneity and results in a relatively even redistribution of bound peptide over the planar bilayer and the fibrils. Physical properties of the lipid fibrils suggest that they have a tubular structure. Our data demonstrate that the peptide-lipid interactions alone can provide a driving force for the spontaneous membrane shape transformations leading to tubule outgrowth and elongation. Further experiments revealed the importance of positive curvature strain in the tubulation process as well as the sufficient positive charge on the peptide (≥+2). The observed membrane transformations could provide a simplified in vitro model for morphogenesis of intracellular tubular structures and intercellular connections. PMID:16997872

  1. 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

  2. Patched targeting peptides for imaging and treatment of hedgehog positive breast tumors.

    PubMed

    Smith, Daniel; Kong, Fanlin; Yang, David; Larson, Richard; Sims-Mourtada, Jennifer; Woodward, Wendy A

    2014-01-01

    High tumor hedgehog expression is correlated with poor prognosis in invasive ductal carcinoma. Peptides which bind the patched receptor have recently been reported to have a growth inhibitory effect in tumors with activated hedgehog signaling. We sought to examine growth inhibition with these peptides in breast cancer cells and use these peptides as molecular imaging probes to follow changes in hedgehog expression after chemotherapy. Significant growth inhibition was observed in breast cancer cell lines treated with PTCH-blocking peptides. Significant in vitro uptake was observed with both FITC- and (99m)Tc-EC-peptide conjugates. In vivo imaging studies displayed greater accumulation of (99m)Tc-labeled peptides within tumors as compared to adjacent muscle tissue. Patched receptor expression increased after treatment and this correlated with an increase in tumor radiotracer uptake. These studies suggest that peptides which bind the sonic hedgehog docking site in patched receptor correlate with patched expression and can be used to image patched in vivo. Further, our data suggest that radiolabeled peptides may enable us to examine the activity of the hedgehog signaling pathway and to evaluate response to anti-cancer therapies.

  3. [Hydrolysis of peptides by immobilized bacterial peptide hydrolases].

    PubMed

    Nekliudov, A D; Deniakina, E K

    2004-01-01

    The feasibility of hydrolysis of a mixture of peptides with an enzyme from the bacterium Xanthomonas rubrilineans, displaying a peptidase activity and immobilized on aluminum oxide, was studied. Kinetic schemes and equations allowing for approaching quantitative description of peptide hydrolysis in complex mixtures containing free amino acids and peptides were obtained. It was demonstrated that as a result of hydrolysis, the content of free amino acids in hydrolysates decreased 2.5- to 3-fold and the molecular weight of the constituent peptides, 2-fold.

  4. 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

  5. Fully automated software solution for protein quantitation by global metabolic labeling with stable isotopes.

    PubMed

    Bindschedler, L V; Cramer, R

    2011-06-15

    Metabolic stable isotope labeling is increasingly employed for accurate protein (and metabolite) quantitation using mass spectrometry (MS). It provides sample-specific isotopologues that can be used to facilitate comparative analysis of two or more samples. Stable Isotope Labeling by Amino acids in Cell culture (SILAC) has been used for almost a decade in proteomic research and analytical software solutions have been established that provide an easy and integrated workflow for elucidating sample abundance ratios for most MS data formats. While SILAC is a discrete labeling method using specific amino acids, global metabolic stable isotope labeling using isotopes such as (15)N labels the entire element content of the sample, i.e. for (15)N the entire peptide backbone in addition to all nitrogen-containing side chains. Although global metabolic labeling can deliver advantages with regard to isotope incorporation and costs, the requirements for data analysis are more demanding because, for instance for polypeptides, the mass difference introduced by the label depends on the amino acid composition. Consequently, there has been less progress on the automation of the data processing and mining steps for this type of protein quantitation. Here, we present a new integrated software solution for the quantitative analysis of protein expression in differential samples and show the benefits of high-resolution MS data in quantitative proteomic analyses.

