Alkylation of Galectin-1 with Iodoacetamide and Mass Spectrometric Mapping of the Sites of Incorporation

Abstract

Galectins can display unique sensitivity to oxidative changes that result in significant conformational alterations that prevent carbohydrate recognition. While a variety of approaches can be utilized to prevent galectin oxidation, several of these require inclusion of reducing agents that not only prevent galectins from undergoing oxidative inactivation but can also interfere with normal redox potentials required for fundamental cellular processes. To overcome the limitations associated with placing cells in an artificial reducing environment, cysteine residues on galectins can be directly alkylated with iodoacetamide to form a stable thioether adduct that is resistant to further modification. Iodoacetamide alkylated galectin remains stable over prolonged periods of time and retains the carbohydrate binding and biological activities of the protein. As a result, this approach allows examination of the biological roles of a stabilized form of galectin-1 without introducing the confounding variables that can occur when typical soluble reducing agents are employed.

Keywords: Alkylation; Galectin-1; Mass spectrometry; Oxidation; Reducing agents.