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https://www.readbyqxmd.com/read/27487254/supercharging-reagent-for-enhanced-liquid-chromatographic-separation-and-charging-of-sialylated-and-high-molecular-weight-glycopeptides-for-nanohplc-esi-ms-ms-analysis
#1
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
https://www.readbyqxmd.com/read/27441318/electrothermal-supercharging-of-proteins-in-native-ms-effects-of-protein-isoelectric-point-buffer-and-nanoesi-emitter-tip-size
#2
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
https://www.readbyqxmd.com/read/27224484/saxs-sans-on-supercharged-proteins-reveals-residue-specific-modifications-of-the-hydration-shell
#3
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
https://www.readbyqxmd.com/read/27166796/expression-of-functional-human-sialyltransferases-st3gal1-and-st6gal1-in-escherichia-coli
#4
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
https://www.readbyqxmd.com/read/27093467/mechanism-of-protein-supercharging-by-sulfolane-and-m-nitrobenzyl-alcohol-molecular-dynamics-simulations-of-the-electrospray-process
#5
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
https://www.readbyqxmd.com/read/27064167/self-assembly-of-proteinaceous-multishell-structures-mediated-by-a-supercharged-protein
#6
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
https://www.readbyqxmd.com/read/26965053/complex-coacervation-of-supercharged-proteins-with-polyelectrolytes
#7
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
https://www.readbyqxmd.com/read/26929348/efficient-delivery-of-genome-editing-proteins-using-bioreducible-lipid-nanoparticles
#8
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
https://www.readbyqxmd.com/read/26919868/real-time-hd-exchange-kinetics-of-proteins-from-buffered-aqueous-solution-with-electrothermal-supercharging-and-top-down-tandem-mass-spectrometry
#9
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
https://www.readbyqxmd.com/read/26307743/on-the-role-of-a-direct-interaction-between-protein-ions-and-solvent-additives-during-protein-supercharging-by-electrospray-ionization-mass-spectrometry
#10
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
https://www.readbyqxmd.com/read/26307717/reversed-phase-liquid-chromatography-hyphenated-to-continuous-flow-extractive-desorption-electrospray-ionization-mass-spectrometry-for-analysis-and-charge-state-manipulation-of-undigested-proteins
#11
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
https://www.readbyqxmd.com/read/26307715/integration-of-electrochemistry-with-ultra-performance-liquid-chromatography-mass-spectrometry
#12
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
https://www.readbyqxmd.com/read/26287436/supercharged-fluorescent-protein-as-a-versatile-probe-for-the-detection-of-glycosaminoglycans-in-vitro-and-in-vivo
#13
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
https://www.readbyqxmd.com/read/26273706/a-poly-adp-ribose-polymerase-1-activity-assay-based-on-the-fret-between-a-cationic-conjugated-polymer-and-supercharged-green-fluorescent-protein
#14
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
https://www.readbyqxmd.com/read/26208330/dna-mediated-supercharged-fluorescent-protein-graphene-oxide-interaction-for-label-free-fluorescence-assay-of-base-excision-repair-enzyme-activity
#15
Zhen Wang, Yong Li, Lijun Li, Daiqi Li, Yan Huang, Zhou Nie, Shouzhuo Yao
The interaction between supercharged green fluorescent protein (ScGFP) and graphene oxide (GO) as well as the resulting quenching effect of GO on ScGFP were investigated. Based on this unique quenching effect and the DNA-mediated ScGFP/GO interaction, a label-free fluorescence method has been established for homogeneously assaying the activity and inhibition of base excision repair enzyme.
September 7, 2015: Chemical Communications: Chem Comm
https://www.readbyqxmd.com/read/26028988/increasing-fragmentation-of-disulfide-bonded-proteins-for-top-down-mass-spectrometry-by-supercharging
#16
Jiang Zhang, Rachel R Ogorzalek Loo, Joseph A Loo
The disulfide bond is an important post-translational modification to form and maintain the native structure and biological functions of proteins. Characterization of disulfide bond linkages is therefore of essential interest in the structural elucidation of proteins. Top-down mass spectrometry (MS) of disulfide-bonded proteins has been hindered by relatively low sequence coverage due to disulfide cross-linking. In this study, we employed top-down ESI-MS with Fourier-transform ion cyclotron resonance (FT-ICR) MS with electron capture dissociation (ECD) and collisionally activated dissociation (CAD) to study the fragmentation of supercharged proteins with multiple intramolecular disulfide bonds...
