Reduction Of Disulphide Bonds Research Articles

Systematic analytical workflow for characterisation and identification of partially reduced species in monoclonal antibody manufacturing

Fragmentation is a major degradation pathway ubiquitous to all therapeutic monoclonal antibody (mAb) and therefore, monitored throughout the manufacturing process. Here, we describe a three-step approach to 1) detect, 2) confirm and 3) characterize partially reduced fragment species in an immunoglobulin G1 (IgG1) mAb with prolonged hold time of harvested cell culture fluid (HCCF). Microchip capillary electrophoresis (MCE) and high-performance size exclusion chromatography (HPSEC) were used as fast and efficient screening methods to detect fragmentation. HPSEC was found to be underestimating fragmentation levels. To confirm and characterize the fragments, capillary electrophoresis-sodium dodecyl sulphate (CE-SDS) was employed. Interestingly, the absence of fragments in the reduced CE-SDS analysis suggested partial reduction of disulphide bonds contributing to fragmentation in this mAb lot. This was further confirmed using reverse phase high performance liquid chromatography (RP-HPLC) coupled with mass spectrometry, which established the presence of heavy-heavy-light (HHL), heavy-heavy (HH), light-light dimer (LL), light chain (LC) and half antibody (HL) fragments with good mass accuracy. In this study, we demonstrated a readily applicable systematic strategy to support process development and investigate anomalous events in manufacturing. An additional highlight of this work is the data-driven comprehensive comparison of modern and conventional analytical techniques for fragment analysis.

Direct Detection of Glycated Human Serum Albumin and Hyperglycosylated IgG3 in Serum, by MALDI-ToF Mass Spectrometry, as a Predictor of COVID-19 Severity.

The prefusion spike protein of SARS-CoV-2 binds advanced glycation end product (AGE)-glycated human serum albumin (HSA) and a higher mass (hyperglycosylated/glycated) immunoglobulin (Ig) G3, as determined by matrix assisted laser desorption mass spectrometry (MALDI-ToF). We set out to investigate if the total blood plasma of patients who had recovered from acute respiratory distress syndrome (ARDS) as a result of COVID-19, contained more glycated HSA and higher mass (glycosylated/glycated) IgG3 than those with only clinically mild or asymptomatic infections. A direct serum dilution, and disulphide bond reduction, method was developed and applied to plasma samples from SARS-CoV-2 seronegative (n = 30) and seropositive (n = 31) healthcare workers (HCWs) and 38 convalescent plasma samples from patients who had been admitted with acute respiratory distress (ARDS) associated with COVID-19. Patients recovering from COVID-19 ARDS had significantly higher mass AGE-glycated HSA and higher mass IgG3 levels. This would indicate that increased levels and/or ratios of hyper-glycosylation (probably terminal sialic acid) IgG3 and AGE glycated HSA may be predisposition markers for the development of COVID-19 ARDS as a result of SARS-CoV2 infection. Furthermore, rapid direct analysis of serum/plasma samples by MALDI-ToF for such humoral immune correlates of COVID-19 presents a feasible screening technology for the most at risk; regardless of age or known health conditions.

Trichothiodystrophy hair shafts display distinct ultrastructural features.

Hair shafts from three trichothiodystrophy (TTD) patients with mutations in the ERCC2 (XPD) gene were examined by transmission electron microscopy. TTD is a rare, recessive disorder with mutations in several genes in the DNA repair/transcription pathway, including ERCC2. Unlike previous studies, the hair shafts were examined after relaxation of their structure by partial disulphide bond reduction in the presence of sodium dodecyl sulphate, permitting improved visualization. Compared with hair shafts of normal phenotype, TTD cuticle cells displayed aberrant marginal bands and exocuticle layers. Clusters of cells stained differently (light versus dark) in the cortex of aberrant shafts, and the keratin macrofibrils appeared much shorter in the cytoplasm. Considerable heterogeneity in these properties was evident among samples and even along the length of single hair shafts. The results are consistent with not only a paucity of high sulphur components, such as keratin-associated proteins, but also a profound imbalance in protein content and organization.

Study of structural changes of gluten proteins during bread dough mixing by Raman spectroscopy

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.

The role of sphingolipids in endoplasmic reticulum stress.