  6. Broad characterization of endogenous peptides in the tree shrew visual system.

    PubMed

    Ranc, Vaclav; Petruzziello, Filomena; Kretz, Robert; Argandoña, Enrike G; Zhang, Xiaozhe; Rainer, Gregor

    2012-05-17

    Endogenous neuropeptides, acting as neurotransmitters or hormones in the brain, carry out important functions including neural plasticity, metabolism and angiogenesis. Previous neuropeptide studies have focused on peptide-rich brain regions such as the striatum or hypothalamus. Here we present an investigation of peptides in the visual system, composed of brain regions that are generally less rich in peptides, with the aim of providing the first broad overview of peptides involved in mammalian visual functions. We target three important parts of the visual system: the primary visual cortex (V1), lateral geniculate nucleus (LGN) and superior colliculus (SC). Our study is performed in the tree shrew, a close relative of primates. Using a combination of data dependent acquisition and targeted LC-MS/MS based neuropeptidomics; we identified a total of 52 peptides from the tree shrew visual system. A total of 26 peptides, for example GAV and neuropeptide K were identified in the visual system for the first time. Out of the total 52 peptides, 27 peptides with high signal-to-noise-ratio (>10) in extracted ion chromatograms (EIC) were subjected to label-free quantitation. We observed generally lower abundance of peptides in the LGN compared to V1 and SC. Consistently, a number of individual peptides showed high abundance in V1 (such as neuropeptide Y or somatostatin 28) and in SC (such as somatostatin 28 AA1-12). This study provides the first in-depth characterization of peptides in the mammalian visual system. These findings now permit the investigation of neuropeptide-regulated mechanisms of visual perception.

  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. Biodiscovery of Aluminum Binding Peptides

    DTIC Science & Technology

    2013-08-01

    display scaffold hosted by a phage (virus), bacteria, or yeast cell. This allows the selfsustaining peptide libraries to be rapidly screened for high...scaffold hosted by a phage (virus), bacteria, or yeast cell. This allows the self- sustaining peptide libraries to be rapidly screened for high...removal. An eCPX peptide display library was grown and induced as described in the paragraph above. After rinsing samples briefly in PBS, the aluminum

  9. 21 CFR 225.180 - Labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 4 2014-04-01 2014-04-01 false Labeling. 225.180 Section 225.180 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS: GENERAL CURRENT GOOD MANUFACTURING PRACTICE FOR MEDICATED FEEDS Labeling § 225.180 Labeling. Labels shall...

  10. 21 CFR 225.180 - Labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 4 2011-04-01 2011-04-01 false Labeling. 225.180 Section 225.180 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS: GENERAL CURRENT GOOD MANUFACTURING PRACTICE FOR MEDICATED FEEDS Labeling § 225.180 Labeling. Labels shall...

  11. 21 CFR 225.180 - Labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 4 2013-04-01 2013-04-01 false Labeling. 225.180 Section 225.180 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS: GENERAL CURRENT GOOD MANUFACTURING PRACTICE FOR MEDICATED FEEDS Labeling § 225.180 Labeling. Labels shall...

  12. 21 CFR 225.180 - Labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 4 2012-04-01 2012-04-01 false Labeling. 225.180 Section 225.180 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS: GENERAL CURRENT GOOD MANUFACTURING PRACTICE FOR MEDICATED FEEDS Labeling § 225.180 Labeling. Labels shall...

  13. 21 CFR 225.180 - Labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 4 2010-04-01 2010-04-01 false Labeling. 225.180 Section 225.180 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS: GENERAL CURRENT GOOD MANUFACTURING PRACTICE FOR MEDICATED FEEDS Labeling § 225.180 Labeling. Labels shall...

  14. 21 CFR 895.25 - Labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... eliminated by labeling or a change in labeling, or change in advertising if the device is a restricted device... person(s) responsible for the labeling or advertising of the device specifying: (1) The deception or risk... labeling, or change in advertising if the device is a restricted device, necessary to correct the...

  15. 40 CFR 211.108 - Sample label.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 24 2010-07-01 2010-07-01 false Sample label. 211.108 Section 211.108 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.108 Sample label. Examples of labels conforming to the requirements...

  16. 40 CFR 156.10 - Labeling requirements.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 156.10 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS LABELING REQUIREMENTS FOR PESTICIDES AND DEVICES General Provisions § 156.10 Labeling requirements. (a) General—(1) Contents of the label. Every pesticide product shall bear a label containing the...

  17. 40 CFR 156.10 - Labeling requirements.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 156.10 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS LABELING REQUIREMENTS FOR PESTICIDES AND DEVICES General Provisions § 156.10 Labeling requirements. (a) General—(1) Contents of the label. Every pesticide product shall bear a label containing the...

  18. 21 CFR 606.121 - Container label.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... color scheme may be used for differentiating ABO Blood groups: Blood group Color of label O Blue A... CURRENT GOOD MANUFACTURING PRACTICE FOR BLOOD AND BLOOD COMPONENTS Additional Labeling Standards for Blood and Blood Components § 606.121 Container label. (a) The container label requirements are designed...

