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Covalent labeling

Yunan Zheng, Martin J Gilgenast, Sacha Hauc, Abhishek Chatterjee
Reversible post-translational modification (PTM) is a powerful and ubiquitous mechanism to regulate protein function. The mechanistic basis of the associated functional regulation by PTMs often involves the recruitment of interaction partners that selectively binds the modified protein. Identifying such functionally important protein-protein interactions that are uniquely triggered by PTMs remains difficult due to several technical challenges. To address this, here we develop technology to site-specifically incorporate two distinct noncanonical amino acids into recombinant proteins: one modeling a PTM of interest and the second harboring a photoaffinity probe...
March 15, 2018: ACS Chemical Biology
Fangxu Sun, Alexander A Choi, Ronghu Wu
Fatty acids (FAs) are essential components in cells and are involved in many cellular activities. Abnormal FA metabolism has been reported to be related to human diseases such as cancer and cardiovascular diseases. Identification and quantification of FAs provide insights into their functions in biological systems, but it is very challenging to analyze them due to their structures and properties. In this work, we developed a novel method by integrating tagging FAs with stable isotope labeled aminoxy tandem mass tags (aminoxyTMTs) and mass spectrometric analysis with the positive mode...
March 9, 2018: Journal of Proteome Research
Sultan Khetani, Vinayaraj Ozhukil Kollath, Varun Kundra, Minh Nguyen, Chantel Debert, Arindom Sen, Kunal Karan, Amir Sanati-Nezhad
Glial fibrillary acidic protein (GFAP) is as an intermediate filament protein expressed by certain cells in the central nervous system (CNS). GFAP has been recognized as a reliable biomarker of CNS injury. However, due to the absence of rapid and easy-to-use assays for the detection of CNS injury biomarkers, measuring GFAP levels to identify CNS injury has not attained widespread clinical implementation. In the present work, we developed a polyethylenimine (PEI) coated graphene screen-printed electrode and used it for highly sensitive immunosensing of GFAP...
March 8, 2018: ACS Sensors
Cecilia Samaniego Lopez, Jimena Hebe Martínez, María Laura Uhrig, Federico Coluccio Leskow, Carla Cecilia Spagnuolo
We introduce a novel fluorescent molecular probe able to detect glycoproteins, especially mucins, with high sensitivity and with a turn-on response along with a large Stokes shift (>130 nm), within the biologically active window. The probe contains an aminotricarbocyanine as the fluorescent reporter with a linked benzoboroxole as the recognition unit, which operates through a dynamic covalent reaction between the boronic hemiester residue of the receptor and cis-diols of the analyte. The superior selectivity of the probe is displayed by the labeling of mucins present in Calu-3 cells...
March 7, 2018: Chemistry: a European Journal
Margaret Lin, Denise Krawitz, Matthew D Callahan, Galahad Deperalta, Aaron T Wecksler
We describe epitope mapping data using multiple covalent labeling footprinting-mass spectrometry (MS) techniques coupled with negative stain transmission electron microscopy (TEM) data to analyze the antibody-antigen interactions in a sandwich enzyme-linked immunosorbant assay (ELISA). Our hydroxyl radical footprinting-MS data using fast photochemical oxidation of proteins (FPOP) indicates suppression of labeling across the antigen upon binding either of the monoclonal antibodies (mAbs) utilized in the ELISA...
March 6, 2018: Journal of the American Society for Mass Spectrometry
Eda Yuhana Ariffin, Yook Heng Lee, Dedi Futra, Ling Ling Tan, Nurul Huda Abd Karim, Nik Nuraznida Nik Ibrahim, Asmat Ahmad
A novel electrochemical DNA biosensor for ultrasensitive and selective quantitation of Escherichia coli DNA based on aminated hollow silica spheres (HSiSs) has been successfully developed. The HSiSs were synthesized with facile sonication and heating techniques. The HSiSs have an inner and an outer surface for DNA immobilization sites after they have been functionalized with 3-aminopropyltriethoxysilane. From field emission scanning electron microscopy images, the presence of pores was confirmed in the functionalized HSiSs...
March 5, 2018: Analytical and Bioanalytical Chemistry
Courtney A Follit, Shannon R Woodruff, Pia D Vogel, John G Wise, Nicolay V Tsarevsky
Receptor-independent cellular uptake of small molecule therapeutics is limited by their physical interaction with the negatively charged surface of cellular membranes. Passive diffusion through the hydrophobic membrane bilayer follows this process. Unless specific carriers exist in the biological membrane, such interactions limit therapeutics to those that are hydrophobic with modest positive charge at physiological pH. Small negatively charged molecules are therefore not efficient as therapeutics. To enable delivery of such molecules into eukaryotic cells, cationic branched polymers with tetraalkylammonium pendant groups were synthesized by copolymerization of a functional monomer (glycidyl methacrylate) with degradable and non-degradable divinyl crosslinkers in the presence of an efficient chain transfer agent, CBr4 , followed by reaction of the multiple pendant epoxide groups and most of the alkyl bromide chain ends with amines...
