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Screening Combinatorial Peptide Library for Optimal Enzyme Substrates and High Affinity Protein Ligands

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2003, Doctor of Philosophy, Ohio State University, Chemistry.
A method for the rapid identification of high-affinity ligands was used to study the specificity of the interaction between FHA2 domain of Rad53 and phosphotyrosyl peptide. A phosphotyrosyl (pY) peptide library containing completely randomized residues at positions -2 to +3 relative to the pY was synthesized on TentaGel resin, with a unique peptide sequence on each resin bead. The library was screened against the biotinylated FHA2 domains, and the beads that carry high-affinity ligands of the FHA2 domains were identified using an enzyme-linked assay. Peptide ladder sequencing of the hundreds of selected beads using MALDI-TOF revealed consensus sequences for FHA2 domains. A new method was developed to rapidly sequence support-bound peptides derived from combinatorial peptide library. Support-bound peptides isolated from one-bead-one-compound libraries are subjected to partial Edman degradation in the presence of an N-terminal blocking agent (~5% phenyl isocyanate). Repetition of the degradation reaction results in a series of sequence-specific truncation products (a peptide ladder). The sequence of the full-length peptide is determined by MALDI-TOF analysis of the peptide ladder. During screening of combinatorial libraries for optimal enzyme substrates, the challenge is to differentiate a reaction product(s) from a complex mixture of substrates. We have overcome this problem by partially labeling the substrates with a heavier isotope (heavy/normal isotope = 1:1), so that each member of the substrate library appears as a doublet in ESI-MS spectrum whereas the products appear as singlets in the spectrum, allowing for their unambiguous identification. The strategy has been successfully demonstrated by peptide deformylase screening. This method was further perfected in the screening of the pY library against the catalytic domain of SHP-1. Limited treatment of the library with a PTP removed phosphoryl group from the most preferred substrates to generate products as singlet peaks, which were readily identified in ESI-FTICR-MS spectrum and sequenced by tandem mass spectrometry. Several selected peptides were individually synthesized and assayed against SHP-1 and the kinetic data confirmed the screening results.
Dehua Pei (Advisor)

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Citations

  • Wang, P. (2003). Screening Combinatorial Peptide Library for Optimal Enzyme Substrates and High Affinity Protein Ligands [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1039797438

    APA Style (7th edition)

  • Wang, Peng. Screening Combinatorial Peptide Library for Optimal Enzyme Substrates and High Affinity Protein Ligands. 2003. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1039797438.

    MLA Style (8th edition)

  • Wang, Peng. "Screening Combinatorial Peptide Library for Optimal Enzyme Substrates and High Affinity Protein Ligands." Doctoral dissertation, Ohio State University, 2003. http://rave.ohiolink.edu/etdc/view?acc_num=osu1039797438

    Chicago Manual of Style (17th edition)