  19. 40 CFR 211.104 - Label content.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 25 2011-07-01 2011-07-01 false Label content. 211.104 Section 211.104 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.104 Label content. The following data and information must be on the label of all products for...

  20. 40 CFR 211.104 - Label content.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 26 2013-07-01 2013-07-01 false Label content. 211.104 Section 211.104 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.104 Label content. The following data and information must be on the label of all products for...

  1. 40 CFR 211.104 - Label content.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 25 2014-07-01 2014-07-01 false Label content. 211.104 Section 211.104 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.104 Label content. The following data and information must be on the label of all products for...

  2. 40 CFR 211.104 - Label content.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 26 2012-07-01 2011-07-01 true Label content. 211.104 Section 211.104 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.104 Label content. The following data and information must be on the label of all products for...

  3. 40 CFR 211.104 - Label content.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 24 2010-07-01 2010-07-01 false Label content. 211.104 Section 211.104 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.104 Label content. The following data and information must be on the label of all products for...

  4. How to Read a Nutrition Facts Label

    MedlinePlus Videos and Cool Tools

    ... to 2-Year-Old How to Read a Nutrition Facts Label (Video) KidsHealth > For Parents > How to Read a Nutrition Facts Label (Video) Print A A A en ... nutricionales (video) Most packaged foods come with a Nutrition Facts label. These labels have a lot of ...

  5. 21 CFR 610.60 - Container label.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Container label. 610.60 Section 610.60 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS GENERAL BIOLOGICAL PRODUCTS STANDARDS Labeling Standards § 610.60 Container label. (a) Full label....

  6. N-terminal chemical protein labeling using the naturally split GOS-TerL intein.

    PubMed

    Bachmann, Anne-Lena; Mootz, Henning D

    2017-03-23

    Chemoselective and regioselective chemical protein labeling is of great importance, yet no current technique is sufficiently general and simple to perform. Protein trans-splicing by split inteins can be used to ligate short tags with chemical labels to either the N or the C terminus of a protein. The CysTag approach exploits split intein fragments without a cysteine fused with such a short tag containing a single cysteine that is easily amenable to selective modification using classical cysteine bioconjugation. Labeling of the protein of interest is achieved through transfer of the pre-labeled tag by protein trans-splicing. This protocol keeps other cysteines unmodified. While split inteins for C-terminal CysTag labeling were previously reported, no high-yielding and naturally split intein for N-terminal labeling has been available. In this work, the recently discovered GOS-TerL intein was explored as the only known naturally split intein that both lacks a cysteine in its N-terminal fragment and is active under ambient conditions. Thioredoxin as a model protein and a camelid nanobody were labeled with a synthetic fluorophore by transferring the pre-labeling CysTag in the protein trans-splicing reaction with yields of about 50 to 90%. The short N-terminal intein fragment was also chemically synthesized with a tag to enable protein labeling by semi-synthetic protein trans-splicing. Our results expand the scope of the CysTag labeling strategy, which achieves selective chemical modification without the requirement for sophisticated biorthogonal functional groups and rather builds on the plethora of commercially available reagents directed at the thiol side chain of cysteine. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

  7. 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.

  8. 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.

  9. 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.)

  10. Antitumor Peptides from Marine Organisms

    PubMed Central

    Zheng, Lan-Hong; Wang, Yue-Jun; Sheng, Jun; Wang, Fang; Zheng, Yuan; Lin, Xiu-Kun; Sun, Mi

    2011-01-01

    The biodiversity of the marine environment and the associated chemical diversity constitute a practically unlimited resource of new antitumor agents in the field of the development of marine bioactive substances. In this review, the progress on studies of antitumor peptides from marine sources is provided. The biological properties and mechanisms of action of different marine peptides are described; information about their molecular diversity is also presented. Novel peptides that induce apoptosis signal pathway, affect the tubulin-microtubule equilibrium and inhibit angiogenesis are presented in association with their pharmacological properties. It is intended to provide useful information for further research in the fields of marine antitumor peptides. PMID:22072999

  11. The PeptideAtlas Project.

    PubMed

    Deutsch, Eric W

    2010-01-01

    PeptideAtlas is a multi-species compendium of peptides observed with tandem mass spectrometry methods. Raw mass spectrometer output files are collected from the community and reprocessed through a uniform analysis and validation pipeline that continues to advance. The results are loaded into a database and the information derived from the raw data is returned to the community via several web-based data exploration tools. The PeptideAtlas resource is useful for experiment planning, improving genome annotation, and other data mining projects. PeptideAtlas has become especially useful for planning targeted proteomics experiments.

