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supercharged protein

Piul S Rabbani, Anna Zhou, Zachary M Borab, Joseph A Frezzo, Nikita Srivastava, Haresh T More, William J Rifkin, Joshua A David, Samuel J Berens, Raymond Chen, Sophia Hameedi, Muhammad H Junejo, Camille Kim, Rita A Sartor, Che F Liu, Pierre B Saadeh, Jin K Montclare, Daniel J Ceradini
Therapeutics utilizing siRNA are currently limited by the availability of safe and effective delivery systems. Cutaneous diseases, specifically ones with significant genetic components are ideal candidates for topical siRNA based therapy but the anatomical structure of skin presents a considerable hurdle. Here, we optimized a novel liposome and protein hybrid nanoparticle delivery system for the topical treatment of diabetic wounds with severe oxidative stress. We utilized a cationic lipid nanoparticle (CLN) composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the edge activator sodium cholate (NaChol), in a 6:1 ratio of DOTAP:NaChol (DNC)...
April 3, 2017: Biomaterials
Micah T Donor, Simon A Ewing, Muhammad A Zenaidee, William Alexander Donald, James S Prell
Supercharging electrospray ionization can be a powerful tool for increasing charge states in mass spectra and generating unfolded ion structures, yet key details of its mechanism remain unclear. The structures of highly-extended protein ions and the mechanism of supercharging were investigated using ion mobility-mass spectrometry. Head-to-tail-linked poly-ubiquitins (Ubq1-11) were used to determine size and charge state scaling laws for unfolded protein ions formed by supercharging while eliminating amino acid composition as a potential confounding factor...
April 3, 2017: Analytical Chemistry
Eita Sasaki, Daniel Böhringer, Michiel van de Waterbeemd, Marc Leibundgut, Reinhard Zschoche, Albert J R Heck, Nenad Ban, Donald Hilvert
Proteins that self-assemble into regular shell-like polyhedra are useful, both in nature and in the laboratory, as molecular containers. Here we describe cryo-electron microscopy (EM) structures of two versatile encapsulation systems that exploit engineered electrostatic interactions for cargo loading. We show that increasing the number of negative charges on the lumenal surface of lumazine synthase, a protein that naturally assembles into a ∼1-MDa dodecahedron composed of 12 pentamers, induces stepwise expansion of the native protein shell, giving rise to thermostable ∼3-MDa and ∼6-MDa assemblies containing 180 and 360 subunits, respectively...
March 10, 2017: Nature Communications
Gregory T Roman, James P Murphy
We demonstrate an integrated microfluidic LC device coupled to a QTOF capable of improving sensitivity and linearity for intact protein analysis while also tuning the charge state distributions (CSD) of whole antibodies. The mechanism for sensitivity improvement using microflow ESI is demonstrated by shifting of the CSD to higher charge state, and narrowing of the overall CSD. Both of these aspects serve to improve ion current of the most abundant charge state of antibodies and lead to improvement in sensitivity over high flow ESI by a factor of 15×...
March 2, 2017: Analyst
Yi Zhang, Tingliang Wang, Jiao Wei, Jinguang He, Tao Wang, Ying Liu, Hua Xu, Jiasheng Dong
BACKGROUND: We developed a novel pedicled DIEP flap model in rat to explore the possible remedy for the distal necrosis of the flap. METHODS: A deep inferior epigastric perforator (DIEP) flap, based on the second right cranial perforator (P2) as the main pedicle, was elevated in 48 Sprague-Dawley rats. The rats were randomized into 4 groups: group I, the left P2 remaining intact as supercharging; group II, the left P2 artery alone kept as supercharging; group III, the left P2 vein alone kept as supercharging; group IV, no supercharging...
2017: PloS One
James I Austerberry, Rana Dajani, Stanislava Panova, Dorota Roberts, Alexander P Golovanov, Alain Pluen, Christopher F van der Walle, Shahid Uddin, Jim Warwicker, Jeremy P Derrick, Robin Curtis
The aggregation propensities of a series of single-chain variable fragment (scFv) proteins harbouring supercharged sequences, salt bridges and lysine/arginine-enriched motifs were characterised as a function of pH and ionic strength to isolate the electrostatic contributions. Recent improvements in aggregation predictors rely on using knowledge of native-state protein-protein interactions. Consistent with previous findings, electrostatic contributions to native protein-protein interactions correlate with aggregate growth pathway and rates...
