Tagging of Phosphopeptides by Phosphate Elimination and Michael Addition: The Effect of Nearest Neighbors on the Reaction Kinetics

Kinetics of the base-catalyzed elimination of the phosphate group from short phosphoserinyl peptides and subsequent addition of β-mercaptoethanol to the dehydroalaninyl residue have been studied by following the reactions by RP HPLC in an 8:3:1 (v/v/v) mixture of water, DMSO and ethanol. The data obtained shows that neighboring side-chain-functionalized residues, including Asp, Tyr, Lys, His, Pro and Asn, influence the rate of elimination and addition steps by less than one order of magnitude. The amount of byproducts always remains below 10%, lending support to the applicability of the method as a research tool for functional phosphoproteomics.