Methods for studying the effects of phosphorylation patterns in proteins.

Protein phosphorylation is one of the most common and versatile regulatory mechanisms in cells. Most human proteins are phosphorylated at multiple sites, giving rise to large numbers of possible phosphorylation patterns. Each phosphorylation pattern can lead to a different functional or pathological outcome. Yet, linking defined phosphorylation patterns to specific biological functions remains a major experimental challenge. In this review we describe the main strategies to study phosphorylation patterns at the protein and domain levels and highlight how they complement each other. We first discuss cellular approaches, including phosphomimetics, kinase-based assays, and genetic code expansion, which allow working in a native environment but have their significant drawbacks. We then describe in vitro methods, such as enzymatic phosphorylation and semi-synthetic phosphoproteins generated by ligation, which afford mechanistic insights but result in low yields and are difficult to scale for producing libraries. We focus on synthetic phosphopeptide libraries as tools that offer precise control over the number and position of phosphosites and are uniquely suited for systematic mapping of phosphorylation patterns. This comes at a price of not working at the protein level, but rather at the domain level. Peptide libraries are often used for preliminary identification of key phosphorylations, later studied in detail at the protein level. We conclude that ideally more than one method should be used and that these methods should not be viewed as competing but rather as complementary. A combined use of several of these approaches provides a practical toolbox for dissecting how phosphorylation patterns regulate protein behavior.