Phosphine Hydrochloride Research Articles

Acceleration of nano-surface and molecular-orientation limited (nSMOL) proteolysis with acidified reduction pretreatment for quantification of Tocilizumab

Antibody drugs are effective therapeutic agents and provide treatment for many types of diseases such as cancer and rheumatoid arthritis. Because many antibody drugs are developed and approved, quantification technologies of antibodies are required for drug development and individualized therapy. We recently reported a high reproducible and robust therapeutic drug monitoring method for antibody drugs. This method was developed to be applicable to all type of antibody drugs and to provide accurate quantification values. The method is named nano-surface and molecular-orientation limited (nSMOL) proteolysis. nSMOL limits the access of protease to immunoglobulin G molecules and aims to selectively proteolyze on Fab region of substrate. However, the bioanalysis of Tocilizumab using nSMOL has not been validated. We newly discovered that acidified reduction pretreatment of Tocilizumab promotes digestive efficiency by nSMOL proteolysis. Exposure of Tocilizumab to Tris(2-carboxyethyl)phosphine hydrochloride before nSMOL proteolysis significantly improved the recovery of peptides. Under this condition, the quantification range of Tocilizumab in human serum was from 0.781 to 200 μg/mL. The quantification values of quality control samples fulfilled all guideline criteria for bioanalytical validation. The signature peptide with the highest quantitative sensitivity was on H-chain VTMLR. From these results, it is expected that the pretreatment with TCEP will broaden the application of antibody drugs quantification by nSMOL proteolysis.

Chain-Breaking Phenolic 2,3-Dihydrobenzo[b]selenophene Antioxidants: Proximity Effects and Regeneration Studies.

Phenolic 2,3-dihydrobenzo[b]selenophene antioxidants bearing an OH-group ortho (9), meta (10, 11) and para (8) to the Se were prepared by seleno-Claisen rearrangement/intramolecular hydroselenation. meta-Isomer (11) was studied by X-ray crystallography. The radical-trapping activity and regenerability of compounds 8-11 were evaluated using a two-phase system in which linoleic acid was undergoing peroxidation in the lipid phase while regeneration of the antioxidant by co-antioxidants (N-acetylcysteine, glutathione, dithiothreitol, ascorbic acid, tris(carboxyethyl)phosphine hydrochloride) was ongoing in the aqueous layer. Compound 9 quenched peroxyl radicals more efficiently than α-tocopherol. It also provided the most long-lasting antioxidant protection. With thiol co-antioxidants it could inhibit peroxidation for more than five-fold longer than the natural product. Regeneration was more efficient when the aqueous phase pH was slightly acidic. Since calculated O-H bond dissociation energies for 8-11 were substantially larger than for α-tocopherol, an antioxidant mechanism involving O-atom transfer from peroxyl to selenium was proposed. The resulting phenolic selenoxide/alkoxyl radical would then exchange a hydrogen atom in a solvent cage before antioxidant regeneration at the aqueous lipid interphase.

Improved analysis of anthocyanins and vitamin C in blue-purple potato cultivars

Methods were optimized for extraction and quantification of anthocyanins (ACY) and vitamin C in potatoes. Acidified aqueous methanol (70%) was the optimal extraction solvent and freeze-drying significantly improved the extraction yield of ACY. The content of ACY varied widely in five potato cultivars from 0.42 to 3.18mg/g dry weight, with the latter being the highest value found in the Finnish cultivar ‘Synkeä Sakari’.Compared with dithiothreitol (DTT), tris(2-carboxyethyl) phosphine hydrochloride (TCEP) was more efficient in reducing dehydroascorbic acid (DHA) to ascorbic acid (AA) and for quantifying the content of total ascorbic acid (TAA). For raw potatoes, quantification of TAA after treatment with TCEP was more reliable than a direct analysis of AA, whereas AA can be analyzed directly in steam-cooked samples. The TAA contents in the three potato cultivars were around 0.30–0.35mg/g dry weight. The loss of AA in steam cooking was 24%.