The endoplasmic reticulum (ER) is an important intracellular compartment in eukaryotic cells and has diverse functions, including protein synthesis, protein folding, lipid metabolism and calcium homeostasis. ER functions are disrupted by various intracellular and extracellular stimuli that cause ER stress, including the inhibition of glycosylation, disulphide bond reduction, ER calcium store depletion, impaired protein transport to the Golgi, excessive ER protein synthesis, impairment of ER-associated protein degradation and mutated ER protein expression. Distinct ER stress signalling pathways, which are known as the unfolded protein response, are deployed to maintain ER homeostasis, and a failure to reverse ER stress triggers cell death. Sphingolipids are lipids that are structurally characterized by long-chain bases, including sphingosine or dihydrosphingosine (also known as sphinganine). Sphingolipids are bioactive molecules long known to regulate various cellular processes, including cell proliferation, migration, apoptosis and cell-cell interaction. Recent studies have uncovered that specific sphingolipids are involved in ER stress. This review summarizes the roles of sphingolipids in ER stress and human diseases in the context of pathogenic events.

Impedance Biosensing atop MoS2 Thin Films with Mo−S Bond Formation to Antibody Fragments Created by Disulphide Bond Reduction

AbstractImmobilization of antibody fragments to 3‐phenoxybenzoic acid (3‐PBA), which are created by disulphide bond (S−S) reduction with tris (2‐carboxyethyl) phosphine (TCEP), is reported atop MoS2 and Cu‐doped MoS2 thin films. MoS2 and Cu‐doped MoS2 thin films are electrodeposited using previously reported methods and tested for their ability to immobilize antibody fragments, before and after annealing in Ar at 500 °C for 3 h. This annealing procedure removes excess sulphur in the as‐deposited films, and creates coordinatively unsaturated Mo sites that are highly reactive towards sulphur, as previously reported for MoS2 hydrodesulphurization catalysts. As demonstrated by electrochemical impedance spectroscopy (EIS) measurements, both annealed MoS2 and Cu‐doped MoS2 thin films adsorb antibody fragments through Mo−S bond formation, unlike the as‐deposited films. Impedance detection of 3‐PBA is reported utilizing antibody fragments bound to both materials, with a sensitivity of 2.7×108 Ω cm2 M−1 and a detection limit of 2.5×10−6 M atop MoS2, and a sensitivity of 5.9×108 Ω cm2 M−1 and a detection limit of 3.8×10−6 M atop Cu‐doped MoS2. The rms surface roughness obtained by atomic force microscopy (AFM) measurements atop annealed MoS2 and Cu‐doped MoS2 ranges from 60–140 nm, so the methods described herein are not limited to ultra‐smooth substrates.

A Proteomics Workflow for the Identification of Labile Disulphide Bonds at the Cell Surface.

Reduction of labile disulphide bonds on leukocyte cell surface proteins plays a regulatory role in immune cell activation. Here I describe a method for the fast, efficient, and unbiased purification of cell-surface proteins containing such labile disulphide bonds. Free thiols liberated from the reduction of labile disulphide bonds are labeled with biotin, purified, enriched, and subsequently identified using liquid chromatography coupled to tandem mass spectrometry. Both the proteins containing the labile disulphide bonds and the position of bonds within the protein are revealed, thus providing a valuable addition to the immunology or biochemistry toolkit.

Identification of PDI Substrates by Mechanism-Based Kinetic Trapping.

Protein disulphide isomerase (PDI) is secreted by activated platelets and endothelial cells and is required for thrombus formation upon vascular injury. PDI catalyzes the reduction, oxidation, or isomerization of disulphide bonds in its substrate proteins. The specific substrates of PDI during thrombus formation have largely remained elusive, in part due to the transient nature of the PDI-substrate interaction.To overcome this challenge we have adapted and developed a kinetic substrate trapping strategy to identify extracellular substrates of PDI. By substitution of selected amino acids in the PDI active sites, we have generated PDI variants that form stable complexes with their substrates for subsequent isolation and identification. We here describe the substrate trapping methodology in detail, including generation and characterization of PDI variants, kinetic trapping experiments, and isolation and identification of bound substrates. The protocol can be adapted for most any biological fluid or sample, and can be applied to other extracellular thiol isomerases.