  12. Antitumor peptides from marine organisms.

    PubMed

    Zheng, Lan-Hong; Wang, Yue-Jun; Sheng, Jun; Wang, Fang; Zheng, Yuan; Lin, Xiu-Kun; Sun, Mi

    2011-01-01

    The biodiversity of the marine environment and the associated chemical diversity constitute a practically unlimited resource of new antitumor agents in the field of the development of marine bioactive substances. In this review, the progress on studies of antitumor peptides from marine sources is provided. The biological properties and mechanisms of action of different marine peptides are described; information about their molecular diversity is also presented. Novel peptides that induce apoptosis signal pathway, affect the tubulin-microtubule equilibrium and inhibit angiogenesis are presented in association with their pharmacological properties. It is intended to provide useful information for further research in the fields of marine antitumor peptides.

  13. An unexpected cell-penetrating peptide from Bothrops jararaca venom identified through a novel size exclusion chromatography screening.

    PubMed

    Sciani, Juliana Mozer; Vigerelli, Hugo; Costa, André Santos; Câmara, Diana Aparecida Dias; Junior, Paulo Luiz-de-Sá; Pimenta, Daniel Carvalho

    2017-01-01

    Efficient drug delivery systems are currently one of the greatest challenges in pharmacokinetics, and the transposition of the gap between in vitro candidate molecule and in vivo test drug is, sometimes, poles apart. In this sense, the cell-penetrating peptides (CPP) may be the bridge uniting these worlds. Here, we describe a technique to rapidly identify unlabeled CPPs after incubation with liposomes, based on commercial desalting (size exclusion) columns and liquid chromatography-MS/MS, for peptide de novo sequencing. Using this approach, we found it possible to identify one new CPP - interestingly, a classical bradykinin-potentiating peptide - in the peptide-rich low molecular mass fraction of the Bothrops jararaca venom, which was also able to penetrate live cell membranes, as confirmed by classical approaches employing fluorescence-labeled analogues of this CPP. Moreover, both the labeled and unlabeled CPPs caused no metabolic, cell-cycle or morphologic alterations, proving to be unmistakably cargo deliverers and not drugs themselves. In sum, we have developed and validated a method for screening label-free peptides for CPP activity, regardless of their biological origin, which could lead to the identification of new and more efficient drug delivery systems. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

  14. Fluorescent amino acid undergoing excited state intramolecular proton transfer for site-specific probing and imaging of peptide interactions.

    PubMed

    Sholokh, Marianna; Zamotaiev, Oleksandr M; Das, Ranjan; Postupalenko, Viktoriia Y; Richert, Ludovic; Dujardin, Denis; Zaporozhets, Olga A; Pivovarenko, Vasyl G; Klymchenko, Andrey S; Mély, Yves

    2015-02-12

    Fluorescent amino acids bearing environment-sensitive fluorophores are highly valuable tools for site-selective probing of peptide/ligand interactions. Herein, we synthesized a fluorescent l-amino acid bearing the 4'-methoxy-3-hydroxyflavone fluorophore (M3HFaa) that shows dual emission, as a result of an excited state intramolecular proton transfer (ESIPT). The dual emission of M3HFaa was found to be substantially more sensitive to hydration as compared to previous analogues. By replacing the Ala30 and Trp37 residues of a HIV-1 nucleocapsid peptide, M3HFaa was observed to preserve the peptide structure and functions. Interaction of the labeled peptides with nucleic acids and lipid vesicles produced a strong switch in their dual emission, favoring the emission of the ESIPT product. This switch was associated with the appearance of long-lived fluorescence lifetimes for the ESIPT product, as a consequence of the rigid environment in the complexes that restricted the relative motions of the M3HFaa aromatic moieties. The strongest restriction and thus the longest fluorescence lifetimes were observed at position 37 in complexes with nucleic acids, where the probe likely stacks with the nucleobases. Based on the dependence of the lifetime values on the nature of the ligand and the labeled position, two-photon fluorescence lifetime imaging was used to identify the binding partners of the labeled peptides microinjected into living cells. Thus, M3HFaa appears as a sensitive tool for monitoring site selectively peptide interactions in solution and living cells.