June 2017: Journal of Drug Delivery Science and Technology
Maarten L J Doornbos, Xuesong Wang, Sophie C Vermond, Luc Peeters, Laura Pérez-Benito, Andrés A Trabanco, Hilde Lavreysen, José María Cid, Laura H Heitman, Gary Tresadern, Adriaan P IJzerman
Covalent labelling of G protein-coupled receptors (GPCRs) by small molecules is a powerful approach to understand binding modes, mechanism of action, pharmacology and even facilitate structure elucidation. We report the first covalent positive allosteric modulator (PAM) for a class C GPCR, the mGlu2 receptor. Three putatively covalent mGlu2 PAMs were designed and synthesized. Pharmacological characterization identified 2 to bind the receptor covalently. Computational modelling combined with receptor mutagenesis revealed T7917...
March 1, 2018: Journal of Medicinal Chemistry
Tongbo Wu, Yufei Yang, Wei Chen, Jiayu Wang, Ziyu Yang, Shenlin Wang, Xianjin Xiao, Mengyuan Li, Meiping Zhao
Lambda exonuclease (λ exo) plays an important role in the resection of DNA ends for DNA repair. Currently, it is also a widely used enzymatic tool in genetic engineering, DNA-binding protein mapping, nanopore sequencing and biosensing. Herein, we disclose two noncanonical properties of this enzyme and suggest a previously undescribed hydrophobic interaction model between λ exo and DNA substrates. We demonstrate that the length of the free portion of the substrate strand in the dsDNA plays an essential role in the initiation of digestion reactions by λ exo...
February 27, 2018: Nucleic Acids Research
Hui Wang, Yang Liu, Gang Liu
Mercury (Hg) and its compounds, originating from a variety of nature and anthropogenic source, are ubiquitous in the natural environment, which cause severe environmental contamination and pose irreversible harm to human health. Fast and accurate sensing approach is of significant importance for mercury detection. Here, a label-free biosensor using Hg(II)-induced cleavage of phosphorothioate (PS) modified RNA was exploited. We designed a specific single-stranded DNA embedded four PS-modified RNA (Hg-DPR) to improve the cleavage reaction yield, and then Hg-DPR was covalently linked with single-walled carbon nanotube field effect transistor (SWNTs/FET) via a peptide bond...
February 28, 2018: ACS Sensors
Jianxin Guan, Chuancheng Jia, Yanwei Li, Zitong Liu, Jinying Wang, Zhongyue Yang, Chunhui Gu, Dingkai Su, Kendall N Houk, Deqing Zhang, Xuefeng Guo
Single-molecule detection can reveal time trajectories and reaction pathways of individual intermediates/transition states in chemical reactions and biological processes, which is of fundamental importance to elucidate their intrinsic mechanisms. We present a reliable, label-free single-molecule approach that allows us to directly explore the dynamic process of basic chemical reactions at the single-event level by using stable graphene-molecule single-molecule junctions. These junctions are constructed by covalently connecting a single molecule with a 9-fluorenone center to nanogapped graphene electrodes...
February 2018: Science Advances
Ran Cheng, Wakana Mori, Longle Ma, Mireille Alhouayek, Akiko Hatori, Yiding Zhang, Daisuke Ogasawara, Gengyang Yuan, Zhen Chen, Xiaofei Zhang, Hang Shi, Tomoteru Yamasaki, Lin Xie, Katsushi Kumata, Masayuki Fujinaga, Yuji Nagai, Takafumi Minamimoto, Mona Svensson, Lu Wang, Yunfei Du, Mary Jo Ondrechen, Neil Vasdev, Benjamin F Cravatt, Christopher Fowler, Ming-Rong Zhang, Steven H Liang
Monoacylglycerol lipase (MAGL) is the principle enzyme for metabolizing the endogenous cannabinoid ligand, 2-arachidonylglycerol (2-AG). Blockade of MAGL increases 2-AG levels, resulting in subsequent activation of the endocannabinoid system, and has emerged as a novel therapeutic strategy to treat drug addiction, inflammation and neurodegenerative diseases. Herein we report a new series of MAGL inhibitors, which were radiolabeled by site-specific labeling technologies, including 11C-carbonylation and spirocyclic iodonium ylide (SCIDY) radiofluorination...
February 26, 2018: Journal of Medicinal Chemistry
Guoneng Cai, Zhengzhong Yu, Rongrong Ren, Dianping Tang
A competitive-displacement reaction strategy based on target-induced dissociation of gold nanoparticles-coated graphene nanosheet (AuNPs/GN) from CdS quantum dots-functionalized mesoporous titanium dioxide (CdS QDs/TiO2) was designed for the sensitive photoelectrochemical (PEC) aptasensing of prostate-specific antigen (PSA) through the exciton-plasmon interaction (EPI) between CdS QDs and AuNPs. To construct such an aptasensing system, capture DNA was initially conjugated covalently onto CdS QDs/TiO2-modified electrode, and then AuNPs/GN-labeled PSA aptamer was bound onto biofunctionalized CdS QDs/TiO2 via hybridization chain reaction of partial bases with capture DNA...