Evaluation of CdSe/ZnS quantum dot–half anti CD3 antibody–conjugate using confocal laser scanning microscopy

ABSTRACTWe investigated the cellular uptake phenomena of half anti CD3 antibody–CdSe/ZnS quantum dot–conjugate into the live cell by using the confocal laser scanning microscopy. The anti CD3 antibody was efficiently reduced when tris 2–carboxyethyl phosphine hydrochloride was used as a reductant. The molecular weight of obtained half antibody was confirmed by the non–reduction SDS page, and the optimum concentration was 5 mM for obtaining the half antibody. Finally, the confocal laser scanning microscopy image indicates that the CdSe/ZnS quantum dot–conjugate was taken up into the live cell spontaneously by connecting the reduced half antibody around the CdSe/ZnS quantum dot.

MiRNA Quantitation with Microelectrode Sensors Enabled by Enzymeless Electrochemical Signal Amplification.

Quantification of circulating microRNAs (miRNAs) is of great interest because of their potentials as disease biomarkers. Currently, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microarray are considered mainstream techniques for miRNA identification and quantitation. However, these techniques are challenged by the low levels and wide dynamic range (from aM to nM) of miRNAs in a physiological sample, as well as the difficulty in the implementation in point-of-care settings. Here, we describe a one-step label-free electrochemical sensing technique by assembling a triple-stem DNA-redox probe structure on a gold microelectrode and introducing a reductant, tris(2-carboxyethyl) phosphine hydrochloride (TCEP) in the detection buffer solution to achieve ultrasensitive miRNAs detection with a detection limit of 0.1 fM.

Surface modification of keratin fibers through step-growth dithiol-diacrylate thiol-ene click reactions

This research proposes a novel surface modification method of keratin fibers by thiol-ene click reaction. In the first step, tris (2-carboxyethyl) phosphine hydrochloride (TCEP) was applied on wool samples to generate thiols by controlled reduction of cystine disulfide bonds in keratin. Then, as-prepared thiol-acrylate quaternary ammioum salt with divinyl terminated was grafted to the reduced keratin through thiol-ene click chemistry combining with a step-growth dithiol-diacrylate reaction. The reaction was confirmed by SEM, FTIR and TGA analysis. Antibacterial activity, antistatic property and hydrophilicity of treated wool fabric were assessed. The results demonstrated that the treatment can impart wool fabric with good antibacterial, antistatic properties and hydrophilicity.

Determination of Anabolic Hormones in Porcine Hair by High-Performance Liquid Chromatography–Mass Spectrometry

ABSTRACTA sensitive, rapid, efficient, and reliable method is reported for the determination of testosterone, 17α-methyltestosterone, trenbolone, trenbolone acetate, boldenone, nandrolone phenylpropionate, nandrolone, and norethindrone in porcine hair. This protocol involves treating 100 mg of pulverized hair by mild digestion using tris(2-carboxyethyl)phosphine hydrochloride and subsequent extraction of the anabolic hormones with methyl tert-butyl ether. The separation of the analytes was performed using acetonitrile and 0.1% aqueous formic acid. Quantitative analysis was performed by high-performance liquid chromatography–tandem triple-quadrupole mass spectrometry with positive electrospray ionization. This method is accurate, offers good sensitivity and high recovery, allowing the determination of anabolic hormones in porcine hair.

Novel bioadhesive polymers as intra-articular agents: Chondroitin sulfate-cysteine conjugates