February 1, 2017: European Journal of Pharmaceutics and Biopharmaceutics
Chia-Wei Lin, Micha A Haeuptle, Markus Aebi
Recent developments in proteomic techniques have led to the development of mass spectrometry (MS)-based methods to characterize site-specific glycosylation of proteins. However, appropriate analytical tools to characterize acidic and high-molecular-weight (hMW) glycopeptides are still lacking. In this study, we demonstrate that the addition of supercharging reagent, m-nitrobenzyl alcohol (m-NBA), into mobile phases greatly facilitates the analysis of acidic and hMW glycopeptides. Using commercial glycoproteins, we demonstrated that in the presence of m-NBA the charge state of sialylated glycopeptides increased and the chromatographic separation of neutral and acidic glycopeptides revealed a remarkable improvement...
September 6, 2016: Analytical Chemistry
Daniel N Mortensen, Evan R Williams
The extent of charging resulting from electrothermal supercharging for protein ions formed from various buffered aqueous solutions using nanoESI emitters with tip diameters between ∼1.5 μm and ∼310 nm is compared. Charging increases with decreasing tip size for proteins that are positively charged in solution but not for proteins that are negatively charged in solution. These results suggest that Coulombic attraction between positively charged protein molecules and the negatively charged glass surfaces in the tips of the emitters causes destabilization and even unfolding of proteins prior to nanoESI...
October 7, 2016: Analyst
Henry S Kim, Anne Martel, Eric Girard, Martine Moulin, Michael Härtlein, Dominique Madern, Martin Blackledge, Bruno Franzetti, Frank Gabel
Water molecules in the immediate vicinity of biomacromolecules, including proteins, constitute a hydration layer characterized by physicochemical properties different from those of bulk water and play a vital role in the activity and stability of these structures, as well as in intermolecular interactions. Previous studies using solution scattering, crystallography, and molecular dynamics simulations have provided valuable information about the properties of these hydration shells, including modifications in density and ionic concentration...
May 24, 2016: Biophysical Journal
Maria Elena Ortiz-Soto, Jürgen Seibel
Sialyltransferases (STs) are disulfide-containing, type II transmembrane glycoproteins that catalyze the transfer of sialic acid to proteins and lipids and participate in the synthesis of the core structure oligosaccharides of human milk. Sialic acids are found at the outermost position of glycostructures, playing a key role in health and disease. Sialylation is also essential for the production of recombinant therapeutic proteins (RTPs). Despite their importance, availability of sialyltransferases is limited due to the low levels of stable, soluble and active protein produced in bacterial expression systems, which hampers biochemical and structural studies on these enzymes and restricts biotechnological applications...
2016: PloS One
Haidy Metwally, Robert G McAllister, Vlad Popa, Lars Konermann
Electrospray ionization (ESI) allows the production of intact gas-phase ions from proteins in solution. Nondenaturing solvent conditions usually culminate in low ESI charge states. However, many mass spectrometric applications benefit from protein ions that are more highly charged. One way to boost protein charge is the addition of supercharging agents (SCAs) such as sulfolane or m-nitrobenzyl alcohol (m-NBA) to the aqueous solution. The supercharging mechanism remains controversial. We use molecular dynamics (MD) simulations to examine how SCAs affect the behavior of ESI nanodroplets...
May 17, 2016: Analytical Chemistry
Eita Sasaki, Donald Hilvert
Engineered variants of the capsid-forming enzyme lumazine synthase can exploit electrostatic interactions to encapsulate complementarily charged guest macromolecules. Here we investigate the effect of ionic strength and cargo molecules on assembly of AaLS-13, a negatively supercharged lumazine synthase protein cage, and we show that multishell structures are produced upon mixing the capsid core with free capsomers and a positively supercharged variant of the green fluorescent protein GFP(+36). The assembly process is mediated by favorable electrostatic interactions between the negatively charged capsid shells/capsomers and GFP(+36) molecules, and it is therefore strongly dependent on ionic strength...
July 7, 2016: Journal of Physical Chemistry. B
Allie C Obermeyer, Carolyn E Mills, Xue-Hui Dong, Romeo J Flores, Bradley D Olsen
Complexation of proteins with polyelectrolytes or block copolymers can lead to phase separation to generate a coacervate phase or self-assembly of coacervate core micelles. However, many proteins do not coacervate at conditions near neutral pH and physiological ionic strength. Here, protein supercharging is used to systematically explore the effect of protein charge on the complex coacervation with polycations. Four model proteins were anionically supercharged to varying degrees as quantified by mass spectrometry...
