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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:
Affinity Modification: Physical Chemistry
Published in Dmitri G. Knorre, Valentin V. Vlassov, Affinity Modification of Biopolymers, 1989
Dmitri G. Knorre, Valentin V. Vlassov
Iodoacetamide was chosen as a reference compound. At identical 0.415 mM concentrations of CXL and ICH2CONH2, the ratio of reaction rates is 9.1.382 Since the Kx for CXL was found to be 0.9 mM−1 at the same conditions, the maximal efficiency value achieved at low concentrations is
Gas Phase Sequence Analysis of Proteins/Peptides
Published in Ajit S. Bhown, Protein/Peptide Sequence Analysis: Current Methodologies, 1988
Alkylation of the protein with iodoacetamide gives 5-carboxamidomethylcysteine (CAM-Cys). PTH-CAMCys elutes just before DMPTU. About 50% of PTH-CAMCys is degraded by deamination to yield PTH-CMCys in the conversion flask under typical conversion conditions. Additional PTH-CMCys can result from deamination of CM-cysteinyl residues during purification or handling of the protein prior to sequencing.
TMT-Based proteomics analysis of LPS-induced acute lung injury
Published in Experimental Lung Research, 2021
Shengsong Chen, Yi Zhang, Qingyuan Zhan
Samples containing 200 µg of protein were added to 30 μl SDT buffer (4% SDS, 100 mM DTT, 150 mM Tris-HCl, pH 8.0, Sangon Biotech, China). The detergent, DTT and other low-molecular-weight components were removed by repeated ultrafiltration (Sartorius, 30 kD) using UA buffer (8 M urea, 150 mM Tris-HCl, pH 8.5, Sangon Biotech, China). Then, to block reduced cysteine residues, 100 μl of iodoacetamide (100 mM IAA in UA buffer, Sangon Biotech, China) was added, and the samples were incubated for 30 min in the dark. The filters were washed with 100 µl of UA buffer three times and then 100 µl of 0.1 M TEAB buffer (Sigma, USA) twice. Finally, the protein suspensions were digested with 4 μg of trypsin (Promega, USA) in 40 μl of 0.1 M TEAB buffer (Thermo Fisher Scientific, USA) overnight at 37 °C, and the resulting peptides were collected as the filtrate. The peptide content in a 0.1% (g/l) solution was estimated by UV light spectral density at 280 nm using an extinction coefficient of 1.1 calculated on the basis of the frequency of tryptophan and tyrosine in vertebrate proteins.
Human ESC-sEVs alleviate age-related bone loss by rejuvenating senescent bone marrow-derived mesenchymal stem cells
Published in Journal of Extracellular Vesicles, 2020
Liangzhi Gong, Bi Chen, Juntao Zhang, Yongjin Sun, Ji Yuan, Xin Niu, Guowen Hu, Yu Chen, Zongping Xie, Zhifeng Deng, Qing Li, Yang Wang
100 μg of proteins for each sample were incorporated into 30 μl SDT buffer (4% SDS, 100 mM DTT, 150 mM Tris-HCl pH 8.0). The detergent, DTT and other low-molecular-weight components were removed using UA buffer (8 M Urea, 150 mM Tris-HCl pH 8.0) by repeated ultrafiltration (Microcon units, 10 kD). Then, 100 μl iodoacetamide (100 mM IAA in UA buffer) was added to block reduced cysteine residues and the samples were incubated for 30 min in darkness. The filters were washed with 100 μl UA buffer three times and then 100 μl 25 mM NH4HCO3 buffer twice. Finally, the protein suspensions were digested with 4 μg trypsin (Promega) in 40 μl 25 mM NH4HCO3 buffer overnight at 37°C, and the resulting peptides were collected as a filtrate. The peptides of each sample were desalted on C18 Cartridges (EmporeTM SPE Cartridges C18 (standard density), bed I.D. 7 mm, volume 3 ml, Sigma), concentrated by vacuum centrifugation and reconstituted in 40 μl of 0.1% (v/v) formic acid. The peptide content was estimated by UV light spectral density at 280 nm using an extinction coefficient of 1.1 of 0.1% (g/l) solution.
In situ antibody phage display yields optimal inhibitors of integrin α11/β1
Published in mAbs, 2020
Eugenio Gallo, Abdellali Kelil, Peter E. Bayliss, Ajitha Jeganathan, Olga Egorova, Lynda Ploder, Jarrett J. Adams, Patricia Giblin, Sachdev S. Sidhu
For immunoprecipitation (IP) of cell-surface protein, 107 lifted and dissociated CAF094 cells were incubated with 500 nM Fab protein in DPBS containing 10 mM CaCl2 and 5 mM MgCl2, for 1 hour at 4°C. Cells were washed with PBS and lysed using IP lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1.0% IGEPAL CA-630, 0.25% Na-deoxycholate, 1 mM EDTA, and protease inhibitor cocktail (Roche)) for 15 minutes at 4°C, and centrifuged at 12000 x g for 5 minutes at 4°C. The supernatant was incubated with 30 μl of sepharose protein-A beads (GE Healthcare) for 1 hour at 4°C. The beads were washed three times with lysis buffer, once with PBS, and resuspended in 22 μl 10 mM glycine, pH 1.5. After 5 minutes, the supernatant was collected and neutralized with 2.2 μl 1 M Tris, pH 8.8. DTT was added to a final concentration of 10 mM. The sample was incubated at 40°C for 1 hour and cooled to room temperature. Iodoacetamide was added to a final concentration of 20 mM, and the sample was incubated at room temperature in the dark for 30 minutes. Trypsin (1 µg, Promega) was added and the sample was incubated overnight at 37°C. Peptides were purified using C18 tips and analyzed on a linear ion trap-Orbitrap hybrid analyzer (LTQ-Orbitrap, ThermoFisher) outfitted with a nanospray source and EASY-nLC split-free nano-LC system (ThermoFisher).