The aim of this study was to generate and characterize a chondroitin sulfate-cysteine conjugate (CS-cys) as a novel bioadhesive agent for intra-articular use. Mucoadhesive properties of synthesized CS-cys were investigated by rheological measurement of polymer-mucus mixture and rotating cylinder method, while bioadhesive features of CS-cys on porcine articular cartilage were evaluated via tensile studies. Thiolation was achieved by attachment of l-cysteine to CS via amide bond formation mediated by carbodiimide as a coupling reagent. The conjugate exhibited 421.17±35.14μmol free thiol groups per gram polymer. The reduced CS-cys displayed 675.09±39.67μmol free thiol groups per gram polymer after disulfide bonds reduction using tris(2-carboxyethyl)phosphine hydrochloride. The increase in dynamic viscosity of thiolated CS due to oxidative disulfide bond formation was demonstrated using capillary viscometer. The combination of CS-cys and mucus led to 4.57-fold increase in dynamic viscosity in comparison with mucus control. Furthermore, adhesion time to porcine mucosa of CS-cys-based test disk was enhanced by 2.48-fold compared to unmodified CS as measured by rotating cylinder method suggesting the interaction between thiomers and mucus gel layer via disulfide bonds formation. Tensile studies of thiolated CS on porcine articular cartilage showed 5.37- and 1.76-fold increase in the total work of adhesion and the maximum detachment force, respectively, in comparison with unmodified CS indicating bioadhesive features of CS-cys. Cytotoxicity of CS-cys was assessed in Caco-2 cells and rat primary articular chondrocytes using MTT and LDH release assay, thereby showing the safety of CS-cys at a concentration of 0.25% (w/v) in Caco-2 cells. Furthermore, 0.1% of CS-cys was found non-toxic to rat primary articular chondrocytes. According to these results, CS-cys provides improved bioadhesive properties that might be useful as an intra-articular agent for treatment of osteoarthritis.

Quantitative proteomics analysis of eggshell membrane proteins during chick embryonic development

The avian eggshell membrane (ESM) is a meshwork made up of highly cross-linked protein fibers and it is a scaffold upon which biomineralization of the eggshell is initiated. The ESM and associated shell participates in embryonic development by providing physical and chemical protection against pathogen invasion. We performed quantitative proteomic analysis of ESM proteins on multiple days during the three phases of embryonic development. The ESMs were stripped from both fertilized and unfertilized eggs at different days of incubation, and solubilized in a novel manner using TCEP-HCl (Tris (2-carboxyethyl) phosphine hydrochloride). The changes in ESM proteins between occurred during incubation were analyzed. Bioinformatics analysis revealed that of the 12 functional protein clusters identified, protease inhibitors were present at all phases of chick development. A group of proteins involved in calcium binding and oxygen transport were only present during the second phase. Extracellular matrix, cell adhesion proteins related to the vascularization of chorioallantoic membrane (CAM), antimicrobial proteins and proteins involved in the binding and transport of lipids were found in the second and third phases of development. These findings provide insight into the functionality and evolving nature of ESM associated proteins involved in chick embryonic development. Biological significanceThe eggshell membranes (ESMs) are a fibrous scaffold that consists of highly crosslinked collagens (types I, V but mainly X), glycoproteins and CREMPs (cysteine-rich eggshell membrane proteins). The ESMs aid in the development of the chick embryo and protect it against pathogen invasion. This biopolymeric fibrous net functions as a platform for nucleation of the calcitic eggshell which provides a primary physical barrier against bacterial ingress. Comparative proteomic analyses of proteins in the ESMs from fertilized eggs and unfertilized eggs showed changes in their levels which varied between the specific phases of chick embryogenesis across 19days of incubation. Bioinformatics characterization of these ESM proteins provides understanding of their evolving nature during chick embryonic development.

Microelectrode miRNA sensors enabled by enzymeless electrochemical signal amplification.

Better detections of circulating microRNAs (miRNAs) as disease biomarkers could advance diseases diagnosis and treatment. Current analysis methods or sensors for research and applications are challenged by the low concentrations and wide dynamic range (from aM to nM) of miRNAs in a physiological sample. Here, we report a one-step label-free electrochemical sensor comprising a triple-stem DNA-redox probe structure on a gold microelectrode. A new signal amplification mechanism without the need of a redox enzyme is introduced. The novel strategy overcomes the fundamental limitations of microelectrode DNA sensors that fail to generate detectable current, which is primarily due to the limited amount of redox probes in response to the target analyte binding. By employing a reductant, tris(2-carboxyethyl) phosphine hydrochloride (TCEP) in the detection buffer solution, each redox molecule on the detection probe is cyclically oxidized at the electrode and reduced by the reductant; thus, the signal is amplified in situ during the detection period. The combined merits in the diagnosis power of cyclic voltammetry and the high sensitivity of pulse voltammetry enable parallel analysis for method validation and optimization previously inaccessible. As such, the detection limit of miRNA-122 was 0.1 fM via direct readout, with a wide detection range from sub fM to nM. The detection time is within minutes, which is a significant improvement over other macroscopic sensors and other relevant techniques such as quantitative reverse transcription polymerase chain reaction (qRT-PCR). The high selectivity of the developed sensors is demonstrated by the discrimination against two most similar family sequences: miR-122-3p present in serum and 2-mismatch synthetic RNA sequence. Interference such as nonspecific adsorption, a common concern in sensor development, is reduced to a negligible amount by adopting a multistep surface modification strategy. Importantly, unlike qRT-PCR, the microelectrochemical sensor offers direct absolute quantitative readout that is amenable to clinical and in-home point-of-care (POC) applications. The sensor design is flexible, capable of being tailored for detection of different miRNAs of interest. Combined with the fact that the sensor was constructed at microscale, the method can be generalized for high throughput detection of miRNA signatures as disease biomarkers.

