Cysteine with iodoacetamide

Denature the proteins, and open disulfide bond by DTT (dithiothreithol), and then alkylate the cysteines with iodoacetamide. 

The next step in sample preparation for MS analysis is reduction of intramolecular disulfide bonds by using reducing agents, such as dithiothreitol (DTT), dithioerythritol (DTE), tributylphosphine (TBP), tris(2-carboxyethyl)phosphine (TCEP), or P-mercaptoethanol (BME). The reduced disulfide bonds should be blocked by alkylatiou to prevent refolding. For this type of reaction, the typical procedures iuvolve carbamidomethylation of cysteines with iodoacetamide or pyridylethylation. 

Draw a simple mechanism for the reaction of a cysteine sulfhydryl group with iodoacetamide. 

Scheme 1. Reduction of homodimer peptide linked via a disulfide bridge with dithio-threitol (DTT) and alkylation of the resulting thiol groups of both cysteines with either acrylamide (upper reaction) or iodoacetamide (lower reaction).

Ramseier U, Chang JY. Modification of cysteine residues with N-methyl iodoacetamide. Analyt. Biochem. 1994 221 231-233. Bernhard WR. Differential modification of metallothionein with iodoacetamide. Methods Enzymol. 1991 205 426-433. 

It is well known that blocking of the free sulfhydryl, Cys-34, with iodoacetamide, cysteine, or glutathione prevents the occurrence of mixed disulfides in aged albumin, as well as the occurrence of the albumin dimer (Peters, 1985). In the structure, Cys-34 is not in close proximity to any disulfide, consequently, it is difficult to imagine its intramolecular participation without substantial conformational change of the albumin molecule. However, it could perhaps participate in the formation of mixed disulfides by catalyzing the reaction via intermolecu-lar interaction, because Cys-34 is known to have an unusually low p/CsH (Pedersen and Jacobsen, 1980) (see Section III,C,1). 

Human GH is a single chain polypeptide composed of 191 amino acid residues cross-linked by two disulfide bridges between cysteine residues at 58 and 165 and 182, and 189, respectively (Figure 3). Reduction of both disulfide bridges and alkylation with iodoacetamide yields the tetra-S-carbamidomethylated hGH which retains full biological activity in animals and humans, and its secondary and tertiary structure are indistinguishable from the structure of the intact honnone (40). 

Their positions in the primary structure have been determined as Cys-46 and Cys-174, respectively (Section II,B). Reactive cysteine residues have also been alkylated with iodoacetamide iHO), modified with spin-labeled reagents HI) and with a mercury substituted salicylate molecule 107). In the latter case the X-ray analysis has shown that two molecules are bound to Cys-132 and Cys-240, respectively. In the homologous rat enzyme other cysteine residues, now known to be ligands to the second zinc atom (Section II,C,3,b), have been differentially carboxymethylated 52). 

Table 12 Identification of G3PC ARATH (GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE, CYTOSOUC), Species Arabidopsis thaliana, SWISS-PROT (SP) entry P25858 using different PMF tools. The restricting parameters used were Arabidopsis thaliana for the species, a minimum number of 4 matched masses, a maximal tolerance for masses of 60 ppm, at most one missed cleavage of tryptic peptides allowed, and the modifications accepted were oxidised methionines and cysteines treated with iodoacetamide to form carboxyamidomethyl cysteines. The databases queried by each program are listed in the right column. In the score column, the first value is the score of the first candidate protein, followed by either the score of the second candidate protein (if the first one is the correct one)...

Reaction of cysteine with alkylating agents yields thioethers. lodoacetic acid, iodoacetamide, dimethylaminoazobenzene iodoacetamide, ethyl-enimine and 4-vinylpyridine are the most commonly used alkylating agents ... 

PKSs employ subtle variants of KS domains to fulfil specific functions within the biosynthetic cluster. Many of the loading modnles from c -AT PKSs utilise a KS domain with a glutamine residue in place of the active site cysteine, and are therefore termed KS domains [67]. It has been shown that alkylation of standard elongating KS domains with iodoacetamide structurally converts the cysteine residne into a moiety with similar chanical characteristics of a KS domain [68]. 

Ozawa, H. (1967) Bridging reagent for protein. II. The reaction of N,N -polymethylenebis(iodoacetamide) with cysteine and rabbit muscle aldolase./. Biochem. (Tokyo) 62, 531. 

Distilled 4-VP (see Note 1). Keep the solution under nitrogen atmosphere in a deep freezer. Iodoacetamide is also frequently used. An addition of 107 or 57 Da per alkylated cysteine residue is observed with vinylpyridine and iodoacetamide, respectively. Cysteine residues derivatized with V-melhyl iodoacetamide can be also analyzed by the Edman sequencing (no PTH standard commercially available). 

Fig. 3. Automatically acquired MALDI-TOF/TOF MS/MS spectra of a tryptic peptide with m/z 1136.67 (upper panel) and of the same peptide after derivatization with SPITC at m/z 1393.11 (lower panel) using CHCA matrix. Whereas the unmodified peptide displays a rather complex spectrum with intense b- and y-ions in the same mass range, the lower spectrum shows a complete and dominant y-series allowing an easy manual sequence interpretation from m/z 189.09 to m/z 1178.32. The peptide sequence LRGGLC YLGK was deduced from the y-series with guanidated Lys (homoarginine = Flar) and N-terminal derivatization with SPITC. The cysteine residue was reduced and alkylated with DTT and iodoacetamide (M = 160.03 g/mol).

Functionalisation of the complexes with reactive groups a number of metal complexes have been modified with (1) an NHS ester, isothiocyanate, and aldehyde, which can react readily with amines of lysine and the A-terminal of proteins and (2) iodoacetamide and maleimide that can react with sulfhydryls of the cysteine residue [3-5, 10]. These facile reactions lead to covalent attachment of luminescent complexes to the target proteins and amine-/sulfhydryl-modified oligonucleotides. 

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