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Synthetic combinatorial peptide libraries and their application in decoding biological interactions

Sweeney, Michael Cameron

Abstract Details

2005, Doctor of Philosophy, Ohio State University, Ohio State Biochemistry Program.
The synthesis of peptides was revolutionized by the adoption of solid-phase synthetic techniques. Subsequent improvement, evolution, and refinement of this chemical technique has allowed research into areas of biology not previously accessible with such speed and breadth. Because of the efficiency and flexibility of the chemistry involved in peptide synthesis, libraries representing millions of unique natural, modified, or unnatural peptides can be constructed rapidly and in high enough purity as to obviate the need for purification. In this work, libraries were synthesized for screening against individual protein domains in an effort to both determine the preferred peptidyl binding partner types for each, as well as to establish an optimized, broadly applicable methodology for screening other domains. One of the problems encountered during the development of the screening methodology was the low success-rate of sequence determination for the peptides selected by each domain. Herein we report the successful modification of the peptide ladder mass spectrometry sequencing technique referred to as partial Edman degradation (PED). Success-rates were improved to greater than 90% for full-length sequencing determination of peptide up to 8-mers, even for more difficult phosphotyrosine (pY)-containing peptides. As a result of this improvement, three pY-binding Src Homology 2 (SH2) domains and two N-terminus binding Baculoviral Inhibitor-of-Apoptosis Repeat (BIR) domains were screened against their respective libraries and the preferred ligand types for each was determined. The advantage of sequencing by the PED method became especially clear in the case of the N-terminal SH2 (N-SH2) domain of Src Homology 2 Protien Tyrosine Phosphatase 2 (SHP-2) as previously unidentified sub-classes of binding consensus motifs were distinguishable due to the discreet nature of the sequencing technique. This work demonstrates the usefulness and potential generality of peptide library screening by this method.
Dehua Pei (Advisor)

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Citations

  • Sweeney, M. C. (2005). Synthetic combinatorial peptide libraries and their application in decoding biological interactions [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1118952919

    APA Style (7th edition)

  • Sweeney, Michael. Synthetic combinatorial peptide libraries and their application in decoding biological interactions. 2005. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1118952919.

    MLA Style (8th edition)

  • Sweeney, Michael. "Synthetic combinatorial peptide libraries and their application in decoding biological interactions." Doctoral dissertation, Ohio State University, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=osu1118952919

    Chicago Manual of Style (17th edition)