Thiol Modification Research Articles

Improved Mucoadhesion, Permeation and In Vitro Anticancer Potential of Synthesized Thiolated Acacia and Karaya Gum Combination: A Systematic Study.

Thiolation of polymers is one of the most appropriate approaches to impart higher mechanical strength and mucoadhesion. Thiol modification of gum karaya and gum acacia was carried out by esterification with 80% thioglycolic acid. FTIR, DSC and XRD confirmed the completion of thiolation reaction. Anticancer potential of developed thiomer was studied on cervical cancer cell lines (HeLa) and more than 60% of human cervical cell lines (HeLa) were inhibited at concentration of 5 µg/100 µL. Immobilized thiol groups were found to be 0.8511 mmol/g as determined by Ellman’s method. Cytotoxicity studies on L929 fibroblast cell lines indicated thiomers were biocompatible. Bilayered tablets were prepared using Ivabradine hydrochloride as the model drug and synthesized thiolated gums as mucoadhesive polymer. Tablets prepared using thiolated polymers in combination showed more swelling, mucoadhesion and residence time as compared to unmodified gums. Thiol modification controlled the release of the drug for 24 h and enhanced permeation of the drug up to 3 fold through porcine buccal mucosa as compared to tablets with unmodified gums. Thiolated polymer showed increased mucoadhesion and permeation, anticancer potential, controlled release and thus can be utilized as a novel excipient in formulation development.

3-Chloro-5-Substituted-1,2,4-Thiadiazoles (TDZs) as Selective and Efficient Protein Thiol Modifiers.

The study of cysteine modifications has gained much attention in recent years. This includes detailed investigations in the field of redox biology with focus on numerous redox derivatives like nitrosothiols, sulfenic acids, sulfinic acids and sulfonic acids resulting from increasing oxidation, S-lipidation, and perthiols. For these studies selective and rapid blocking of free protein thiols is required to prevent disulfide rearrangement. In our attempt to find new inhibitors of human histone deacetylase 8 (HDAC8) we discovered 5-sulfonyl and 5-sulfinyl substituted 1,2,4-thiadiazoles (TDZ), which surprisingly show an outstanding reactivity against thiols in aqueous solution. Encouraged by these observations we investigated the mechanism of action in detail and show that these compounds react more specifically and faster than commonly used N-ethyl maleimide, making them superior alternatives for efficient blocking of free thiols in proteins. We show that 5-sulfonyl-TDZ can be readily applied in commonly used biotin switch assays. Using the example of human HDAC8, we demonstrate that cysteine modification by a 5-sulfonyl-TDZ is easily measurable using quantitative HPLC/ESI-QTOF-MS/MS, and allows for the simultaneous measurement of the modification kinetics of seven solvent-accessible cysteines in HDAC8.

Bioinspired metal-organic framework-based liquid-infused surface (MOF-LIS) with corrosion and biofouling prohibition properties

Corrosion and biofouling are two main deterioration effects for shortening the service life of metal material in marine environment. In this study, targeting corrosion and biofouling of Cu, a bioinspired liquid-infused surface (LIS) coating based on metal organic frameworks (MOFs) is constructed onto Cu by sequential steps, including oxidation, coordination reaction, thiol modification and infusion process. The microstructure, chemical composition and wettability of the coating are uncovered through different characterization techniques, such as scanning electron microscopy, high-resolution transmission electron microscopy, electron diffraction, X-ray photoelectron spectroscopy and so forth. Corrosion durability test results show that MOF-LIS performs high corrosion inhibition to underneath Cu. As a representative guiding parameter, Rct of MOF-LIS reaches 5.26 × 107 Ω cm2, which is much higher than that of bare Cu (694.6 Ω cm2), illustrating a corrosion inhibition efficiency up to 99.999% for Cu metal. Using diatoms as the probe organisms, after being immersed in seawater for 8 d, the attaching diatoms density on MOF-LIS (7.33 × 104 cells/cm2) is much less than bare Cu surface (3.95 × 108 cells/cm2), illustrating the high biofouling inhibition efficiency. Therefore, the versatile properties enable MOF-LIS as a coating having potential usage for anti-corrosion and anti-biofouling in the marine environment.