  15. Covalent surface chemistry gradients for presenting bioactive peptides.

    PubMed

    Kipper, Matt J; Kleinman, Hynda K; Wang, Francis W

    2007-04-15

    The activation of surfaces by covalent attachment of bioactive moieties is an important strategy for improving the performance of biomedical materials. Such techniques have also been used as tools to study cellular responses to particular chemistries of interest. The creation of gradients of covalently bound chemistries is a logical extension of this technique. Gradient surfaces may permit the rapid screening of a large range of concentrations in a single experiment. In addition, the biological response to the gradient itself may provide new information on receptor requirements and cell signaling. The current work describes a rapid and flexible technique for the covalent addition of bioactive peptide gradients to a surface or gel and a simple fluorescence technique for assaying the gradient. In this technique, bioactive peptides with a terminal cysteine are bound via a heterobifunctional coupling agent to primary amine-containing surfaces and gels. A gradient in the coupling agent is created on the surfaces or gels by varying the residence time of the coupling agent across the surface or gel, thereby controlling the extent of reaction. We demonstrate this technique using poly(l-lysine)-coated glass surfaces and fibrin gels. Once the surface or gel has been activated by the addition of the coupling agent gradient, the bioactive peptide is added. Quantitation of the gradient is achieved by measuring the reaction kinetics of the coupling agent with the surface or gel of interest. This can be done either by fluorescently labeling the coupling agent (in the case of surfaces) or by spectrophotometrically detecting the release of pyridine-2-thione, which is produced when the thiol-reactive portion of the coupling agent reacts. By these methods, we can obtain reasonably precise estimates for the peptide gradients without using expensive spectroscopic or radiolabeling techniques. Validation with changes in fibroblast cell migration behavior across a bioactive peptide

  16. 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.

  17. 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

  18. Peptide Amyloid Surface Display

    PubMed Central

    2015-01-01

    Homomeric self-assembly of peptides into amyloid fibers is a feature of many diseases. A central role has been suggested for the lateral fiber surface affecting gains of toxic function. To investigate this, a protein scaffold that presents a discrete, parallel β-sheet surface for amyloid subdomains up to eight residues in length has been designed. Scaffolds that present the fiber surface of islet amyloid polypeptide (IAPP) were prepared. The designs show sequence-specific surface effects apparent in that they gain the capacity to attenuate rates of IAPP self-assembly in solution and affect IAPP-induced toxicity in insulin-secreting cells. PMID:25541905

  19. Plant antimicrobial peptides.

    PubMed

    Nawrot, Robert; Barylski, Jakub; Nowicki, Grzegorz; Broniarczyk, Justyna; Buchwald, Waldemar; Goździcka-Józefiak, Anna

    2014-05-01

    Plant antimicrobial peptides (AMPs) are a component of barrier defense system of plants. They have been isolated from roots, seeds, flowers, stems, and leaves of a wide variety of species and have activities towards phytopathogens, as well as against bacteria pathogenic to humans. Thus, plant AMPs are considered as promising antibiotic compounds with important biotechnological applications. Plant AMPs are grouped into several families and share general features such as positive charge, the presence of disulfide bonds (which stabilize the structure), and the mechanism of action targeting outer membrane structures.

  20. 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

  1. 49 CFR 172.430 - POISON label.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 2 2013-10-01 2013-10-01 false POISON label. 172.430 Section 172.430... SECURITY PLANS Labeling § 172.430 POISON label. (a) Except for size and color, the POISON label must be as follows: EC02MR91.029 (b) In addition to complying with § 172.407, the background on the POISON label...

  2. 49 CFR 172.430 - POISON label.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 2 2010-10-01 2010-10-01 false POISON label. 172.430 Section 172.430... SECURITY PLANS Labeling § 172.430 POISON label. (a) Except for size and color, the POISON label must be as follows: EC02MR91.029 (b) In addition to complying with § 172.407, the background on the POISON label...

  3. 49 CFR 172.430 - POISON label.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 2 2012-10-01 2012-10-01 false POISON label. 172.430 Section 172.430... SECURITY PLANS Labeling § 172.430 POISON label. (a) Except for size and color, the POISON label must be as follows: EC02MR91.029 (b) In addition to complying with § 172.407, the background on the POISON label...

  4. 49 CFR 172.430 - POISON label.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 2 2014-10-01 2014-10-01 false POISON label. 172.430 Section 172.430... SECURITY PLANS Labeling § 172.430 POISON label. (a) Except for size and color, the POISON label must be as follows: EC02MR91.029 (b) In addition to complying with § 172.407, the background on the POISON label...

  5. 49 CFR 172.430 - POISON label.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 2 2011-10-01 2011-10-01 false POISON label. 172.430 Section 172.430... SECURITY PLANS Labeling § 172.430 POISON label. (a) Except for size and color, the POISON label must be as follows: EC02MR91.029 (b) In addition to complying with § 172.407, the background on the POISON label...