Rapid charge variant analysis of monoclonal antibodies to support lead candidate biopharmaceutical development

Biopharmaceuticals are complex therapeutic proteins produced in living cells that undergo a variety of enzymatic and non-enzymatic posttranslational modifications both intracellularly and also following secretion into the condition media. Characterization of these posttranslational modifications is a regulatory requirement to ensure that the molecule meets the required levels of quality to ensure patient safety. Ion exchange chromatography, particularly cation exchange, is routinely used for the determination of the charge variant profile of monoclonal antibodies (mAbs) using either an increasing concentration of salt or the generation of a pH gradient to facilitate elution of the mAb charge variants from the cation exchange phase. In this study, salt and pH gradient elution modes were compared to develop an optimized separation for the mAb standard reference material from NIST on a strong cation exchange phase. Separation using the pH gradient approach was found to outperform salt gradient separation. The developed pH gradient method was transformed into an ultra-fast separation method to facilitate rapid molecular profiling and triage during lead candidate and cell line development activities. The ultrafast method was validated and showed excellent performance for linearity and precision as well as applicability for the analysis of a variety of mAbs with different pI-values. The method has proven suitable for the integration into process analytical technology (PAT) frameworks and was found to be powerful in combination with automated data analysis strategies for obtaining low end-to-end processing time. This was demonstrated by the acquisition and analysis of in total 45 runs within only 5 h. The method was next applied for profiling in-house produced candidate biosimilars of trastuzumab and enabled the assessment of the charge variant profile of these candidates in <25 min. Differences identified for trastuzumab expressed using a stable CHO cell line were found to result from incomplete cleavage of the signal peptide from the light chain as determined using high resolution reversed-phase LC-MS following digestion with IdeS protease and disulphide bond reduction. The proposed method is well suited for molecular triage during cell line development or indeed for potential process analytical technology application during larger scale manufacture.

Raman study of the cleavage of disulphide bonds in albumin, chymotrypsin, and thrombin

The structural changes resulting from the reduction of disulphide bonds in three globular proteins (albumin, chymotrypsin and thrombin) in aqueous solutions are investigated using Raman spectroscopy. Changes in SS and SH vibrational bands are used for direct observation of the disulphide bond reduction. The corresponding changes in secondary structure and H-bonding of tyrosines are observed. It is shown that the unfolding pathway after the reduction of disulphide bridges in albumin depends on the reducing agent. The most developed changes are observed for the band assigned to the ggg conformation of the disulphide bridge in two similar serine proteases (thrombin and chymotrypsin) but the secondary structure of thrombin is much more stable against the reduction of the disulphide bonds.

Disulphide Bond Reduction of RNase A by Drug Metosartan a Comparative Study

Disulphide Bond Reduction of RNase A by Drug Metosartan a Comparative Study

Electrochemical Protein Cleavage in a Microfluidic Cell for Proteomics Studies

Electrochemical protein digestion prior to mass spectrometric analysis is a purely instrumental approach to protein identification, offering reduced consumption of chemicals and shorter analysis times compared to the use of enzymes and chemical cleavage agents. Here we demonstrate the possibilities of site-specific peptide bond cleavage and disulphide bond reduction in a microfluidic electrochemical cell. The use of microfluidics in this context is beneficial for increased electrochemical cleavage yields, small sample volumes and the possibility of rapid on-line analysis, thereby providing a versatile tool for routine proteomics studies.

Composition and properties of peptides that survive standardised in vitro gastro-pancreatic digestion of bovine milk

There are conflicting results on the release and conservation of bioactive protein sequences during in vitro digestion. Only the peptides that are released and survive may exert their action. Peptides <3 kDa were determined by mass spectrometry after standardised in vitro gastro-pancreatic digestion of bovine milk. At a degree of digestion of 30.7%, 384 peptides were obtained from 2 to 25 residues in length. Five other peptides were recovered after reduction of disulphide bonds. Due to the presence of proline residues at the last or second last position at the C-terminus, a high number of peptides with ACE-inhibitory activity were obtained. Peptides with opioid, antimicrobial, immunomodulatory and antithrombotic activities were recovered, with many precursors and degradative products. Peptides with opioid and antimicrobial activities may be the result of evolutionary processes. The peptides obtained were similar to those released in the human jejunum.

