•A chromatographic HILICCAD method to measure levels of mAb trisulfide modifications.•Comparable results to the alternative method—peptide map with LC/MS detection.•Method applicable to ...cysteine-linked ADC production and cell culture process development.
A robust, high-throughput method using hydrophilic interaction liquid chromatography (HILIC) coupled with a charged aerosol detector (CAD) is reported as a novel approach for trisulfide quantitation in monoclonal antibodies (mAbs). The products of mAb reduction using tris(2-carboxyethyl)phosphine (TCEP) include a species (TCEP(S)) that is stoichiometrically produced from trisulfides. The TCEP reaction products are chromatographically separated, detected, and quantified by the HILICCAD method. The method was qualified to quantify trisulfides across a range of 1–40% (mol trisulfide/mol mAb). In all tested matrix components, assay linearity and intermediate precision were established with correlation coefficients (R2)>0.99, and relative standard deviations (RSD)<10%. A method comparability study was performed using peptide mapping LC–MS as an orthogonal measurement. For the range of 1–40% trisulfides, the analysis demonstrates that, on average, HILICCAD reads between 0.95 and 1.10 times the value of LC–MS with 95% confidence. Applications of the HILICCAD method include trisulfide determination in purified mAbs to be used in the production of cysteine-linked antibody-drug conjugates, and in cell culture development studies to understand sources of, and strategies for control of, trisulfides.
•Raman spectroscopy was used to study gluten structure changes during mixing.•Raman spectroscopy measurements were performed directly in the dough matrix.•A thiol blocking reagent was used as a ...reducing agent to reduce disulphide bonds.•Intermolecular disulphide bonds are decisive for the stability of the gluten network.•Hydrophobic bonds are important in the structural evolution of dough during mixing.
The aim of the present study was to evaluate Raman spectroscopy in determining changes that occur in the structure of gluten proteins induced during bread dough mixing. Raman spectra were measured directly within the dough. Three particular phases of mixing were studied: under-mixing, optimum mixing and over-mixing. A thiol blocking reagent, Tris(2-carboxyethyl)phosphine (TCEP) was then used to reduce disulphide bonds within proteins to confirm the important role of disulphide bridges in gluten network formation. For the control dough, the most important changes occurred during the optimum mixing phase when an increase in intermolecular disulphide bonds, anti-parallel β-sheet and α-helix structures was observed, combined with the hydrophobic burial of tryptophan and tyrosine residues. The addition of TCEP appeared to effectively reduce the formation of intermolecular disulphide bonds, anti-parallel β-sheet and α-helix structures and lead to a more disordered secondary protein structure.
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•The oxidation of TCEP by IrCl62− has been investigated kinetically over a wide pH range.•Observed second-order rate constant vs pH profile has been established.•Reaction mechanism ...involving phosphine radical cations is proposed and convincing.•The reactivity of the TCEP species towards an oxidant is deciphered for the first time.
Despite of wide applications of tris(2-carboxyethyl)phosphine (TCEP) as a reductant, the reductions of single electron oxidants by TCEP are poorly understood mechanistically. Herein the reduction of IrCl62− by TCEP was thus investigated kinetically over a wide pH range at 1.0 M ionic strength. Overall second-order kinetics was proved for the reduction, being first-order with respect to both Ir(IV) and TCEP, respectively. The redox stoichiometry was determined to be ΔIr(IV):ΔTCEP = 2:1 and the corresponding phosphine oxide (TCEPO) was identified as the oxidation product of TCEP by 1H NMR spectroscopy. A reaction mechanism was rationalized in term of parallel reactions of IrCl62− with all the TCEP protolysis species being as the rate-determining steps, in which phosphine radical cations were involved as the transient intermediates. Rate constants of the rate-determining steps were calculated, rendering a reactivity trend for the five TCEP species: P(CH2CH2CO2−)3 ≫ +HP(CH2CH2CO2−)3 ≈ +HP(CH2CH2CO2−)2(CH2CH2CO2H) ≫ +HP(CH2CH2CO2−)(CH2CH2CO2H)2 ≫ +HP(CH2CH2CO2H)3. This is the first time to decipher the reactivity of all the TCEP protolysis species in the redox reactions of TCEP. Additionally, activation parameters were also measured for the reaction of P(CH2CH2CO2−)3 with IrCl62− yielding ΔH5‡ = 26.3 ± 1.4 kJ∙mol−1 and ΔS5‡ = −48 ± 5 J∙K−1∙mol−1; a possible mode of electron transfer is discussed. No doubt, the mechanism and the reactivity derived from the present work can serve as a model for the oxidations of TCEP by other single electron oxidants that are involved in chemically- and biologically-important processes.