  6. MEASURING OF PROTEIN SYNTHESIS USING METABOLIC 2H-LABELING, HIGH-RESOLUTION MASS SPECTROMETRY AND AN ALGORITHM

    PubMed Central

    Kasumov, Takhar; Ilchenko, Sergey; Li, Ling; Rachdaoui, Nadia; Sadigov, Rovshan; Willard, Belinda; McCullough, Arthur J.; Previs, Stephen

    2013-01-01

    We recently developed a method for estimating protin dynamics in vivo with 2H2O using MALDI-TOF MS (Rachdaoui N. et al., MCP, 8, 2653-2662, 2009) and we confirmed that 2H-labeling of many hepatic free amino acids rapidly equilibrated with body water. Although this is a reliable method, it required modest sample purification and necessitated the determination of tissue-specific amino acid labeling. Another approach for quantifying protein kinetics is to measure the 2H-enrichments of body water (precursor) and protein-bound amino acid or proteolytic peptide (product) and to estimate how many copies of deuterium are incorporated into a product. In this study we have used nanospray LTQ-FTICR mass spectrometry to simultaneously measure the isotopic enrichment of peptides and protein-bound amino acids. A mathematical algorithm was developed to aid the data processing. The most notable improvement centers on the fact that the precursor:product labeling ratio can be obtained by measuring the labeling of water and a protein(s) (or peptides) of interest, therein minimizing the need to measure the amino acid labeling. As a proof of principle, we demonstrate that this approach can detect the effect of nutritional status on albumin synthesis in rats given 2H2O. PMID:21256107

  7. Measuring protein synthesis using metabolic ²H labeling, high-resolution mass spectrometry, and an algorithm.

    PubMed

    Kasumov, Takhar; Ilchenko, Serguey; Li, Ling; Rachdaoui, Nadia; Sadygov, Rovshan G; Willard, Belinda; McCullough, Arthur J; Previs, Stephen

    2011-05-01

    We recently developed a method for estimating protein dynamics in vivo with heavy water ((2)H(2)O) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) [16], and we confirmed that (2)H labeling of many hepatic free amino acids rapidly equilibrated with body water. Although this is a reliable method, it required modest sample purification and necessitated the determination of tissue-specific amino acid labeling. Another approach for quantifying protein kinetics is to measure the (2)H enrichments of body water (precursor) and protein-bound amino acid or proteolytic peptide (product) and to estimate how many copies of deuterium are incorporated into a product. In the current study, we used nanospray linear trap Fourier transform ion cyclotron resonance mass spectrometry (LTQ FT-ICR MS) to simultaneously measure the isotopic enrichment of peptides and protein-bound amino acids. A mathematical algorithm was developed to aid the data processing. The most notable improvement centers on the fact that the precursor/product labeling ratio can be obtained by measuring the labeling of water and a protein (or peptide) of interest, thereby minimizing the need to measure the amino acid labeling. As a proof of principle, we demonstrate that this approach can detect the effect of nutritional status on albumin synthesis in rats given (2)H(2)O.

  8. 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

  9. Preparation of Fluorescent Microcystin Derivatives by Direct Arginine Labelling and Their Biological Evaluation.

    PubMed

    Grundler, Verena; Faltermann, Susanne; Fent, Karl; Gademann, Karl

    2015-07-27

    Microcystin is the most prevalent toxin produced by cyanobacteria and poses a severe threat to livestock, humans and entire ecosystems. We report the preparation of a series of fluorescent microcystin derivatives by direct arginine labelling of the unprotected peptides at the guanidinium side chain. This new method allows a simple late-stage diversification strategy for native peptides devoid of protecting groups under mild conditions. A series of fluorophores were conjugated to microcystin-LR in good to very good yield. The fluorescent probes displayed biological activity comparable to that of unlabelled microcystin, in both phosphatase inhibition assays and toxicity tests on the crustacean Thamnocephalus platyurus. In addition, we demonstrate that the fluorescent probes penetrated Huh7 cells. Whole-animal imaging was performed on T. platyurus: labelled compound was mainly observed in the digestive tract.

  10. Resonance energy transfer study of peptide-lipid complexes.

    PubMed

    Gorbenko, G; Saito, H; Molotkovsky, J; Tanaka, M; Egashira, M; Nakano, M; Handa, T

    2001-09-18

    Resonance energy transfer involving tryptophan as a donor and anthrylvinyl-labeled phosphatidylcholine (AV-PC), 3-methoxybenzanthrone (MBA) and 8-anilino-1-naphthalene sulfonic acid (ANS) as acceptors has been examined to obtain information on the structure of peptide-lipid systems consisting of 18A or Ac-18A-NH(2) peptides and large unilamellar phosphatidylcholine vesicles. The lower and upper limits for the tryptophan distance from the bilayer midplane have been assessed in terms of the models of energy transfer in two-dimensional systems, taking into account orientational effects. Evidence for the existence of preferential orientations of Ac-18A-NH(2) with respect to the lipid-water interface has been obtained.