Penicillamine attenuates agglutination of ram spermatozoa in capacitating media through the reduction of disulphide bonds on a sperm binding protein

Penicillamine attenuates agglutination of ram spermatozoa in capacitating media through the reduction of disulphide bonds on a sperm binding protein

D-penicillamine prevents ram sperm agglutination by reducing the disulphide bonds of a copper-binding sperm protein.

Head-to-head agglutination of ram spermatozoa is induced by dilution in the Tyrode's capacitation medium with albumin, lactate and pyruvate (TALP) and ameliorated by the addition of the thiol d-penicillamine (PEN). To better understand the association and disassociation of ram spermatozoa, we investigated the mechanism of action of PEN in perturbing sperm agglutination. PEN acts as a chelator of heavy metals, an antioxidant and a reducing agent. Chelation is not the main mechanism of action, as the broad-spectrum chelator ethylenediaminetetraacetic acid and the copper-specific chelator bathocuproinedisulfonic acid were inferior anti-agglutination agents compared with PEN. Oxidative stress is also an unlikely mechanism of sperm association, as PEN was significantly more effective in ameliorating agglutination than the antioxidants superoxide dismutase, ascorbic acid, α-tocopherol and catalase. Only the reducing agents cysteine and DL-dithiothreitol displayed similar levels of non-agglutinated spermatozoa at 0 h compared with PEN but were less effective after 3 h of incubation (37 °C). The addition of 10 µM Cu(2+) to 250 µM PEN + TALP caused a rapid reversion of the motile sperm population from a non-agglutinated state to an agglutinated state. Other heavy metals (cobalt, iron, manganese and zinc) did not provoke such a strong response. Together, these results indicate that PEN prevents sperm association by the reduction of disulphide bonds on a sperm membrane protein that binds copper. ADAM proteins are possible candidates, as targeted inhibition of the metalloproteinase domain significantly increased the percentage of motile, non-agglutinated spermatozoa (52.0% ± 7.8) compared with TALP alone (10.6% ± 6.1).

Microwave-Alkali Treatment of Chicken Feathers for Protein Hydrolysate Production

Conversion of chicken feathers into valuable products like protein and amino acids is very challenging due to the rigid structure of keratins extensively cross-linked by disulphide, hydrogen and hydrophobic bonds. An efficient treatment is necessary for reducing the disulphide bonds and increasing the feathers solubilisation. This study investigated the effects of microwave-alkali treatment on disulphide bond reduction and morphological changes of chicken feathers for protein hydrolysate production. Feathers were treated at different sodium hydroxide concentrations (0.1, 0.5, 1.0, 1.5, 2.0 M), various microwave power levels (100, 300, 450, 600, 800 W) and residence times (2, 4, 6, 8, 10 min). The most efficient conditions for microwave-alkali treatment of feathers were (1) 10 min; (2) 0.5 M NaOH and (3) 800 W which produced 24.72 mM thiol and 26.74 mg/mL protein. In comparison to the autoclave-alkali and the conventional heating-alkali method, the microwave-alkali treatment denatured the feather keratins and reduced the disulphide bonds in feathers to a greater extent. Scanning electron microscope and fourier transform infrared analyses showed that the structure of the microwave-alkali treated feathers was highly disrupted and significantly changed from fibers into an amorphous structure. Based on the amino acid profile, the protein hydrolysate from the microwave-alkali treatment contained a significantly higher concentration of amino acids (69.4 mg/g of feathers) compared to the autoclave-alkali (19.0 mg/g of feathers) and the conventional heating-alkali (27.8 mg/g of feathers) treatments. Microwave-alkali treatment was more efficient than conventional treatments in breaking down the disulphide bonds, disrupting the feather structure and producing protein hydrolysate.

Functionality of the storage proteins in gluten-free cereals and pseudocereals in dough systems

The dough functionality of the storage proteins in “gluten-free” grains has been studied for almost 25 years. Zein, maize prolamin, when isolated as α-zein can form a wheat gluten-like visco-elastic dough when mixed with water above its glass transition temperature. There is good evidence that its dough-forming properties are related to a change in protein conformation from α-helix to β-sheet and association of the molecules into fibrils. Stabilisation of β-sheet structure and visco-elasticity can be enhanced by inclusion of a co-protein. No other isolated cereal or pseudocereal storage protein has been shown to form a visco-elastic dough. Many treatments have been applied to improve “gluten-free” storage protein functionality, including acid/base, deamidation, cross-linking by oxidising agents and transglutaminase, proteolysis, disulphide bond reduction and high pressure treatment. Such treatments have some limited positive benefits on batter-type dough functionality, but none is universally effective and the effects seem to be dependent on the composition and structure of the particular storage protein. Research into mutants where prolamin synthesis is altered appears to be promising in terms of improved dough functionality and scientific understanding. Research into how treatments affect the functionality and structure of isolated storage proteins from “gluten-free” grains other than maize is required.

