High throughput sequencing of cyclic peptide immobilized on a gel-type single bead

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.

A novel cyclic peptide library immobilized on gel‐type beads focusing on rapid construction and characterization for comprehensive drug discovery

“…The OPOB‐library was previously constructed in which peptides were cyclized with a disulfide bond between two D‐cysteines and internal loops consisting of six residues randomized with 19 proteinogenic amino acids other than cysteine and 5 non‐proteinogenic amino acids, the diversity of which was ca 200 million (Nokihara et al, 2016). Previously time‐consuming Edman degradation was applied to sequencing of peptides with a disulfide bridge (Nokihara et al, 1992), hence we have encountered a phenomenon that the amino‐terminus of the selected bead did not accept Edman‐degradation due to blockade of the amino group during screening.…”

“…Previously time‐consuming Edman degradation was applied to sequencing of peptides with a disulfide bridge (Nokihara et al, 1992), hence we have encountered a phenomenon that the amino‐terminus of the selected bead did not accept Edman‐degradation due to blockade of the amino group during screening. Then, an improved method has been established by the partial hydrolysis in combination with mass spectrometric analyses (Nokihara et al, 2016). However still difficulties were remaining, that is, the yield of partial hydrolysis was sequence dependent and termini of the selected single bead are unclear, thus often numerous candidates were required in the second library construction for confirmation of the sequence of the target.…”

“…The peptide library prepared on the basis of “one peptide immobilized on one bead (OPOB)” concept is one of the useful sources for the discovery of biologically active compounds (Lam, 1991). We have established a general production method of high quality and uniform composition of OPOB as well as a convenient method for analysis of selected one bead by using mass spectrometry instead of traditional Edman degradation (Nokihara et al, 2016). Although diversity of an OPOB library is relatively limited in number compared with that of a genetic library, flexibility in choice of chemical reactions and building blocks increases diversity in quality or per space, and a useful lead molecule can be obtained if the library is constructed in consideration of drug‐likeness and previous results in peptide screening.…”

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

“…In our previous report 19 natural and 5 non-proteinogenic amino acids were used as building blocks for the investigation of protein-protein interactions and discovery of drug candidates. The hexapeptide containing two D-cystines form a ring with 24 possible building units can generate 24 6 peptides, diversity: ca 200 million, reported by Nokihara, K. et al (2016). In that work restrictions of Edman-degradation had been overcome by partial hydrolysis followed by mass spectrometric analyses, although a further problem concerning the cleavability during hydrolyses as this is sequence dependent and, in some cases, resulted in unde ned termini.…”

“…The structure elucidation can also be performed by ESI-LC/MS, although, the amino acids having the same mass used as building blocks in OPOB-library construction such as Leu, Ile and Nle (113.0841 Da) as well as Val and Nva (99.0684 Da) can not be differentiated. For this reason we preferred to use a MALDI-TOF-MS/MS which allows the method of a high-energy collision-induced dissociation as previously reported byNokihara, K. et al (2016). Since the mass spectrum of the peptide liberated by NTCB gave relatively simple pro les consisting mainly of b-ions, it was easy to discriminate between them by examining the dions associated with the b-ions.…”