Functional evaluation of an electrophilic focused library to identify a covalent inhibitor against intrinsically disordered circadian clock transcription factors
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Iodoacetamide
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Diphtheria toxin was modified at one or both of its cysteine disulfide bridges by iodoacetamide, methylmethanethiosulfonate, and atomic mercury. The products of these reactions were characterized and tested for toxicity in vitro and in vivo. All were toxic in vitro, but had lost almost all cytotoxic activity toward HeLa cells. It was possible to show from in vivo protection experiments that modification of the cysteine disulfide in the B-chain interfered with cell surface binding, while modification of the cysteine disulfide linking the A and B domains inhibits a step subsequent to binding in the intoxication process. The latter finding supports a functional role for this interdomain cysteine disulfide in the membrane transport process.
Iodoacetamide
Diphtheria Toxin
HeLa
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If cysteine or cystine are identified in a protein, they require modification if they are to be quantified. Thiol groups may be blocked by a variety of reagents, including iodoacetic acid and iodoacetamide. Iodoacetate produces the S-carboxymethyl derivative of cysteine, effectively introducing new negative charges into the protein. Where such a charge difference is undesirable, iodoacetamide may be used to produce S-carboxyamidomethylcysteine (on acid hydrolysis, as for amino acid analysis, this yields S-carboxymethylcysteine). The charge difference between these two derivatives has been utilized in a method to quantify the number of cysteine residues in a protein ([1], see Chapter 83).
Iodoacetamide
Iodoacetic acid
Thiol
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Reactive oxygen species \(ROS) are increasingly recognised as important signalling molecules that act through the oxidation of protein cysteine residues.Comprehensive identi cation of redox-regulated proteins and pathways is crucial to understand ROS-mediated events.Identifying cysteine oxidation on a whole-proteome scale remains a technical challenge due to the low abundance of oxidised thiols.Redox proteomics techniques therefore use multistep enrichment protocols, but these have inherent limitations and inform only on the enriched proteome.We developed stable isotope cysteine labelling with iodoacetamide \(SICyLIA), a simple, unbiased, and robust mass spectrometry-based work ow for thiol oxidation analysis.SICyLIA does not require enrichment steps and achieves unbiased proteome-wide sensitivity.We applied SICyLIA to diverse cellular models and primary tissues and generated the most indepth thiol oxidation pro les to date.Our results demonstrate that acute and chronic oxidative stress causes oxidation of distinct metabolic proteins, indicating that cysteine oxidation plays a key role in the metabolic adaptation to redox stress.Analysis of mouse kidneys showed oxidation of proteins circulating in bio uids, through which cellular redox stress can affect whole-body physiology.Obtaining accurate peptide oxidation pro les from complex organs using SICyLIA holds promise for future analysis of patient-derived samples to study human pathologies.Reagents • Sodium dodecyl sulfate \(SDS) • Iodoacetamide light \( 12 C 2 H 4 INO, Sigma-Aldrich \(Merck)) • Iodoacetamide heavy \( 13 C 2 D 2 H 2 INO, Sigma-Aldrich \(Merck)) • Phosphate buffered saline \(PBS) • Bicinchoninic acid \(BCA) assay kit \(Thermo Scienti c) • Ammonium bicarbonate \(Ambic) • Dithiothreitol \(DTT) • N-ethylmaleimide \(NEM) • Trichloroacetic acid \(TCA) • Urea • Endoproteinase Lys-C \(mass spectrometry grade, Alpha laboratories) • Trypsin \(mass spectrometry grade, Promega) • Tri uoroacetic acid \(TFA) • Acetic acid • Acetonitrile \(ACN) • Formic acid • LC-MS grade water **Reagent
Iodoacetamide
Proteome
Isotopic labeling
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INTRODUCTIONIntact interchain and/or intrachain disulfide linkage in a protein can present problems during proteolytic or chemical fragmentation procedures. Disulfide bonds are commonly cleaved by reducing cystine to yield cysteine residues. However, cysteine residues are highly reactive, which can complicate sequence work. In this protocol, the intact protein (>1 mg) is reduced and then S-alkylated with iodoacetic acid (or iodoacetamide). The resulting cysteine derivative S-carboxymethylcysteine (or S-carboximadomethylcysteine) is easily detectable during chemical sequencing.
Iodoacetamide
Iodoacetic acid
Disulfide Linkage
Residue (chemistry)
Derivative (finance)
Chemical modification
Cleavage (geology)
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Iodoacetamide
Maleimide
Cysteine Metabolism
Affinity labeling
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If cysteine or cystine is identified in a protein it requires modification in order to be quantified. Thiol groups may be blocked by a variety of reagents including iodoacetic acid and iodoacetamide. Iodoacetate produces the S-carboxymethyl derivative of cysteine, effectively introducing new negative charges into the protein. Where such a charge difference is undesirable, iodoacetamide may be used to derivatize cysteine to S-carboxyamidomethylcysteine (on acid hydrolysis, as for amino acid analysis, this yields S-carboxymethylcysteine). The charge difference between these two derivatives has been utilized in a method to quantify the number of cysteine residues in a protein ([1], see Chapter 89 ).
Iodoacetamide
Iodoacetic acid
Thiol
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Iodoacetamide
Acceptor
Reactivity
Thiol
Stoichiometry
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Iodoacetamide
Thiol
Isotopic labeling
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Iodoacetamide
Residue (chemistry)
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Iodoacetamide
Reactivity
Proteome
Posttranslational modification
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