N‐t‐butyliodoacetamide and iodoacetanilide: two new cysteine alkylating reagents for relative quantitation of proteins

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Abstract
The synthesis and application of two new alkylating reagents, N‐tert‐butyl‐2‐iodoacetamide (N‐t‐butyliodoacetamide) and 2‐iodo‐N‐phenylacetamide (iodoacetanilide), are described. N‐t‐Butyliodoacetamide and iodoacetanilide were synthesised to purity in their d0‐light and in their respective d9‐ and d5‐heavy forms. The newly synthesised reagents are covalently bound to peptides containing cysteines via an alkylation reaction. The mass differences of 5 and 9 Da avoid possible problems of overlapping isotope distribution. For each alkylated cysteine a peptide mass increases, respectively, by a multiple of 113 and 133 Da for the d0‐light form of N‐t‐butyliodoacetamide and iodoacetanilide. These reagents can therefore replace common alkylating reagents in existing proteomics‐based applications. Alkylated peptides increase in mass in the same mass range as amino acids and remain suitable for tandem mass spectrometry (MS/MS) data acquisition and analysis. The compounds are simple to use and derivatisation is based on widely applied alkylating procedures. Preliminary results show that these reagents can be applied for both protein quantitation and identification by peptide mass finger printing and/or MS/MS techniques. Using these chemicals and the suggested workflow enables the quantitative analysis of the whole protein sample and realises access to peptides that may contain potential post‐translational modifications. Other approaches that incorporate a matrix‐assisted laser desorption/ionisation (MALDI) interface prior to MS can take advantage of these chemicals, such as the molecular scanner. Copyright © 2003 John Wiley & Sons, Ltd.