Regulation of Derlin-1-mediated degradation of NADPH oxidase partner p22phox by thiol modification

The transmembrane protein p22phox heterodimerizes with NADPH oxidase (Nox) 1–4 and is essential for the reactive oxygen species-producing capacity of oxidases. Missense mutations in the p22phox gene prevent the formation of phagocytic Nox2-based oxidase, which contributes to host defense. This results in chronic granulomatous disease (CGD), a severe primary immunodeficiency syndrome. In this study, we characterized missense mutations in p22phox (L51Q, L52P, E53V, and P55R) in the A22° type (wherein the p22phox protein is undetectable) of CGD. We demonstrated that these substitutions enhanced the degradation of the p22phox protein in the endoplasmic reticulum (ER) and the binding of p22phox to Derlin-1, a key component of ER-associated degradation (ERAD). Therefore, the L51-L52-E53-P55 sequence is responsible for protein stability in the ER. We observed that the oxidation of the thiol group of Cys-50, which is adjacent to the L51-L52-E53-P55 sequence, suppressed p22phox degradation. However, the suppression effect was markedly attenuated by the serine substitution of Cys-50. Blocking the free thiol of Cys-50 by alkylation or C50S substitution promoted the association of p22phox with Derlin-1. Derlin-1 depletion partially suppressed the degradation of p22phox mutant proteins. Furthermore, heterodimerization with p22phox (C50S) induced rapid degradation of not only Nox2 but also nonphagocytic Nox4 protein, which is responsible for redox signaling. Thus, the redox-sensitive Cys-50 appears to determine whether p22phox becomes a target for degradation by the ERAD system through its interaction with Derlin-1.

Site-Specific Fluorescent Labeling of the Cysteine-Rich Toxin, DkTx, for TRPV1 Ion Channel Imaging and Membrane Binding Studies.

Peptide toxins secreted by venomous animals bind to mammalian ion channel proteins and modulate their function. The high specificity of these toxins for their target ion channels enables them to serve as powerful tools for ion channel biology. Toxins labeled with fluorescent dyes are employed for the cellular imaging of channels and also for studying toxin-channel and toxin-membrane interactions. Several of these toxins are cysteine-rich, rendering the production of properly folded fluorescently labeled toxins technically challenging. Herein, we evaluate a variety of site-specific protein bioconjugation approaches for producing fluorescently labeled double-knot toxin (DkTx), a potent TRPV1 ion channel agonist that contains an uncommonly large number of cysteines (12 out of a total of 75 amino acids present in the protein). We find that popular cysteine-mediated bioconjugation approaches are unsuccessful as the introduction of a non-native cysteine residue for thiol modification leads to the formation of misfolded toxin species. Moreover, N-terminal aldehyde-mediated bioconjugation approaches are also not suitable as the resultant labeled toxin lacks activity. In contrast to these approaches, C-terminal bioconjugation of DkTx via the sortase bioconjugation technology yields functionally active fluorescently labeled DkTx. We employ this labeled toxin for imaging rat TRPV1 heterologously expressed in Xenopus laevis oocytes, as well as for performing membrane binding studies on giant unilamellar vesicles composed of different lipid compositions. Our studies set the stage for using fluorescent DkTx as a tool for TRPV1 biology and provide an informative blueprint for labeling cysteine-rich proteins.

Switch off/switch on of a cysteinyl protease as a way to preserve the active catalytic group by modification with a reversible covalent thiol modifier: Immobilization of ficin on vinyl-sulfone activated supports

The immobilization of ficin (a cysteinyl proteases) on vinyl sulfone agarose produced its almost full inactivation. It was observed that the incubation of the free and immobilized enzyme in β-mercaptoethanol produced a 20 % of enzyme activity recovery, suggesting that the inactivation due to the immobilization could be a consequence of the modification of the catalytic Cys. To prevent the enzyme inactivation during the immobilization, switching off of ficin via Cys reaction with dipyridyl-disulfide was implemented, giving a reversible disulfide bond that produced a fully inactive enzyme. The switch on of ficin activity was implemented by incubation in 1 M β-mercaptoethanol. Using this strategy to immobilize the enzyme on vinyl sulfone agarose beads, the expressed activity of the immobilized ficin could be boosted up to 80 %. The immobilized enzyme presented a thermal stabilization similar to that obtained using ficin-glyoxyl-agarose beads. This procedure may be extended to many enzymes containing critical Cys, to permit their immobilization or chemical modification.