Rate turnover in mechano-catalytic coupling: A model and its microscopic origin.

A novel aspect in the area of mechano-chemistry concerns the effect of external forces on enzyme activity, i.e., the existence of mechano-catalytic coupling. Recent experiments on enzyme-catalyzed disulphide bond reduction in proteins under the effect of a force applied on the termini of the protein substrate reveal an unexpected biphasic force dependence for the bond cleavage rate. Here, using atomistic molecular dynamics simulations combined with Smoluchowski theory, we propose a model for this behavior. For a broad range of forces and systems, the model reproduces the experimentally observed rates by solving a reaction-diffusion equation for a "protein coordinate" diffusing in a force-dependent effective potential. The atomistic simulations are used to compute, from first principles, the parameters of the model via a quasiharmonic analysis. Additionally, the simulations are also used to provide details about the microscopic degrees of freedom that are important for the underlying mechano-catalysis.

Over-expression of Trxo1 increases the viability of tobacco BY-2 cells under H2O2 treatment.

Reactive oxygen species (ROS), especially hydrogen peroxide, play a critical role in the regulation of plant development and in the induction of plant defence responses during stress adaptation, as well as in plant cell death. The antioxidant system is responsible for controlling ROS levels in these processes but redox homeostasis is also a key factor in plant cell metabolism under normal and stress situations. Thioredoxins (Trxs) are ubiquitous small proteins found in different cell compartments, including mitochondria and nuclei (Trxo1), and are involved in the regulation of target proteins through reduction of disulphide bonds, although their role under oxidative stress has been less well studied. This study describes over-expression of a Trxo1 for the first time, using a cell-culture model subjected to an oxidative treatment provoked by H2O2. Control and over-expressing PsTrxo1 tobacco (Nicotiana tabacum) BY-2 cells were treated with 35 mm H2O2 and the effects were analysed by studying the growth dynamics of the cultures together with oxidative stress parameters, as well as several components of the antioxidant systems involved in the metabolism of H2O2. Analysis of different hallmarks of programmed cell death was also carried out. Over-expression of PsTrxo1 caused significant differences in the response of TBY-2 cells to high concentrations of H2O2, namely higher and maintained viability in over-expressing cells, whilst the control line presented a severe decrease in viability and marked indications of oxidative stress, with generalized cell death after 3 d of treatment. In over-expressing cells, an increase in catalase activity, decreases in H2O2 and nitric oxide contents and maintenance of the glutathione redox state were observed. A decreased content of endogenous H2O2 may be responsible in part for the delayed cell death found in over-expressing cells, in which changes in oxidative parameters and antioxidants were less extended after the oxidative treatment. It is concluded that PsTrxo1 transformation protects TBY-2 cells from exogenous H2O2, thus increasing their viability via a process in which not only antioxidants but also Trxo1 seem to be involved.

Expression, characterization and crystal structure of thioredoxin from Schistosoma japonicum.

Schistosoma japonicum, a human blood fluke, causes a parasitic disease affecting millions of people in Asia. Thioredoxin-glutathione system of S. japonicum plays a critical role in maintaining the redox balance in parasite, which is a potential target for development of novel antischistosomal agents. Here we cloned the gene of S. japonicum thioredoxin (SjTrx), expressed and purified the recombinant SjTrx in Escherichia coli. Functional assay shows that SjTrx catalyses the dithiothreitol (DTT) reduction of insulin disulphide bonds. The coupling assay of SjTrx with its endogenous reductase, thioredoxin glutathione reductase from S. japonicum (SjTGR), supports its biological function to maintain the redox homeostasis in the cell. Furthermore, the crystal structure of SjTrx in the oxidized state was determined at 2.0 Å resolution, revealing a typical architecture of thioredoxin fold. The structural information of SjTrx provides us important clues for understanding the maintenance function of redox homeostasis in S. japonicum and pathogenesis of this chronic disease.