A novel sequence analysis for one cyclic peptide immobilized on a single bead by MALDI-TOF-MS/MS focusing on discovery of medicinal medium sized molecules based on protein-protein interactions

Abstract One cyclic peptide immobilized on one single gel-type bead have been attracting attention as resources for the discovery of 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 unclear termini on partial hydrolysis, which resulted efficacy problems for construction of a later second library for which numbers 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 sequencing. The present method is useful for investigation of protein-protein interactions as well as discovery of drug candidates.