Potential of mercapto montmorillonites for immobilization remediation of Hg-contaminated paddy soil: Perspective from soil environmental quality

Consideration of the high exposure risk of mercury (Hg) in paddy system, the development highly-efficient and environmental-friendly amendments for the immobilization remediation of Hg-contaminated paddy soil is of significant importance for mitigating Hg-related health risks. In this study, the potential of 2-mercaptoethylammonium intercalation, 3-mercaptopropyltrimethoxysilane covalent grafting and mechanochemical grafting mercapto montmorillonites (referred as ISH-Mt, GSH-Mt and BSH-Mt, respectively) for immobilization remediation of Hg contaminated paddy soil and its effects on soil environmental quality were investigated. Results indicated that the application of 0.1–1% (m/m) mercapto Mts exhibited much higher immobilization efficiency for Hg than pristine Mt., in which soil HCl and Na2S2O3 extractable Hg content were decreased by 5.13–62.44% and 8.03–82.24% as compared to CK. Hg fractions in mercapto Mts amended soils shifted form exchangeable and carbonate-bound to organic-bound as results of complexation by the loaded thiol groups. The response of soil environmental quality indicators to amendments varied with the preparation methods of mercapto Mts. Compared to CK, ISH-Mt exhibited greater effects on soil physicochemical properties than GSH-Mt and BSH-Mt, such as the decreased pH and the substantially increased EC (9.85–29.00%) and available S content (5.13–41.09%), implied the potential risk of soil acidification and thiol modifier leaching. Application of mercapto Mts enhanced soil enzyme activities of dehydrogenase, arylsulfatase and fluorescein diacetate hydrolase by 14.63–27.55%, 2.39–5.41% and 25.64–39.72%, respectively. While, GSH-Mt caused greater disturbance on bacterial community than ISH-Mt and BSH-Mt for the reason that GSH-Mt changed the composition of predominant phyla, increased the abundance of methylators Anaerolinea and Geobacter and exhibited the most biomarkers (15) and differential metabolic pathways (44). Overall consideration Hg immobilization potential and secondary environment risk, BSH-Mt was a promising immobilization amendment for the remediation of Hg-contaminated paddy soil.

Interfacial engineering of metallic rhodium by thiol modification approach for ambient electrosynthesis of ammonia

Interfacial engineering of metallic rhodium by thiol modification approach for ambient electrosynthesis of ammonia

Thiol Modifications in the Extracellular Space-Key Proteins in Inflammation and Viral Infection.

Posttranslational modifications (PTMs) allow to control molecular and cellular functions in response to specific signals and changes in the microenvironment of cells. They regulate structure, localization, stability, and function of proteins in a spatial and temporal manner. Among them, specific thiol modifications of cysteine (Cys) residues facilitate rapid signal transduction. In fact, Cys is unique because it contains the highly reactive thiol group that can undergo different reversible and irreversible modifications. Upon inflammation and changes in the cellular microenvironment, many extracellular soluble and membrane proteins undergo thiol modifications, particularly dithiol–disulfide exchange, S-glutathionylation, and S-nitrosylation. Among others, these thiol switches are essential for inflammatory signaling, regulation of gene expression, cytokine release, immunoglobulin function and isoform variation, and antigen presentation. Interestingly, also the redox state of bacterial and viral proteins depends on host cell-mediated redox reactions that are critical for invasion and infection. Here, we highlight mechanistic thiol switches in inflammatory pathways and infections including cholera, diphtheria, hepatitis, human immunodeficiency virus (HIV), influenza, and coronavirus disease 2019 (COVID-19).

