Reduction and S-Carboxymethylation of Proteins: Large-Scale Method
This protocol was adapted from “Peptide Mapping and Sequence Analysis of Gel-Resolved Protein,” Chapter 7, in Proteins and Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.INTRODUCTION
Intact 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.
