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The Modification of Cysteine
Published in Roger L. Lundblad, Chemical Reagents for Protein Modification, 2020
The α-halo acids decompose in water, with the rate being far more rapid at alkaline pH. In the case of iodoacetic acid, the products are iodide and glycolic acid. We recrystallize the commercially obtained reagents and store over P2O5. The compounds are readily soluble in water. In the case of the free acid, it is useful to dissolve the compound in base prior to addition to the reaction mixture. In the case of α-haloacetyl derivatives, the resultant S-carboxymethylcysteine is easily quantitated by amino acid analysis.
Reactivities of Amino Acids and Proteins with Iodine
Published in Erwin Regoeczi, Iodine-Labeled Plasma Proteins, 2019
Alkylation of the sulfur (i.e., the covalent attachment of a hydrocarbon sequence to it) is another way of elucidating methionine’s role in proteins. As explained further below, this technique is also important for the quantitation of methionine sulfoxide. Methyl iodide (CH3I), iodoacetic acid (CH2ICOOH), and iodoacetamide (CH2ICONH2) are some of the commonly used alkylating agents. The “carboxymethylation” reaction with iodoacetic acid, yielding the carboxymethylsulfonium salt of methionione, takes place as follows:
Biochemical Characterization of an Immunoglobulin Lambda I Light Chain Amyloid Protein Isolated From Formalin-Fixed Paraffin-Embedded Tissue Sections
Published in Gilles Grateau, Robert A. Kyle, Martha Skinner, Amyloid and Amyloidosis, 2004
M. Yazaki, J.J. Liepnieks, M.D. Benson
Ten 4μm thick approximately 2cm2 sections of formalin-fixed cardiac tissue on glass slides were deparaffinized in xylene, rehydrated sequentially in 100%, 95%, 80% ethanol and distilled water, and air dried. Tissue was scraped from the slides and 1ml of 6M guanidine hydrochloride, 0.5M Tris pH8.2 containing 10mg dithiothreitol/ml and 1mg EDTA/ml was added. After incubation at 37°C for five days, the sample was alkylated with 24.1mg iodoacetic acid and filtered through a 0.2μm filter. 100μl was applied to a ProSorb PVDF cartridge for sequence analysis. The remainder was exhaustively dialyzed in Spectra/Por 6 dialysis tubing against water and lyophilized. The dried sample was suspended in 1ml of 10% acetic acid. Five percent was analyzed by SDS-PAGE and silver staining. Sixty percent was digested with trypsin, fractionated by reverse phase HPLC on a synchropak RP-8 column (100 × 4.6mm), and eluted peaks analyzed by Edman degradation. Twenty-five percent was digested with pyroglutamate aminopeptidase, applied to a ProSorb cartridge, and analyzed by Edman degradation.
Characterization and prediction of positional 4-hydroxyproline and sulfotyrosine, two post-translational modifications that can occur at substantial levels in CHO cells-expressed biotherapeutics
Published in mAbs, 2019
Oksana Tyshchuk, Christoph Gstöttner, Dennis Funk, Simone Nicolardi, Stefan Frost, Stefan Klostermann, Tim Becker, Elena Jolkver, Felix Schumacher, Claudia Ferrara Koller, Hans Rainer Völger, Manfred Wuhrer, Patrick Bulau, Michael Mølhøj
The antibodies were denatured and reduced in 0.3 M Tris-HCl pH 8, 6 M guanidine-HCl and 20 mM dithiothreitol (DTT) at 37°C for 1 h, and alkylated by adding 40 mM iodoacetic acid (C13: 99%) (Sigma-Aldrich) at room temperature in the dark for 15 min. Excess iodoacetic acid was inactivated by adding DTT to 40 mM. The alkylated fusion protein was buffer exchanged using NAP5 gel filtration columns, and a proteolytic digestion with trypsin was performed in 50 mM Tris-HCl, pH 7.5 at 37°C for 16 h. The reaction was stopped by adding formic acid to 0.4% (v/v). Thermolysin digests were performed as described by Tyshchuk et al.11 Digested samples were stored at −80°C and analyzed by UPLC-MS/MS using a nanoAcquity UPLC (Waters) and an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). About 2.5 µg digested fusion protein was injected in 5 µL. Chromatographic separation was performed by reversed-phase on a BEH300 C18 column (1 × 150 mm, 1.7 µm) or a CSH130 C18 column, 1 × 150 mm, 1.7 µm (Waters) using mobile phase A and B containing 0.1% (v/v) formic acid in UPLC grade water and acetonitrile, respectively, 60 µL/min flow rate, 50°C column temperature, and the following gradient: 1% mobile phase B [0–3 min], 1% to 40% mobile phase B [3–93 min], 40% to 99% mobile phase B [93–94 min], 99% mobile phase B [94–96 min], 99% to 1% mobile phase B [96–97 min], and 1% mobile phase B [97–105 min]. Two injections of mobile phase A were performed between sample injections using a similar 50 min gradient up to 99% mobile phase B to prevent carry-over between samples. Synthetic peptides were spiked into to digests at different levels.