The TetR-type regulator AtsR is involved in multidrug response in Corynebacterium glutamicum

BackgroundThe TetR (tetracycline repressor) family is one of the major transcription factor families that regulate expression of genes involved in bacterial antimicrobial resistance systems. NCgl0886 protein, designated as AtsR, is a member of the TetR family identified in Corynebacterium glutamicum, which is conserved in several species of the genera Corynebacterium, also including the well-known pathogen C. diphtheriae. AtsR is located at no far upstream of the identically oriented ncgl0884 gene, encoding a putative multidrug efflux pump protein, and in the same operon with ncgl0887, encoding a resistance, nodulation and cell division (RND) superfamily drug exporter. However, the role of AtsR is not clearly understood.ResultsHere we showed that dimeric AtsR directly repressed the expression of the ncgl0887-atsR operon, as well as indirectly controlled the ncgl0884 transcription. Antibiotics and toxic compounds induced the expression of ncgl0887-atsR operon. A perfect palindromic motif (5΄-TGCAA-N2-TTGCA-3΄; 12 bp) was identified in the upstream region of ncgl0887-atsR operon. Electrophoretic mobility shift assays (EMSAs) demonstrated specific binding of AtsR to this motif, and hydrogen peroxide (H2O2) blocked binding. H2O2 oxidized cysteine residues to form Cys123-Cys187 intermolecular disulfide bonds between two subunits in AtsR dimer, which altered its DNA-binding characteristics and caused its dissociation, thereby leading to derepression of the drug efflux protein. Deletion of ncgl0884 and ncgl0887 increased the susceptibilities of C. glutamicum for several toxic compounds, but overexpression of atsR decreased the drug tolerance of C. glutamicum.ConclusionsOur study revealed that AtsR was a redox regulator that sensed oxidative stress via thiol modification. The results obtained here will contribute to our understanding of the drug response mechanism not only in C. glutamicum but also in the related bacteria C. diphtheriae.

OPDAylation of Thiols of the Redox Regulatory Network In Vitro.

cis-(+)-12-Oxophytodienoic acid (OPDA) is a reactive oxylipin produced by catalytic oxygenation of polyunsaturated α-linolenic acid (18:3 (ω − 3)) in the chloroplast. Apart from its function as precursor for jasmonic acid synthesis, OPDA serves as a signaling molecule and regulator on its own, namely by tuning enzyme activities and altering expression of OPDA-responsive genes. A possible reaction mechanism is the covalent binding of OPDA to thiols via the addition to the C=C double bond of its α,β-unsaturated carbonyl group in the cyclopentenone ring. The reactivity allows for covalent modification of accessible cysteinyl thiols in proteins. This work investigated the reaction of OPDA with selected chloroplast and cytosolic thioredoxins (TRX) and glutaredoxins (GRX) of Arabidopsis thaliana. OPDA reacted with TRX and GRX as detected by decreased m-PEG maleimide binding, consumption of OPDA, reduced ability for insulin reduction and inability to activate glyceraldehyde-3-phosphate dehydrogenase and regenerate glutathione peroxidase (GPXL8), and with lower efficiency, peroxiredoxin IIB (PRXIIB). OPDAylation of certain protein thiols occurs quickly and efficiently in vitro and is a potent post-translational modification in a stressful environment.

Ultrasensitive disposable apatasensor for reagentless electrocatalytic detection of thrombin: An O2-Dependent hemin-G4-aptamer assay on gold screen-printed electrodes

Serine protease thrombin is a strong neurotoxin produced by the injured brain and an Alzheimer's disease biomarker. To address its point-of-care testing (POCT), we adapted the O2-dependent aptamer assay for thrombin to gold screen-printed electrodes (Au SPE). The assay exploits reagentless (with no mediators) electrocatalytic activity of hemin-G4 DNAzyme in O2 reduction. Au SPEs modified with thiolated hemin-conjugated aptamer and PEG showed enhanced electrocatalytic activity in O2 reduction upon thrombin binding to the aptamer, then folding into the electroactive hemin-G4 DNAzyme structure. 0.5 fM thrombin were detected in aerated PBS and artificial cerebrospinal fluid, correspondingly, with the logarithmic linear range extending to 100 fM; dopamine, and uric and ascorbic acids did not interfere with electroanalysis. The disposable aptasensor met basic POCT requirements, with a minimal shelf life of 3 days. However, the reactivity and suitability of the Au SPE surface for thiol binding and electrocatalysis required special surface pre-treatment and modification protocols, and the fundamental problem of a long-term stability of thiol modification on gold should be addressed for practical applications of Au SPE-based apatasensors in POCT.

Complexity of NAC Action as an Antidiabetic Agent: Opposing Effects of Oxidative and Reductive Stress on Insulin Secretion and Insulin Signaling.

