title: A manual sequence method of peptides and phosphopeptides using 4-(1'-cyanoisoindolyl)phenylisothiocyanate.
abstract: A method for sequence analysis and identification of phosphoamino acids in peptides based on high performance liquid chromatography (HPLC) is described. The peptides were derivatized with an Edman type reagent, 4-(1'-cyanoisoindolyl)phenylisothiocyanate (CIPIC) and subsequently cleaved to generate stable and fluorescent 4-(1'-cyanoisoindolyl)phenylthiazolinone (CIP-TZ)-amino acids. Several experimental factors that affected derivatization on membranes were examined. Under the optimized conditions, the CIP-TZ derivatives of Try(p), Thr(p) and Ser(p) were obtained and separated from their parent amino acids with baseline resolution using an isocratic elution system. Up to the 4th residue of phosphorylated pentapeptides was successfully identified, whereas phosphoamino acid residues could not be detected by the conventional procedure using phenylisothiocyanate (PITC). The results demonstrated the potential of CIPIC as a derivatization reagent for peptide sequencing and the applicability of the method for the study and identification of phosphoamino acids in peptides.
authors.affiliation: Faculty of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, Bunkyo-Machi 1-14, Nagasaki 852-8521, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Shibata
authors.forename: Moses N
authors.initials: MN
authors.lastname: Wainaina
authors.forename: Takayuki
authors.initials: T
authors.lastname: Miyoshi
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Kabashima
authors.forename: Masaaki
authors.initials: M
authors.lastname: Kai
mesh_terms: D000097:Acetonitriles / Q000737:chemistry; D000596:Amino Acids / Q000032:analysis / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D007211:Indoles / Q000737:chemistry*; D017879:Isothiocyanates / Q000737:chemistry*; D000432:Methanol / Q000737:chemistry; D010455:Peptides / Q000032:analysis / Q000737:chemistry*; D010748:Phosphopeptides / Q000032:analysis / Q000737:chemistry*; D020539:Sequence Analysis, Protein / Q000379:methods*; D013696:Temperature; D013997:Time Factors
chemical_list: C560731:4-(1'-cyanoisoindolyl)phenylisothiocyanate; D000097:Acetonitriles; D000596:Amino Acids; D007211:Indoles; D017879:Isothiocyanates; D010455:Peptides; D010748:Phosphopeptides; C005441:phenylisothiocyanate; D000432:Methanol; C032159:acetonitrile