A high throughput sequence determination for one cyclic peptide immobilized on a single bead by MALDI-TOF-MS/MS focusing on discovery of interacting peptides and medicinal medium sized molecules

Abstract

One cyclic peptide immobilized on one gel-type bead has been attracting attention for use in the discovery of interacting peptides in protein recognition and/or medicinal medium sized molecules. Although high throughput characterization of peptides has been a bottleneck. Previously we have reported construction of high-quality bead-libraries of which diversity was ca 200 million, and encountered some difficulties such as restrictions of Edman-degradation and sequence dependent cleavability with ill defined termini on partial hydrolysis. This resulted in efficacy problems for construction of a subsequent second library for which the number of candidates were increased. The present work describes direct liberation from beads by a one-pot reaction using 2-nitro-5-thiocyanatobenzoic acid followed by off-line nanoscale liquid chromatography/matrix assisted laser desorption ionization time-of-flight mass spectrometry analyses to realize faster and routine sequence determination of the peptide on the beads. The present method is useful for investigation of protein-protein interactions as well as discovery of drug candidates.