Dysregulated redox balance is involved in the pathogenesis of type 2 diabetes. While the benefit of antioxidants in neutralizing oxidative stress is well characterized, the potential harm of antioxidant-induced reductive stress is unclear. The aim of this study was to investigate the dose-dependent effects of the antioxidant N-acetylcysteine (NAC) on various tissues involved in the regulation of blood glucose and the mechanisms underlying its functions. H2O2 was used as an oxidizing agent in order to compare the outcomes of oxidative and reductive stress on cellular function. Cellular death in pancreatic islets and diminished insulin secretion were facilitated by H2O2-induced oxidative stress but not by NAC. On the other hand, myotubes and adipocytes were negatively affected by NAC-induced reductive stress, as demonstrated by the impaired transmission of insulin signaling and glucose transport, as opposed to H2O2-stimulatory action. This was accompanied by redox balance alteration and thiol modifications of proteins. The NAC-induced deterioration of insulin signaling was also observed in healthy mice, while both insulin secretion and insulin signaling were improved in diabetic mice. This study establishes the tissue-specific effects of NAC and the importance of the delicate maintenance of redox balance, emphasizing the challenge of implementing antioxidant therapy in the clinic.

Redox Proteomics Analysis of Atherosclerotic Aortas: Application of the "OxICAT" Method.

Atherosclerosis development and progression have been linked to vascular reactive oxygen species (ROS). Plaque formation and especially instability, frequently resulting in acute coronary syndromes, have been linked to cell apoptosis and senescence, but also mainly to increased cellular oxidative stress. ROS are characterized by their high chemical reactivity and a resulting short half-life. This high reactivity usually involves reversible and/or irreversible protein modifications and specifically the covalent oxidative modification of cysteine residues. The latter can be used for the identification of protein-chemical footprints, leading to indirect monitoring of ROS. Proteomics and especially liquid chromatography tandem mass spectrometry (LC-MS/MS) approaches have emerged as a powerful tool to identify such protein modifications in biological samples (e.g., body fluids, tissues, cells). Application of a well-established quantitative thiol trapping technique termed OxICAT enables the detection and quantification of oxidative thiol modifications of thousands of proteins in a single experiment. In this chapter, a step-by-step guide for the redox proteomics analysis of atherosclerotic aortas, by utilizing the OxICAT method, as optimized by our group is provided.

Thiol modification of galactoarabinan and its appraisal as controlled release carrier of sofosbuvir

The study shows the development and optimization of thiolated and unthiolated polymer as a carrier of sofosbuvir, an anti-hepatic drug for sustained release. Successful thiolation was confirmed by the appearance of peak at 2518 cm−1 in FTIR and 9.07 mg of thiol group per gram of polymer (found by Ellmans reagent test). The decrease in the moisture content in thiolated polymer by Karl Fischer titration also confirmed the modification of polymer. The unthiolated and thiolated polymer was characterized by Fourier-transform infrared spectroscopy, scanning electron microscopy and thermal analysis, and evaluated for in vitro dissolution release studies and in vivo pharmacokinetics. Pre-formulation and post-formulation tests for tablets were performed. Thiolated (15% and 20%) and unthiolated (15%) polymer possessed excellent flow properties whereas; thiolated polymer (25%) showed sustained release of drug for 4 h in in vitro dissolution profile. Moreover, pharmacokinetic parameters including Cmax, tmax, t1/2 and AUC were determined after administering standard oral solution (SOS) and tablets of sofosbuvir at a single oral dose of 42 mg/kg body weight. Results of in vivo pharmacokinetics (p < 0.0001) have explored that Cmax increased from 503 ng/ml (SOS) to 591 and 653 ng/ml for F and TF ng/ml, tmax from less than an hour (SOS) to 5hrs. The t1/2 was increased from 1hr (SOS) to 5.15 and 7 h for F and TF and observed average AUC was 2104, 5616.5, 7199 ng/ml/hr for standard, F and TF respectively. The observed Area under the curve (AUC) for all three formulations was significantly different (p > 0.05), indicating the noticeable influence of the polymer and its modified form. Based on this study, it can be concluded that thiolated polymer formulation has exhibited sustained release of sofosbuvir with improved pharmacokinetic profile.

Photocurable Coatings Based on Bio-Renewable Oligomers and Monomers.