April 21, 2016: Soft Matter
Ming Wang, John A Zuris, Fantao Meng, Holly Rees, Shuo Sun, Pu Deng, Yong Han, Xue Gao, Dimitra Pouli, Qi Wu, Irene Georgakoudi, David R Liu, Qiaobing Xu
A central challenge to the development of protein-based therapeutics is the inefficiency of delivery of protein cargo across the mammalian cell membrane, including escape from endosomes. Here we report that combining bioreducible lipid nanoparticles with negatively supercharged Cre recombinase or anionic Cas9:single-guide (sg)RNA complexes drives the electrostatic assembly of nanoparticles that mediate potent protein delivery and genome editing. These bioreducible lipids efficiently deliver protein cargo into cells, facilitate the escape of protein from endosomes in response to the reductive intracellular environment, and direct protein to its intracellular target sites...
March 15, 2016: Proceedings of the National Academy of Sciences of the United States of America
Catherine C Going, Zijie Xia, Evan R Williams
Electrothermal supercharging (ETS) with electrospray ionization produces highly charged protein ions from buffered aqueous solutions in which proteins have native folded structures. ETS increases the charge of ribonuclease A by 34%, whereas only a 6% increase in charge occurs for a reduced-alkylated form of this protein, which is unfolded and its structure is ~66% random coil in this solution. These results indicate that protein denaturation that occurs in the ESI droplets is the primary mechanism for ETS. ETS does not affect the extent of solution-phase hydrogen-deuterium exchange (HDX) that occurs for four proteins that have significantly different structures in solution, consistent with a droplet lifetime that is considerably shorter than observable rates of HDX...
June 2016: Journal of the American Society for Mass Spectrometry
Kevin A Douglass, Andre R Venter
The addition of certain reagents during the electrospray ionization mass spectrometry of proteins can shift the protein ion signal charge-state distributions (CSDs) to higher average charge states, a phenomenon known as 'supercharging'. The role of reagent gas-phase basicity (GB) during this process was investigated in both the negative and positive ion modes. Reagents with known or calculated GBs were added individually in equimolar amounts to protein solutions which were subsequently electrosprayed for mass spectrometry analysis...
2015: European Journal of Mass Spectrometry
Li Li, Samuel H Yang, Veronika Vidova, Elisa M Rice, Aruna B Wijeratne, Vladimír Havlíček, Kevin A Schug
The application of continuous flow-extractive desorption electrospray ionization (CF-EDESI), an ambient ionization source demonstrated previously for use with intact protein analysis, is expanded here for the coupling of reversed phase protein separations to mass spectrometry. This configuration allows the introduction of charging additives to enhance detection without affecting the chromatographic separation mechanism. Two demonstrations of the advantages of CF-EDESI are presented in this work. First, a proof-of- principle is presented to demonstrate the applicability of hyphenation of liquid chromatography (LC) to CF- EDESI...
2015: European Journal of Mass Spectrometry
Yi Cai, Qiuling Zheng, Yong Liu, Roy Helmy, Joseph A Loo, Hao Chen
This study presents the development of ultra-performance liquid chromatography (UPLC) mass spectrometry (MS) combined with electrochemistry (EC) for the first time and its application for the structural analysis of proteins/peptides that contain disulfide bonds. In our approach, a protein/peptide mixture sample undergoes a fast UPLC separation and subsequent electrochemical reduction in an electrochemical flow cell followed by online MS and tandem mass spectrometry (MS/MS) analyses. The electrochemical cell is coupled to the mass spectrometer using our recently developed desorption electrospray ionization (DESI) interface...
2015: European Journal of Mass Spectrometry
Wenshuang Wang, Naihan Han, Ruijuan Li, Wenjun Han, Xiaoru Zhang, Fuchuan Li
Glycosaminoglycans (GAGs) are linear acidic heteropolysaccharides that are ubiquitously expressed in animal tissues and participate in various life processes. To date, the detection and visualization of GAGs in complex biological samples and living organisms remain a challenge because of the lack of powerful biocompatible probes. In this study, a superpositively charged green fluorescent protein (ScGFP) was shown great potential in GAG detection for the first time. First, on the basis of the phenomenon of GAGs dose-dependently inhibiting the fluorescence quenching of ScGFP by graphene oxide, a simple and highly sensitive signal-on homogeneous platform was established for detecting and quantifying GAGs, even in complex samples such as heparin in citrated plasma and oversulfated chondroitin sulfate in heparin...
September 15, 2015: Analytical Chemistry
Shiyun Tang, Zhou Nie, Wang Li, Daiqi Li, Yan Huang, Shouzhuo Yao
A label-free and convenient strategy for PARP-1 activity assay and inhibitors assessment has been developed based on the fluorescence resonance energy transfer (FRET) between a cationic conjugated polymer (CCP) and supercharged green fluorescent protein (scGFP).
October 1, 2015: Chemical Communications: Chem Comm
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