Abstract
Relatively larger scale peptide libraries immobilized on a gel-type solid support consisting of 24 natural and non-natural amino acids by the “split and combine method” have been constructed to find interacting molecules. The diversity was ca. 200 millions of hexapeptides with cysteinyl residues forming cyclotide. Selected beads after screening can be sequenced by the conventional Edman degradation, although several restrictions and the problems are known. To resolve these, a novel combinatorial method involving partial acid hydrolysis followed by liquid chromatography with on-line mass spectrometric analyses has been established. Problems were uncovered in an early stage of the process. Uncertain assignment caused by byproducts derived from a cystine residue and other materials could be resolved by optimal hydrolysis conditions and derivatization before mass spectrometric analysis. Discrimination between Leu and Ile could be performed using high energy collision induced dissociation in the high resolution MALDI-TOF-MS/MS. The present optimized protocol is useful for discovery of sequences of interacting molecules and a second library construction.
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Acknowledgments
The authors thank G. Ueta, N. Ono, N. Miyazato, K. Suzuki, M. Miyajima, and Y. Kodama, the Peptide-Vehicle Team, HiPep Laboratories for assistance and to Dr. V. Wray, Helmholtz Centre for Infection Research, Braunschweig, Germany, for linguistic advice. A part of the construction of the peptide-beads was supported by the “Program for Creating Okinawa Innovation of Okinawa Industry Promotion Public Corporation”, Grant-in-Aid for Cabinet Office, Government of Japan and Okinawa Prefecture (2008–2010).
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Influence of PEG derived signals in the MALDI-TOF–MS/MS, AHST-10 system, Ion chromatogram of hydrolysates of Gln and Asn, an example of successful deconvolution, cyclo[c-HFYHVV-c], Discrimination of isomers by MS/MS with HE-CID. This information is available on the publisher’s web site along with the published article. (DOCX 1631 kb)
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Nokihara, K., Kasama, T., Tominaga, Y. et al. High throughput sequencing of cyclic peptide immobilized on a gel-type single bead. Amino Acids 48, 2491–2499 (2016). https://doi.org/10.1007/s00726-016-2269-1
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DOI: https://doi.org/10.1007/s00726-016-2269-1