Due to long-term problems related to environmental protection, economic aspects, and waste management in the chemical industry, it is justified to develop renewable polymers as an alternative to synthetic polymers. Two kinds of acrylic bio-renewable components were used for the modification of acrylated epoxidized soybean oil (AESO). The bio-based compositions used as photocurable binders to obtain the photocurable coatings with satisfactory properties and high bio content were then prepared. The kinetic of curing reaction of the oligomers and monomers towards radical photopolymerization and the properties of the cured coatings were fully investigated; the results are discussed in relation with the compounds’ structures. Important information about how to design and obtain renewable photocurable coatings with satisfactory properties was provided in this study. In this study, AESO resin was modified with renewable oligomer or (math)acrylate monomer to increase the reactivity and reduce the viscosity of the photoreactive system in order to obtain renewable and viable alternatives to petroleum-based polymeric materials with perfect film-forming properties. It turned out that both photopolymerization rate and hardness of cured coatings were increased significantly with the addition of modifiers; the use of a thiol modifier and change of the photoinitiator concentration allowed to improve the adhesion, hardness, and control of the photo-curing process.

Radiation Driven Chemistry in Biomolecules—is (V)UV Involved in the Bioactivity of Argon Jet Plasmas?

Cold physical plasmas, especially noble gas driven plasma jets, emit considerable amounts of ultraviolet radiation (UV). Given that a noble gas channel is present, even the energetic vacuum UV can reach the treated target. The relevance of UV radiation for antimicrobial effects is generally accepted. It remains to be clarified if this radiation is relevant for other biomedical application of plasmas, e.g., in wound care or cancer remediation. In this work, the role of (vacuum) ultraviolet radiation generated by the argon plasma jet kINPen for cysteine modifications was investigated in aqueous solutions and porcine skin. To differentiate the effects of photons of different wavelength and complete plasma discharge, a micro chamber equipped with a MgF2, Suprasil, or Borosilicate glass window was used. In liquid phase, plasma-derived VUV radiation was effective and led to the formation of cysteine oxidation products and molecule breakdown products, yielding sulfite, sulfate, and hydrogen sulfide. At the boundary layer, the impact of VUV photons led to water molecule photolysis and formation of hydroxyl radicals and hydrogen peroxide. In addition, photolytic cleavage of the weak carbon-sulfur bond initiated the formation of sulfur oxy ions. In the intact skin model, protein thiol modification was rare even if a VUV transparent MgF2 window was used. Presumably, the plasma-derived VUV radiation played a limited role since reactions at the boundary layer are less frequent and the dense biomolecules layers block it effectively, inhibiting significant penetration. This result further emphasizes the safety of physical plasmas in biomedical applications.

Nitro-fatty acids: electrophilic signaling molecules in plant physiology.

Nitro fatty acids (NO2-FA)have relevant physiological roles as signaling molecules in biotic and abiotic stress, growth, and development, but the mechanism of action remains controversial. The two main mechanisms involving nitric oxide release and thiol modification are discussed. Fatty acids (FAs) are major components of membranes and contribute to cellular energetic demands. Besides, FAs are precursors of signaling molecules, including oxylipins and other oxidized fatty acids derived from the activity of lipoxygenases. In addition, non-canonical modified fatty acids, such as nitro-fatty acids (NO2-FAs), are formed in animals and plants. The synthesis NO2-FAs involves a nitration reaction between unsaturated fatty acids and reactive nitrogen species (RNS). This review will focus on recent findings showing that, in plants, NO2-FAs such as nitro-linolenic acid (NO2-Ln) and nitro-oleic acid (NO2-OA) have relevant physiological roles as signaling molecules in biotic and abiotic stress, growth, and development. Moreover, since there is controversy on mechanisms of action of NO2-FAs as signaling molecules, we will provide evidence showing why this aspect needs further evaluation.