February 21, 2018: ACS Sensors
Martin Kahms, Jürgen Klingauf
Styryl dyes and genetically encoded pH-sensitive fluorescent proteins like pHluorin are well-established tools for the optical analysis of synaptic vesicle (SV) recycling at presynaptic boutons. Here, we describe the development of a new class of fluorescent probes based on pH-sensitive organic dyes covalently bound to lipids, providing a promising complementary assay to genetically encoded fluorescent probes. These new optical tracers allow a pure read out of membrane turnover during synaptic activity and visualization of multiple rounds of stimulation-dependent SV recycling without genetic perturbation...
2018: Frontiers in Cellular Neuroscience
Jungmin Lee, Xinyi Feng, Ou Chen, Moungi G Bawendi, Jun Huang
We have developed a strategy for synthesizing immediately activable, water-soluble, compact (∼10-12 nm hydrodynamic diameter) quantum dots with a small number of stable and controllable conjugation handles for long distance delivery and subsequent biomolecule conjugation. Upon covalent conjugation with engineered monovalent streptavidin, the sample results in a population consisting of low-valency quantum dots. Alternatively, we have synthesized quantum dots with a small number of biotin molecules that can self-assemble with engineered divalent streptavidin via high-affinity biotin-streptavidin interactions...
February 16, 2018: Nanoscale
Ke Sherry Li, Liuqing Shi, Michael L Gross
Assessment of protein structure and interaction is crucial for understanding protein structure/function relationships. Compared to high-resolution structural tools, including X-ray crystallography, nuclear magnetic resonance (NMR), and cryo-EM, and traditional low-resolution methods, such as circular dichroism, UV-vis, and florescence spectroscopy, mass spectrometry (MS)-based protein footprinting affords medium-to-high resolution (i.e., regional and residue-specific insights) by taking advantage of proteomics methods focused on the primary structure...
February 16, 2018: Accounts of Chemical Research
Alena Semerádtová, Marcel Štofik, Oldřich Neděla, Ondřej Staněk, Petr Slepička, Zdeňka Kolská, Jan Malý
Herein, we report a novel concept of low-cost flexible platform for fluorescence-based biosensor. The surface of polyethylene naphthalate (PEN) foil was exposed to KrF excimer laser through a photolitographic contact mask. Laser initiated surface modification resulted in micro-patterned areas with surface functional groups available for localized covalent immobilization of biotin. High affinity binding protein (albumin-binding domain (ABD) of protein G, Streptococcus G148) recognizing human serum albumin (HSA), genetically fused with streptavidin (SA-ABDwt), was immobilized on the micro-patterned surface through biotin-streptavidin coupling...
February 2, 2018: Colloids and Surfaces. B, Biointerfaces
Andrea Brigitta Alber, David Michael Suter
Proteins are in a dynamic state of synthesis and degradation and their half-lives can be adjusted under various circumstances. However, most commonly used approaches to determine protein half-life are either limited to population averages from lysed cells or require the use of protein synthesis inhibitors. This protocol describes a method to measure protein half-lives in single living adherent cells, using SNAP-tag fusion proteins in combination with fluorescence time-lapse microscopy. Any protein of interest fused to a SNAP-tag can be covalently bound by a fluorescent, cell permeable dye that is coupled to a benzylguanine derivative, and the decay of the labeled protein population can be monitored after washout of the residual dye...
February 4, 2018: Journal of Visualized Experiments: JoVE
Fatemeh Hakimian, Hedayatollah Ghourchian, Azam Sadat Hashemi, Mohammad Reza Arastoo, Mohammad Behnam Rad
An ultrasensitive optical biosensor for microRNA-155 (miR-155) was developed to diagnose breast cancer at early stages. At first, the probe DNA covalently bind to the negatively charged gold nanoparticles (citrate-capped AuNPs). Then, the target miR-155 electrostatically adsorb onto the positively charged gold nanoparticles (polyethylenimine-capped AuNP) surface. Finally, by mixing citrate-capped AuNP/probe and polyethylenimine-capped AuNP/miR-155, hybridization occurs and the optical signal of the mixture give a measure to quantify the miR-155 content...
February 13, 2018: Scientific Reports
Muhammet Aydın, Elif Burcu Aydın, Mustafa Kemal Sezgintürk
Label-free immunosensor based on tetra armed star-shaped poly(glycidylmethacrylate) (Star PGMA ) modified disposable ITO electrode was developed for detection of p53 protein, an important colorectal cancer biomarker. This disposable biosensor was fabricated by spin-coating technique using star-shaped Star PGMA with epoxy side groups. After formation of a stable film with epoxy ends, anti-p53 antibodies were bound to these groups covalently. Stepwise modification of the electrode was followed by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) studies...
February 5, 2018: Biosensors & Bioelectronics
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