Multiphosphorylated peptides: importance, synthetic strategies, and applications for studying biological mechanisms

Abstract

Advances in the synthesis of multiphosphorylated peptides and peptide libraries: tools for studying the effects of phosphorylation patterns on protein function and regulation.

Electrochemical biosensors based on peptide-kinase interactions at the kinase docking site

“…Kinases are sensed based on their catalytic active sites and those may not be very specific for relevant substrates. 38 For example, casein kinase (CK2) phosphorylates at least 160 proteins, making recognition through its catalytic site non-specific. ERK2 also phosphorylates many substrate proteins.…”

Automated Synthesis of Heavily Phosphorylated Peptides

“…First, coupling of mono-protected phosphory-lated amino acids results in low yields, since these amino acids are both bulky and charged and thus suffer from electrostatic repulsion and cause steric hindrance (especially true for pThr). [15] High temperatures, usually achieved by microwave irradiation, and large molar equivalents are used for increasing the coupling efficiency. Second, protected phosphorylated amino acids, especially pSer, are prone to β-elimination when treated with alkaline solution, thus, the basicity, temperature and duration of Fmoc deprotection cycles have to be carefully monitored.…”

“…The Fmoc SPPS approach to phosphopeptide synthesis differs from standard SPPS protocols in two aspects. First, coupling of mono‐protected phosphorylated amino acids results in low yields, since these amino acids are both bulky and charged and thus suffer from electrostatic repulsion and cause steric hindrance (especially true for pThr) [15] . High temperatures, usually achieved by microwave irradiation, and large molar equivalents are used for increasing the coupling efficiency.…”

PermaPhosSer: autonomous synthesis of functional, permanently phosphorylated proteins

“…1,2 In humans, over 75% of proteins are reversibly phosphorylated at one or more sites, with nearly 80% of these being phosphoserine (pSer) modifications, 3 on which we focus here. Two factors have greatly limited efforts to define the interactions and functions of specific phospho-proteins: (1) the challenge of making specific homogeneous, and quantitatively phosphorylated protein forms for in vitro study and (2) the challenge of the transient nature of phosphorylation in the cellular milieu due to phosphatase activity 4,5 . The first challenge has now been well-addressed through genetic code expansion (GCE) systems that enable efficient, translational incorporation of pSer at genetically programmed codons into recombinant proteins produced in E. coli.…”

“…Reversible protein phosphorylation events underlie the exquisitely orchestrated regulation of many signaling pathways, with imbalances in these systems being key signatures of disease. 1,2 In humans, over 75% of proteins are reversibly phosphorylated at one or more sites, with nearly 80% of these being phosphoserine (pSer) modifications, 3 on which we focus here. Two factors have greatly limited efforts to define the interactions and functions of specific phospho-proteins: (1) the challenge of making specific homogeneous, and quantitatively phosphorylated protein forms for in vitro study and (2) the challenge of the transient nature of phosphorylation in the cellular milieu due to phosphatase activity 4,5 .…”