Precise identification of O-linked β-N-acetylglucosamine peptides based on O-mesitylenesulfonylhydroxylamine elimination reaction

氧连接氮乙酰葡萄糖胺(O-GlcNAc)是一种重要的蛋白质翻译后修饰,它在维持机体正常的生命活动中发挥着重要作用。许多研究证实,O-GlcNAc糖基化修饰稳态的破坏与人类多种疾病的发生相关,大规模富集鉴定O-GlcNAc糖基化修饰蛋白有助于发现新的临床疾病诊断标志物。由于O-GlcNAc糖基化修饰丰度较低,形成的糖苷键不稳定,O-GlcNAc糖基化修饰蛋白/肽段的富集鉴定面临一定挑战。近年来,全乙酰化的非天然糖代谢标记技术被广泛应用于O-GlcNAc糖基化修饰蛋白/肽段的富集鉴定。然而,最新的研究发现,在细胞代谢标记过程中,全乙酰化的非天然单糖会同时标记半胱氨酸的巯基而引入半胱氨酸巯基-叠氮糖人为修饰物。该副反应在一定程度上干扰了O-GlcNAc糖基化修饰蛋白/肽段的富集鉴定。鉴于此,研究发展了一种通过三甲基苯磺酰羟胺(MSH)特异性氧化消除半胱氨酸巯基-叠氮糖人为修饰物的方法,进而显著提高O-GlcNAc糖基化修饰肽段的精准鉴定。该方法建立于温和的磷酸钠缓冲液(50 mmol/L, pH=8)体系,利用过量的MSH,于95 ℃避光振荡反应30 min,可完全消除半胱氨酸巯基-叠氮糖人为修饰物。该方法应用于Hela细胞中,可有效消除叠氮全乙酰化半乳糖胺(Ac4GalNAz)代谢产生的半胱氨酸巯基-叠氮糖人为修饰物,从而成功富集鉴定到157条O-GlcNAc糖基化修饰肽段,归属于130个蛋白质。该方法有效去除了半胱氨酸巯基-叠氮糖人为修饰物对代谢标记结果的干扰,为非天然糖代谢标记技术在糖蛋白组学分析中的应用提供了新的研究策略。

Hexaammineruthenium (II)/(III) and Hexacyanoferrate (II)/(III) Redox-Probes for Graphene-Based Inkjet-Printed Electrodes: Benchmarking with Gold and Glassy Carbon Screen Printed Electrodes

Gold and glassy carbon (GC) screen printed electrodes (SPEs) are being widely used for chemical and chemical/biological sensing. Number of commercial sources are available to use gold and GC SPEs off-the-shelf sensors for electrochemical characterization. Cyclic voltammetry (CV), while provides fundamentals of electrode processes pertaining to the charge transfer phenomena, is often obscured by the stability of electrodes due to unfavorable interactions between the redox probes and electrode materials even though gold and GC are excellent choices. Hexaammineruthenium (II)/(III) and Hexacyanoferrat (II)/(III) are the two most extensively used redox probes among several others. However, for example, the later one causes corrosion problems in case of gold electrodes leading to damage in electrode surface modification (such as thiol modification), and thus, providing unreliable results. Therefore, a need to understand the electrode-redox probe interaction and the underlying electrochemical properties is essential.Graphene inkjet-printed electrodes (G-IPEs) have emerged as promising electrochemical sensors that could minimize the cost, ease manufacturing, bring environmental remediation and have potential to revolutionize fabrication of mechanically flexible and bendable electrochemical sensors with number of practical applications such as environmental sensing and biomedical sensing. In addition, due to its exceptionally promising physical properties, graphene will provide multifunctional sensor systems such as electrochemical and strain-based sensors. However, a careful study and one-to-one comparison of G-IPEs with gold and GC SPEs in fundamental performance in terms of their CVs using Hexaammineruthenium (II)/(III) and Hexacyanoferrat (II)/(III) are not available, making it difficult to assess the suitability of use of G-IPEs. In this work, we present a scalable manufacturing of graphene-based inks (pure graphene ink as well as composite inks of graphene with carbon nanotubes, graphene aerosol gels, hexagonal boron nitride), and their additive manufacturing to produce electrochemical electrodes. Detailed cyclic voltammetry study to compare the graphene-based IPEs with gold and GC SPEs and benchmark their electrochemical performance in terms of the charge transfer properties as well as chemical/mechanical stabilities will be presented. Such a study will be greatly beneficial to select the redox probe of choice for graphene-based printed electrochemical sensors.Keywords: Graphene-based inks, Inkjet Printing, Redox-Probes, Cyclic Voltammetry, Charge-Transfer*Corresponding author: srdas@ksu.edu