Engineered and Native Coenzyme B12-dependent Isovaleryl-CoA/Pivalyl-CoA Mutase

Adenosylcobalamin-dependent isomerases catalyze carbon skeleton rearrangements using radical chemistry. We have recently demonstrated that an isobutyryl-CoA mutase variant, IcmF, a member of this enzyme family that catalyzes the interconversion of isobutyryl-CoA and n-butyryl-CoA also catalyzes the interconversion between isovaleryl-CoA and pivalyl-CoA, albeit with low efficiency and high susceptibility to inactivation. Given the biotechnological potential of the isovaleryl-CoA/pivalyl-CoA mutase (PCM) reaction, we initially attempted to engineer IcmF to be a more proficient PCM by targeting two active site residues predicted based on sequence alignments and crystal structures, to be key to substrate selectivity. Of the eight mutants tested, the F598A mutation was the most robust, resulting in an ∼17-fold increase in the catalytic efficiency of the PCM activity and a concomitant ∼240-fold decrease in the isobutyryl-CoA mutase activity compared with wild-type IcmF. Hence, mutation of a single residue in IcmF tuned substrate specificity yielding an ∼4000-fold increase in the specificity for an unnatural substrate. However, the F598A mutant was even more susceptible to inactivation than wild-type IcmF. To circumvent this limitation, we used bioinformatics analysis to identify an authentic PCM in genomic databases. Cloning and expression of the putative AdoCbl-dependent PCM with an α2β2 heterotetrameric organization similar to that of isobutyryl-CoA mutase and a recently characterized archaeal methylmalonyl-CoA mutase, allowed demonstration of its robust PCM activity. To simplify kinetic analysis and handling, a variant PCM-F was generated in which the αβ subunits were fused into a single polypeptide via a short 11-amino acid linker. The fusion protein, PCM-F, retained high PCM activity and like PCM, was resistant to inactivation. Neither PCM nor PCM-F displayed detectable isobutyryl-CoA mutase activity, demonstrating that PCM represents a novel 5'-deoxyadenosylcobalamin-dependent acyl-CoA mutase. The newly discovered PCM and the derivative PCM-F, have potential applications in bioremediation of pivalic acid found in sludge, in stereospecific synthesis of C5 carboxylic acids and alcohols, and in the production of potential commodity and specialty chemicals.

Production of 8-hydroxy-9,12(Z,Z)-octadecadienoic acid from linoleic acid by recombinant cells expressing H1004A-C1006S variant of Aspergillus nidulans diol synthase

An H1004A-C1006S variant of Aspergillus nidulans diol synthase was identified as an 8-dioxygenase. Whole recombinant Escherichia coli cells expressing the double-site variant showed the highest activity for converting linoleic acid to 8-hydroperoxy-9,12(Z,Z)-octadecadienoic acid (8-HPODE), which in turn was reduced to 8-hydroxy-9,12(Z,Z)-octadecadienoic acid (8-HODE) by the addition of a reducing agent. The optimization of 8-HPODE production was performed by response surface methodology, and the optimal conditions were pH 8.3, 27.2°C, 22.7% dimethyl sulfoxide, 27.0gl−1 cells, and 16.5gl−1 linoleic acid in a 100ml baffled flask containing a 10ml reaction mixture with agitation at 236rpm. The reduction of 8-HPODE to 8-HODE was highest when Tris(2-carboxyethyl)phosphine hydrochloride (TECP-HCl) was added at a final concentration of 25mM. Under the optimized reaction and reduction conditions, whole cells expressing H1004A-C1006S variant of A. nidulans diol synthase produced 5.0gl−1 8-HODE for 1h without the formation of di-hydroxy fatty acids. This is the first report of the biotechnological production of 8-HODE.