February 1, 2015: International Journal of Mass Spectrometry
https://www.readbyqxmd.com/read/25916598/supercharging-by-m-nba-improves-etd-based-quantification-of-hydroxyl-radical-protein-footprinting
#17
Xiaoyan Li, Zixuan Li, Boer Xie, Joshua S Sharp
Hydroxyl radical protein footprinting (HRPF) is an MS-based technique for analyzing protein structure based on measuring the oxidation of amino acid side chains by hydroxyl radicals diffusing in solution. Spatial resolution of HRPF is limited by the smallest portion of the protein for which oxidation amounts can be accurately quantitated. Previous work has shown electron transfer dissociation (ETD) to be the most reliable method for quantifying the amount of oxidation of each amino acid side chain in a mixture of peptide oxidation isomers, but efficient ETD requires high peptide charge states, which limits its applicability for HRPF...
August 2015: Journal of the American Society for Mass Spectrometry
https://www.readbyqxmd.com/read/25808179/rethinking-the-capsid-proteins-of-enveloped-viruses-multifunctionality-from-genome-packaging-to-genome-transfection
#18
REVIEW
João M Freire, Nuno C Santos, Ana Salomé Veiga, Andrea T Da Poian, Miguel A R B Castanho
Regardless of the debate on whether there is a place for viruses in the tree of life, it is consensual that they co-evolve with their hosts under the pressure of genome minimization. The abundance of multifunctional viral structural proteins is a consequence of this pressure. The molecular key to multifunctionality is the existence of intrinsically disordered domains together with ordered domains in the same protein. Capsid proteins, the hallmark of viruses, are not exceptions because they have coexisting ordered and disordered domains that are crucial for multifunctionality...
June 2015: FEBS Journal
https://www.readbyqxmd.com/read/25762331/arresting-amyloid-with-coulomb-s-law-acetylation-of-als-linked-sod1-by-aspirin-impedes-aggregation
#19
Alireza Abdolvahabi, Yunhua Shi, Nicholas R Rhodes, Nathan P Cook, Angel A Martí, Bryan F Shaw
Although the magnitude of a protein's net charge (Z) can control its rate of self-assembly into amyloid, and its interactions with cellular membranes, the net charge of a protein is not viewed as a druggable parameter. This article demonstrates that aspirin (the quintessential acylating pharmacon) can inhibit the amyloidogenesis of superoxide dismutase (SOD1) by increasing the intrinsic net negative charge of the polypeptide, i.e., by acetylation (neutralization) of multiple lysines. The protective effects of acetylation were diminished (but not abolished) in 100 mM NaCl and were statistically significant: a total of 432 thioflavin-T amyloid assays were performed for all studied proteins...
March 10, 2015: Biophysical Journal
https://www.readbyqxmd.com/read/25753972/increasing-protein-charge-state-when-using-laser-electrospray-mass-spectrometry
#20
COMPARATIVE STUDY
Santosh Karki, Paul M Flanigan, Johnny J Perez, Jieutonne J Archer, Robert J Levis
Femtosecond (fs) laser vaporization is used to transfer cytochrome c, myoglobin, lysozyme, and ubiquitin from the condensed phase into an electrospray (ES) plume consisting of a mixture of a supercharging reagent, m-nitrobenzyl alcohol (m-NBA), and trifluoroacetic acid (TFA), acetic acid (AA), or formic acid (FA). Interaction of acid-sensitive proteins like cytochrome c and myoglobin with the highly charged ES droplets resulted in a shift to higher charge states in comparison with acid-stable proteins like lysozyme and ubiquitin...
May 2015: Journal of the American Society for Mass Spectrometry
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