  11. Peptide aldehyde inhibitors of hepatitis A virus 3C proteinase.

    PubMed

    Malcolm, B A; Lowe, C; Shechosky, S; McKay, R T; Yang, C C; Shah, V J; Simon, R J; Vederas, J C; Santi, D V

    1995-06-27

    Picornaviral 3C proteinases are a group of closely related thiol proteinases responsible for processing of the viral polyprotein into its component proteins. These proteinases adopt a chymotrypsin-like fold [Allaire et al. (1994) Nature 369, 72-77; Matthews et al. (1994) Cell 77, 761-771] and a display an active-site configuration like those of the serine proteinases. Peptide-aldehydes based on the preferred peptide substrates for hepatitis A virus (HAV) 3C proteinase were synthesized by reduction of a thioester precursor. Acetyl-Leu-Ala-Ala-(N,N'-dimethylglutaminal) was found to be a reversible, slow-binding inhibitor for HAV 3C with a Ki* of (4.2 +/- 0.8) x 10(-8) M. This inhibitor showed 50-fold less activity against the highly homologous human rhinovirus (strain 14) 3C proteinase, whose peptide substrate specificity is slightly different, suggesting a high degree of selectivity. NMR spectrometry of the adduct of the 13C-labeled inhibitor with the HAV-3C proteinase indicate that a thiohemiacetal is formed between the enzyme and the aldehyde carbon as previously noted for peptide-aldehyde inhibitors of papain [Lewis & Wolfenden (1977) Biochemistry 16,4890-4894; Gamcsik et al. (1983) J. Am. Chem. Soc. 105, 6324-6325]. The adduct can also be observed by electrospray mass spectrometry.

  12. 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.

  13. 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.

  14. 2D DIGE saturation labeling for minute sample amounts.

    PubMed

    Arnold, Georg J; Fröhlich, Thomas

    2012-01-01

    The 2D DIGE technique, based on fluorophores covalently linked to amino acid side chain residues and the concept of an internal standard, has significantly improved reproducibility, sensitivity, and the dynamic range of protein quantification. In saturation DIGE, sulfhydryl groups of cysteines are labeled with cyanine dyes to completion, providing a so far unraveled sensitivity for protein detection and quantification in 2D gel-based proteomic experiments. Only a few micrograms of protein per 2D gel facilitate the analysis of about 2,000 analytes from complex mammalian cell or tissue samples. As a consequence, 2D saturation DIGE is the method of choice when only minute sample amounts are available for quantitative proteome analysis at the level of proteins rather than peptides. Since very low amounts of samples have to be handled in a reproducible manner, saturation DIGE-based proteomic experiments are technically demanding. Moreover, successful saturation DIGE approaches require a strict adherence to adequate reaction conditions at each step. This chapter is dedicated to colleagues already experienced in 2D PAGE protein separation and intends to support the establishment of this ultrasensitive technique in proteomic workgroups. We provide basic guidelines for the experimental design and discuss crucial aspects concerning labeling chemistry, sample preparation, and pitfalls caused by labeling artifacts. A detailed step-by-step protocol comprises all aspects from initial sample preparation to image analysis and statistical evaluation. Furthermore, we describe the generation of preparative saturation DIGE gels necessary for mass spectrometry-based spot identification.

  15. One-step 18F labeling of biomolecules using organotrifluoroborates

    PubMed Central

    Liu, Zhibo; Lin, Kuo-Shyan; Bénard, François; Pourghiasian, Maral; Kiesewetter, Dale O; Perrin, David M; Chen, Xiaoyuan

    2017-01-01

    Herein we present a general protocol for the functionalization of biomolecules with an organotrifluoroborate moiety so that they can be radiolabeled with aqueous 18F fluoride (18F−) and used for positron emission tomography (PET) imaging. Among the β+-emitting radionuclides, fluorine-18 (18F) is the isotope of choice for PET, and it is produced, on-demand, in many hospitals worldwide. Organotrifluoroborates can be 18F-labeled in one step in aqueous conditions via 18F–19F isotope exchange. This protocol features a recently designed ammoniomethyltrifluoroborate, and it describes the following: (i) a synthetic strategy that affords modular synthesis of radiolabeling precursors via a copper-catalyzed ‘click’ reaction; and (ii) a one-step 18F-labeling method that obviates the need for HPLC purification. Within 30 min, 18F-labeled PET imaging probes, such as peptides, can be synthesized in good chemical and radiochemical purity (>98%), satisfactory radiochemical yield of 20–35% (n > 20, non-decay corrected) and high specific activity of 40–111 GBq/µmol (1.1–3.0 Ci/µmol). The entire procedure, including the precursor preparation and 18F radiolabeling, takes 7–10 d. PMID:26313478

