High throughput sequencing of cyclic peptide immobilized on a gel-type single bead.
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.
HiPep Laboratories, Nakatsukasa-cho 486-46, Kamigyo-ku, Kyoto, 602-8158, Japan. noki@hipep.jp.
Kiyoshi
K
Nokihara
Research Center for Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan.
Takeshi
T
Kasama
HiPep Laboratories, Nakatsukasa-cho 486-46, Kamigyo-ku, Kyoto, 602-8158, Japan.
Yuki
Y
Tominaga
HiPep Laboratories, Nakatsukasa-cho 486-46, Kamigyo-ku, Kyoto, 602-8158, Japan.
Atsushi
A
Kitagawa
HiPep Laboratories, Nakatsukasa-cho 486-46, Kamigyo-ku, Kyoto, 602-8158, Japan.
Akiyoshi
A
Hirata
HiPep Laboratories, Nakatsukasa-cho 486-46, Kamigyo-ku, Kyoto, 602-8158, Japan.
Takafumi
T
Ohyama
Imtakt Corp., Chudoji Minamicho 134, Shimogyo-ku, Kyoto, 600-8813, Japan.
Itaru
I
Yazawa
D019151:Peptide Library; D010456:Peptides, Cyclic / Q000737:chemistry / Q000235:genetics*; D020539:Sequence Analysis, Protein / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D019151:Peptide Library; D010456:Peptides, Cyclic
Cyclic octapeptide; Mass spectrometry; Nano-LC; One peptide on one bead; Partial acid hydrolysis; de novo sequencing