Enhancement of the Musca domestica hytrosavirus infection with orally delivered reducing agents

House flies (Musca domestica L.) throughout the world are infected with the salivary gland hypertrophy virus MdSGHV (Hytrosaviridae). Although the primary route of infection is thought to be via ingestion, flies that are old enough to feed normally are resistant to infection per os, suggesting that the peritrophic matrix (PM) is a barrier to virus transmission. Histological examination of the peritrophic matrix of healthy flies revealed a multilaminate structure produced by midgut cells located near the proventriculus. SEM revealed the PM to form a confluent sheet surrounding the food bolus with pores/openings less than 10nm in diameter. TEM revealed the PM to be multilayered, varying in width from 350 to 900nm, and generally thinner in male than in female flies. When flies were fed on the reducing agents dithiothetriol (DTT) or tris (2-caboxyethyl)phosphine hydrochloride (TCEP) for 48h before per os exposure to the virus, infection rates increased 10- to 20-fold compared with flies that did not receive the reducing agent treatments. PM’s from flies treated with DTT and TCEP displayed varying degrees of disruption, particularly in the outer layer, and lacked the electron-dense inner layer facing the ectoperitrophic space. Both drugs were somewhat toxic to the flies, resulting in>40% mortality at doses greater than 10mM (DTT) or 5mM (TCEP). DTT increased male fly susceptibility (55.1% infected) more than that of females (7.8%), whereas TCEP increased susceptibility of females (42.9%) more than that of males (26.2%). The cause for the sex differences in response to oral exposure the reducing agents is unclear. Exposing flies to food treated with virus and the reducing agents at the same time, rather than pretreating flies with the drugs, had no effect on susceptibility to the virus. Presumably, the reducing agent disrupted the enveloped virus and acted as a viricidal agent. In summary, it is proposed that the reducing agents influence integrity of the PM barrier and increase the susceptibility of flies to infection by MdSGHV.

Fully validated LC–MS/MS method for quantification of homocysteine concentrations in samples of human serum: A new approach

Reported homocysteine (HCY) concentrations in human serum show poor concordance amongst laboratories due to endogenous HCY in the matrices used for assay calibrators and QCs. Hence, we have developed a fully validated LC-MS/MS method for measurement of HCY concentrations in human serum samples that addresses this issue by minimising matrix effects. We used small volumes (20μL) of 2% Bovine Serum Albumin (BSA) as surrogate matrix for making calibrators and QCs with concentrations adjusted for the endogenous HCY concentration in the surrogate matrix using the method of standard additions. To aliquots (20μL) of human serum samples, calibrators or QCs, were added HCY-d4 (internal standard) and tris-(2-carboxyethyl) phosphine hydrochloride (TCEP) as reducing agent. After protein precipitation, diluted supernatants were injected into the LC-MS/MS. Calibration curves were linear; QCs were accurate (5.6% deviation from nominal), precise (CV%≤9.6%), stable for four freeze-thaw cycles, and when stored at room temperature for 5h or at -80°C (27 days). Recoveries from QCs in surrogate matrix or pooled human serum were 91.9 and 95.9%, respectively. There was no matrix effect using 6 different individual serum samples including one that was haemolysed. Our LC-MS/MS method has satisfied all of the validation criteria of the 2012 EMA guideline.