•CuCl solutions efficiently trap and stabilize H2S and sulfhydryls from headspaces.•Trapped material is quantitatively released with TCEP and brine dilution.•Wines in anoxia at 50 °C release up to ...400 μg/L of H2S and 58 μg/L of MeSH in 68 days.•Differences between released and accumulated amounts reveal high reactivity.•The TCEP-BCDA test, even with trapping solutions, does not work with red wines.
Some relevant food systems release tiny amounts of sulfidic gases, whose measurement is difficult because of their inherent instability. The present paper demonstrates that Cu(I) solutions trap quantitatively and stabilize sulfidic gases. Once trapped, the gases remain stable for weeks at 4 °C and at least 8 days at 75 °C. Trapped gases can be quantitatively released with tris(2-carboxyethyl) phosphine (TCEP) and brine dilution and then determined by GC. Trapping solutions, placed in 20-mL opened vials housed in 100 mL hermetically-sealed flasks containing wine in anoxia, have been used to monitor the release of sulfidic gases by wines, revealing that at 50 °C, up to 400 μg/L of H2S and 58 μg/L of MeSH can be released in 68 days, and 3–5 times more at 75 °C in 28 days. The possibility to differentiate between released and accumulated amounts provides key clues to understanding the fate of sulfidic gases in wine and other food systems.
Oocyte in vitro maturation (IVM) is a crucial process that determines subsequent in vitro embryo production. The present study investigated the effects of the antioxidant tris (2-carboxyethyl) ...phosphine hydrochloride (TCEP-HCL) on the in vitro maturation of porcine oocytes and in vitro developmental competence of fertilized embryos. Oocytes were matured in IVM medium based on four concentration groups of TCEP-HCL (0, 50, 100, and 200 μM) treatment. 100 μM TCEP-HCL treatment significantly increased the oocyte first polar body extrusion rate, monospermy rate and subsequent in vitro fertilized embryo developmental capacity (cleavage rate, blastocyst formation rate, and blastocyst total cell number) compared to those in the control group. Furthermore, 100 μM TCEP-HCL treatment significantly reduced the levels of reactive oxygen species, significantly increased glutathione levels and mitochondrial content compared to those in the control group. Moreover, 100 μM TCEP-HCL treatment significantly decreased the oocyte apoptosis, blastocyst apoptosis compared to that in the controls. In summary, these results indicate that 100 μM TCEP-HCL treatment improves the quality and developmental capacity of in vitro-fertilized embryos by decreasing oxidative stress in porcine oocytes.
•TCEP-HCL reduces oxidative stress in in vitro-matured oocytes.•TCEP-HCL treatment alleviates polyspermy during in vitro fertilization.•TCEP-HCL improves in vitro fertilized embryo developmental capacity and quality.
Stem cell‐based therapy has been highlighted as a potential avenue to promote tissue regeneration, where stimulation of stem cells to differentiate into the targeted cell type is essential. One of ...the factors that induce stem cells to differentiate is their surrounding microenvironment. In this study, the correlation between mild reductant and early osteogenic commitment was evaluated. A cell surface‐reducing microenvironment significantly silenced the transforming growth factor (TGF)‐β signaling pathway of mesenchymal stem cells (MSCs), followed by increased focal adhesion and inhibition of cell membrane protein dimerization. Furthermore, in vivo transplantation of MSCs exposed to the reducing microenvironment resulted in an early osteogenic commitment and neobone formation. Thus, these results highlight the potential of cell surface‐reducing microenvironment to influence early osteogenic commitment.