  16. Mapping of radiolabeled peptides derived from proteolysis of polypeptides bound to nitrocellulose after Western blotting

    SciTech Connect

    Carrey, E.A.; Hardie, D.G.

    1986-11-01

    Sections of nitrocellulose containing bound /sup 32/P-labeled polypeptides were excised from Western blots and exhaustively digested by trypsin in order to analyze the distribution of phosphorylation sites between the products of limited proteolysis of the multifunctional protein CAD. Using the criterion of analytical isoelectric focusing, the /sup 32/P-peptides obtained by this method were found to be similar, although not identical, to peptides obtained by a more conventional digestion of trichloroacetic acid precipitates. Digestion on Western blots is more straightforward than electrophoretic elution of individual gel slices, gives better recoveries than direct digestion of gel slices, and is particularly suitable for peptide mapping of small peptides which bind to nitrocellulose but would diffuse out of polyacrylamide gels during the commonly used fixing and staining procedures.

  17. Rapid and one-step radiofluorination of bioactive peptides: potential PET radiopharmaceuticals.

    PubMed

    AlJammaz, I; Al-Otaibi, B; Abousekhrah, A; Okarvi, S

    2014-09-01

    In an attempt to develop a new and rapid method for labeling peptides with (18)F, we have synthesized MUC1-[(18)F]SFB and BBN-[(18)F]SFB peptide conjugates using a convenient and one-step simple reactions. Radiochemical yields for MUC1-[(18)F]SFB and BBN-[(18)F]SFB peptide conjugates were greater than 70% in less than 30 min synthesis time, thus amenable for automation for the radiofluorination of peptides. in vitro tests on T47D breast cancer cells showed that the significant amounts of the radioconjugates were associated with cell fractions and held sufficient affinities and specificities toward T47D cell line. These radioconjugates may be useful as molecular probes for detecting and staging of breast cancer and monitoring tumor response to treatment.

  18. Treatment of B-cell lymphoma using peptides. A novel concept.

    PubMed Central

    Lam, K S

    1993-01-01

    Combination chemotherapy remains the major current treatment of non-Hodgkin's lymphoma. B-cell lymphoma often has tumor-specific surface immunoglobulins called idiotypes. Clinical trials using murine monoclonal anti-idiotype antibodies as a targeting approach have shown some success. I describe a novel concept of using idiotype-specific peptides as an alternative targeting approach for the treatment of B-cell lymphoma. In brief, octapeptides that bind to the surface idiotype of the B-cell lymphoma are isolated from a large synthetic peptide library (10(6) to 10(7) peptides). Once the sequence of a tumor-specific octapeptide ligand is defined, large quantities can be synthesized and conjugated with a radionuclide (such as iodine 131). This should permit highly specific destruction of lymphoma cells that bind the labeled peptide. The theoretic advantages of this approach over the previous use of anti-idiotype antibodies are addressed. Images PMID:8342262

  19. 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.

  20. Metrics for Labeled Markov Systems

    NASA Technical Reports Server (NTRS)

    Desharnais, Josee; Jagadeesan, Radha; Gupta, Vineet; Panangaden, Prakash

    1999-01-01

    Partial Labeled Markov Chains are simultaneously generalizations of process algebra and of traditional Markov chains. They provide a foundation for interacting discrete probabilistic systems, the interaction being synchronization on labels as in process algebra. Existing notions of process equivalence are too sensitive to the exact probabilities of various transitions. This paper addresses contextual reasoning principles for reasoning about more robust notions of "approximate" equivalence between concurrent interacting probabilistic systems. The present results indicate that:We develop a family of metrics between partial labeled Markov chains to formalize the notion of distance between processes. We show that processes at distance zero are bisimilar. We describe a decision procedure to compute the distance between two processes. We show that reasoning about approximate equivalence can be done compositionally by showing that process combinators do not increase distance. We introduce an asymptotic metric to capture asymptotic properties of Markov chains; and show that parallel composition does not increase asymptotic distance.