Surface modification of cowpea chlorotic mottle virus capsids via a copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction and their adhesion behavior with HeLa cells

A copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction was exploited for the surface modification of cowpea chlorotic mottle virus (CCMV). The exposed carboxyl residues of the CCMV capsids were modified with an alkyne and then further modified with an azide, using a triazole connection in the presence of CuSO4, tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and a bathocuproin disulfonic acid disodium salt (BCDS). Fluorogenic coumarin was successfully grafted onto the CCMV capsids and monitored by fast protein liquid chromatography (FPLC) and UV-irradiated SDS-PAGE. An oligo-ethylene glycol (OEG) short chain and an Arg-Gly-Asp (RGD) peptide were also connected to the CCMV capsids via the CuAAC reaction. Size-exclusion FPLC, transmission electron microscopy (TEM), and dynamic light scattering (DLS) analyses confirmed the modification and integrity of the viral capsids. Interestingly, OEG-CCMV displayed a unique phenomenon of connected bridges with the intact capsids crosslinked to each other. Coumarin-CCMV, OEG-CCMV, and RGD-CCMV were absorbed onto APTES slides for cell binding with HeLa cells. The opposite adhesion behavior of OEG-CCMV and RGD-CCMV indicated the inhibition effect of OEG and the promotion effect of RGD for cell attachment. This provides a generalized method for chemical modification of the surface of virus capsids with multivalent ligands, which demonstrates the potential applications in bioimaging, tissue engineering, and drug delivery.

Determination of Lipoic Acid in the form of 2-S-pyridinium Derivative by High-performance Liquid Chromatography with Ultraviolet Detection

We have developed a sensitive, simple and cheap method for determination of lipoic acid in urine and dietary supplement tablets. The method is based on conversion of lipoic acid to its thiol counterpart – dihydrolipoic acid – by reductive cleavage with tris(2-carboxyethyl)phosphine hydrochloride prior to precolumn derivatization with 1-benzyl-2- chloropyridinium bromide, followed by ion-pairing reversed-phase liquid chromatography separation and ultraviolet detection of its 2-S-pyridinium derivative at 321 nm. Bathochromic shift from the reagent absorption maximum at 275 nm to that of derivative maximum at 321 nm, taking place during reaction, is analytically advantageous. In developing this method the following parameters were investigated and optimized: the time, pH and reagent excess in the derivatization step and mobile phase buffer concentration, pH, organic modifier and column temperature in the separation step. Calibration plot, understood as the relationship between instrument response and known concentration of the analyte, demonstrated linearity of results when applied to normal urine spiked with growing amounts of lipoic acid within the tested range 0.2 – 50 µmol/L. The method was validated for urine samples received from healthy donors and urine concentration of lipoic acid was monitored after oral administration of 600 mg of lipoic acid.

Purification and partial characterization of a novel fibrinogenase from the venom of Deinagkistrodon acutus: Inhibition of platelet aggregation

A novel fibrinogenase, DAnase, was purified from the venom of Deinagkistrodon acutus by a combination of anion and cation exchange chromatography. Unlike other fibrinogenases which are usually single polypeptide chain proteins, the enzyme was a disulfide-linked dimer with an isoelectric point of 6.03 and an apparent molecular weight of 25kDa on SDS–polyacrylamide gel electrophoresis. DAnase showed α-fibrinogenase activity devoid of fibrinolytic activity. It hydrolyzed rapidly the Aα-chain of fibrinogen and followed by the Bβ-chain and did not cleave the γ-chain. It also exhibited arginine esterase activity. The fibrinogenolytic and arginine esterase activities were completely inhibited by phenylmethanesulfonyl fluoride or tris-(2-carboxyethyl)phosphine hydrochloride, but not by EDTA, indicating that DAnase is a serine protease requiring disulfide bridge(s) for its activity. The protease strongly inhibited ADP-induced platelet aggregation in human platelet-rich plasma but was lack of ADPase activity, indicating that its fibrinogenolytic activity is involved in its inhibition of ADP-induced platelet aggregation. DAnase was devoid of hemorrhagic activity and Factor XIII activation activity. DAnase may have a potential clinical application for the therapy of thrombosis disease.

