Substrate Profiling of Protein Tyrosine Phosphatase PTP1B by Screening a Combinatorial Peptide Library

Abstract

Protein tyrosine phosphatases (PTPs) are a large family of enzymes that catalyze the hydrolytic removal of the phosphoryl group from phosphotyrosyl (pY) proteins. To date, the in vivo substrates and physiological functions of PTPs remain poorly defined. In this work, we have developed a novel combinatorial library method to systematically determine the substrate specificity of PTPs. A one-bead-one-compound peptide library containing five randomized residues, Fmoc-XXXXXpYAA (where X is norleucine or 17 proteinogenic amino acids excluding Tyr, Cys, and Met), was chemically synthesized on 90-μm TentaGel resin by the split-and-pool method. Limited treatment of the library with a PTP removed the phosphoryl group from beads that carry the most preferred substrates. The exposed tyrosine side chain was selectively oxidized into an orthoquinone by the treatment with tyrosinase in the presence of atmospheric oxygen. The orthoquinone was then selectively derivatized with biotin-hydrazide, followed by on-bead colorim...