This paper presented an electrochemical aptasensor for dopamine detection with high sensitivity and selectivity. The electrochemical signal was amplified by electrochemical-chemical redox cycling ...with tris(2-carboxyethyl)phosphine (TCEP) as the reducing reagent. Specifically, dopamine captured by the aptamer-covered gold nanoparticles-modified carbon glass electrode was cycled by TCEP after its electrochemical oxidization, enabling an increase in the anodic current. Because of the high specificity and strong binding affinity of aptamer to dopamine, the sensor is fairly selective in not responding to common interferences. The results of chronoamperometry indicated that this aptasensor allowed for the detection of dopamine in a linear concentration range of 5nM to 0.5μM. The detection limit was estimated to be 1.8nM. Additionally, the aptasensor was successfully applied to determine dopamine spiked in the serum sample and gave recoveries ranging from 94.6 to 107.2%. This work would also be valuable for development of new types of electrochemical sensors for determination of other electroactive substances by marrying specific receptors and effective reducing or oxidizing reagents.
•We reported an electrochemical aptasensor for dopamine detection.•The signal was amplified by electrochemical-chemical redox cycling.•Tris(2-carboxyethyl)phosphine was used as the reducing agent to regenerate dopamine.•The linear range is 5nM–0.5μM and the detection limit is 1.8nM.
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•A novel redox cycling was reported and used for colorimetric immunoassay.•Pyrroloquinoline quinone (PQQ) promoted the reduction of Fe(III)-ferrozine by tris(2-carboxyethyl)phosphine ...(TCEP).•Mesoporous silica nanoparticle was used as the carrier of PQQ and recognition antibody.•The signal was monitored by the generation of colorful Fe(II)-ferrozine.•A detection limit of 1 pg/mL was obtained for prostate-specific antigen detection.
This work reported a novel redox cycling in which pyrroloquinoline quinone (PQQ) promoted the reduction of Fe(III)-ferrozine by tris(2-carboxyethyl)phosphine (TCEP). Specifically, PQQ was reduced into pyrroloquinoline quinol (PQQH2) by TCEP, and then the produced PQQH2 reduced colorless Fe(III)-ferrozine into dull red Fe(II)-ferrozine. The redox cycling was employed for development of colorimetric immunosensors through the generation of colorful Fe(II)-ferrozine. Mesoporous silica nanoparticle (MSN) was used as the carrier for both PQQ and recognition antibody through electrostatic interactions. Magnetic bead (MB) was used as the support for the immobilization of capture antibody. After the sandwich-type immunoreactions and magnetic separation, the MSN-PQQ nanolabels on the MB surface triggered the production of Fe(II)-ferrozine. The limit of detection was found to be 1 pg/mL with prostate specific antigen (PSA) as the model target. The result for the analysis of serum sample is in agreement with that achieved by the commercial enzyme-linked immunosorbent assay kits. The proposed immunosensor obviated the use of enzyme molecules for signal amplification and did not require expensive instruments for signal readout. This work should be valuable for the design of novel nanolabels and the proposed sensing strategy by the redox cycling could be applied to develop more sensitive biosensors.
Disulfide-bonded thiols in malt and hops were first identified as possible precursors of thiols in beer. The presence of disulfide-bonded 3-mercaptohexan-1-ol (3MH) was confirmed in malt and hops by ...observing an 8.9–9.9 times increase in the 3MH concentration in hopped water and unhopped wort after the reduction using tris(2-carboxyethyl)phosphine (TCEP), a reducing agent specific for disulfide bonds. The presence of disulfide-bonded 4-mercapto-4-methylpentan-2-one (4MMP) was confirmed in hops by observing 2.1 and 5.1 times increase in the 4MMP concentration after reduction in hopped water. Proteins, peptides, and amino acids having sulfhydryl groups or other thiol substances were assumed to form disulfide bonds with polyfunctional thiols in malt and hops. The release of thiols by the reduction of disulfide-bonded thiols during fermentation was first identified. A 65–82% of disulfide-bonded 3MH were reduced during fermentation, and as a result, concentrations of 3MH in hopped water and unhopped wort increased by 9.5–14.2 times during fermentation.