Modifying Surface Resistivity and Liquid Moisture Management Property of Keratin Fibers through Thiol–Ene Click Reactions

This paper reports on a new method for improving the antistatic and liquid moisture management properties of keratinous materials. The method involves the generation of thiols by controlled reduction of cystine disulfide bonds in keratin with tris(2-carboxyethyl) phosphine hydrochloride and subsequent grafting of hydrophilic groups onto the reduced keratin by reaction with an acrylate sulfonate or acrylamide sulfonate through thiol-ene click chemistry. The modified substrates were characterized with Raman spectroscopy and scanning electron microscopy and evaluated for their performance changes in liquid moisture management, surface resistivity, and wet burst strength. The results have revealed that the thiol-acrylate reaction is more efficient than the thiol-acrylamide reaction, and the keratinous substrate modified with an acrylate sulfonate salt exhibits significantly improved antistatic and liquid moisture management properties.

Poly[N-(2-hydroxypropyl)methacrylamide] nanogels by RAFT polymerization in inverse emulsion

Inverse emulsion reversible addition-–fragmentation chain transfer (RAFT) polymerization has been used to generate N-(2-hydroxypropyl)methacrylamide (HPMA) based nanoparticles and reduction-sensitive nanogels with diameters of 100–200 nm. Inverse emulsion RAFT homopolymerization of HPMA afforded nanoparticles composed of linear PHPMA macromolecules with relatively narrow molecular weight distributions (Mw/Mn = 1.35–1.55). Copolymerization of HPMA with N,N′-bis(acryloyl)cystamine (BAC) at BAC to chain transfer agent ratios >1.5 resulted in crosslinked nanoparticles that could be isolated and redispersed in water. At smaller BAC to chain transfer agent ratios, non-crosslinked nanoparticles composed of (hyper)branched poly(N-(2-hydroxypropyl)methacrylamide-co-N,N′-bis(acryloyl)cystamine) copolymers were obtained. Addition of protein to the aqueous phase during the inverse emulsion copolymerization of HPMA and BAC allowed preparation of stimuli-responsive, protein-loaded nanogels, which were shown to release their payload under reductive conditions, e.g. upon exposure to tris(2-carboxyethyl)phosphine hydrochloride. These reduction-sensitive nanogels are of interest, e.g. as carriers for the delivery of proteins.

Fabrication of a TCEP-immobilised monolithic silica microchip for reduction of disulphide bonds in proteins

Protein identification by mass spectrometry forms the cornerstone of proteomics. Commonly, the identification of proteins is based on digestion of proteins into peptides using proteolytic enzymes. Cleavage of the disulphide bonds in proteins before enzymatic digestion and mass spectral analysis is important in order to facilitate the accessibility of the enzyme to cleave the proteins into peptides. As a result, the protein sequence coverage will be increased. In this study, a novel approach for immobilisation of the reducing reagent on the surface of a silica-based monolith in order to use it for reduction of disulphide bonds in proteins was successfully developed. This was carried out by silanisation of the surface of the silica-based monolith with (3-aminopropyl)triethoxysilane (APTES), followed by immobilisation of the reducing reagent, tris(2-carboxyethyl)phosphine hydrochloride (TCEP) on the surface of the amino-bonded silica monolith. The fabricated monolith was characterised using SEM analysis, EDX analysis, IR spectroscopy, and BET model. The performance of the fabricated glass microchip containing the TCEP-immobilised silica monolith to reduce the disulphide bonds in proteins was checked by injection of 100 μL of denatured insulin inside the microchip using a syringe pump at a flow rate of 10 μL min−1, followed by sealing both ends of the ETFE tubes with Blu-Tak. The microchip was kept in a humidified chamber for 30 min at 60 °C. After the reduction reaction, the reduced cysteine residues were alkylated with IAA at 60 °C for 30 min, followed by using a MALDI-TOF-MS instrument for qualitative confirmation. The results show that the fabricated microchip-based silica monolith has the ability to reduce disulphide bonds in insulin. In addition, the method is simple, reduces the risk of contamination, and results in lower amounts of the sample and reagents compared with the conventional techniques for proteomics sample preparation. A future study investigating reduction of the disulphide bonds in proteins from a real sample using this new microfluidic device would be very interesting.