Defect-induced electronic states amplify the cellular toxicity of ZnO nanoparticles
Published in Nanotoxicology, 2020
Indushekhar Persaud, Achyut J. Raghavendra, Archini Paruthi, Nasser B. Alsaleh, Valerie C. Minarchick, James R. Roede, Ramakrishna Podila, Jared M. Brown
RAECs were exposed to 20 µg/mL of pristine, annealed, oxidized, or reduced ZnO NPs for 6, 12, and 24 h. The cell fraction was collected and prepared for high-pressure liquid chromatography (HPLC) measurements of the thiol/disulfide couples concentration to determine the cellular redox potential. The procedures were based on that of Jones et al. (Jones and Liang 2009). Briefly, the cell pellet was resuspended in 0.5 mL of 5% perchloric acid (PCA)/0.2 M boric acid/10 μM ϒ-Glu-Cys and then sonicated. Samples were centrifuged at 13,000×g for 2 min and 300 µl of the supernatant was placed in a new tube. The tubes with the pellets were aspirated and stored for protein quantification by bicinchoninic acid (BCA) protein assay. Derivation of samples to extract the thiol/disulfide couples was performed on the collected supernatant. For each sample, 60 μL of 9.3 mg/mL iodoacetic acid was added, and the pH was adjusted to 8.8–9.2 with 1 M KOH. The samples were incubated for 20 min at room temperature after which 300 μl of 20 mg/mL of dansyl chloride was added to each sample. The samples were placed in the dark overnight. For each sample, 500 μL of chloroform was added, vortexed, and centrifuged at 13,000×g for 2 min to separate the aqueous and organic layers. HPLC was performed on an amino column with a Supelcosil™ LC-NH2 25 cm × 4.6 mm, 5 μm column (Supleco, Bellefonte, PA) with an Agilent 1200 HPLC equipped with a fluorimeter. The concentration of glutathione, its disulfide form GSSG, cysteine (Cys), and cystine (CySS) was determined from the scans. The Nernst equation was used to calculate the redox potential of the thiol/disulfide ratio:
N-acetyl cysteine treatment mitigates biomarkers of oxidative stress in different tissues of bile duct ligated rats
Published in Stress, 2021
Mohammad Mehdi Ommati, Ali Amjadinia, Khadijeh Mousavi, Negar Azarpira, Akram Jamshidzadeh, Reza Heidari
The reduced (GSH) and oxidized (GSSG) glutathione content in different tissues of cirrhotic animals was assessed by the HPLC analysis of deproteinized samples (TCA 50% w: v) after derivatization with iodoacetic acid and fluoro-2,4-dinitrobenzene (Meeks & Harrison, 1991). The technique is based on the formation of S-carboxymethyl derivatives of free thiol groups with iodoacetic acid, followed by the change of free amino groups to 2,4-dinitrophenyl derivatives by reaction with fluoro-2,4-dinitrobenzene (FDNB) (Meeks & Harrison, 1991). The HPLC system consisted of a 25 cm NH2 column (Bischoff chromatography, Leonberg, Germany), the flow rate 1 mL/min, and a UV detector (λ = 252 nm) (Meeks & Harrison, 1991). The mobile phases involved buffer A (Water: Methanol; 1:4 v: v) and buffer B (Acetate buffer: Buffer A; 1:4 v: v). A gradient method with a steady increase of buffer B to 95% in 20 min was used (Meeks & Harrison, 1991). GSSG and GSH were used as external standards. Tissue samples (200 mg) were homogenized in Tris-HCl buffer (250 mM; pH = 7.4; 4 °C), and 500 µL of TCA (50% w: v) was added. Samples were incubated for 15 min on ice. Afterward, samples were mixed well and centrifuged (15,000 g, 15 min, 4 °C). Then, the NaOH: NaHCO3 (2 M: 2 M) solution was added (≈300 µL) to 1 mL of the supernatant until the gas production was subsided. Afterward, 100 µL of iodoacetic acid (1.5% w: v in water) was added, and samples were incubated in the dark (1 h, 4 °C). Then, 500 µL of 2, 4-dinitrofluorobenzene (DNFB; 1.5% w: v in absolute ethanol) was added and incubated in the dark (25 °C, at least for 24 h). Finally, samples were centrifuged (15,000 g, 15 min), and 25 µL of the supernatant was injected into the described HPLC system (Meeks & Harrison, 1991; Truong et al., 2006).