Synthetic approaches to protein phosphorylation

•Classical site-directed mutagenesis is limited in studying protein phosphorylation.•We discuss synthetic, semisynthetic, and genetic methods to make phosphoproteins.•New techniques for cellular transduction of phosphoproteins are promising. Reversible protein phosphorylation is critically important in biology and medicine. Hundreds of thousands of sites of protein phosphorylation have been discovered but our understanding of the functions of the vast majority of these post-translational modifications is lacking. This review describes several chemical and biochemical methods that are under development and in current use to install phospho-amino acids and their mimics site-specifically into proteins. The relative merits of total chemical synthesis, semisynthesis, and nonsense suppression strategies for studying protein phosphorylation are discussed in terms of technical simplicity, scope, and versatility.