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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010A Method for Screening Peptides Bound to EGFR by UsingMultiple Fluorescent Amino Acids as Fluorescent TagsMizuki Kitamatsu, Takahiro Yamamoto, and Masahiko SisidoDepartment of Medical and Bioengineering, Graduate School of Natural Science andTechnology, Okayama University, 3-1-1 Tsushimanaka, Okayama, 700-0082, JapanIntroductionA peptide library is generally used for screening peptides bound to a target protein. Thephage display method [1] and the one-bead–one-compound method [2,3] are widely usedmethods for screening with a peptide library. However, the methods have a drawback in thecase of screening for short peptides because of the need to fix the short peptides on largecarriers like phages and beads. The carriers often interact with targets nonspecifically. Toovercome this drawback, we have developed a new screening method without large carriers[4]. In this method, a peptide library is labeled with multiple fluorescent amino acids. Thepeptides binding to targets are detected quantitatively by specific fluorescence from thefluorescent amino acids labeling on peptides. In this study, we screened 8-mer peptidesbound to epidermal growth factor receptor (EGFR) by this method. EGFR is a receptortyrosine kinase over-expressed on surface of many human cancers. Therefore, an EGFRbindingpeptide can be regarded as a significant target of tumor and it is an attractivemedical tool.Results and DiscussionTwo hundred twenty-five peptides were prepared by solid-phase peptide synthesis. Thesequence is shown in Figure 1. Y 1 and Y 2 indicate 15 D-amino acids other than Cys, Gly,Leu, Glu and Gln. X indicates equimolar mixture of these amino acids. Fl indicates afluorescent amino acid. Fifteen fluorescent amino acids were used in this study (Figure 1).AcHNOCOOHN2 HFlOHNOCOOHN2 HO6 OHNO XHO Y 1 HO XHO XNNNNHNNNN2XHO XHO YHOH2 X OHNOHNOHNOHNOHNOHNOHNOOOHNOOOHHmc(327/454)OONHONCm3(450/488)OOHNOOMoc(351/404)OOONHOCoc(353/428)OOOHNOOHHoc(410/510)ONH +NHONH@52(455/519)HNAcd(385/419)HNOHNOHNOHNOHNOHNOHNOHNOHNOOOCmr(328/392)OHNONOOMac(385/476)OONNHAca(388/422)NHNOODec(429/473)OOHNOONS39(385/552)ON +OHNCF 3NOHOHNOOCOOHFamSO 3 H(501/525)A43(410/450)ONHNOCOOHO N +Tmr(552/577)Fig. 1. Chemical structure of the peptide modified with a fluorescent amino acid andchemical structures of fluorescent amino acids. Values in a parenthesis indicate maximumexcitation/emission wavelengths obtained from the 2D-FL spectrum of each fluorescentamino acid in 70 mM HEPES buffer (pH 7.4)/methanol (1/1 (v/v)) at room temperature.296