title: LC-UV/MS characterization and DOE optimization of the iodinated peptide obestatin.
abstract: The aim of this study was to investigate the direct iodination of the recently discovered peptide obestatin by LC-UV/ESI ion trap MS analysis. The influence of selected reaction parameters on obestatin iodination by chloramine-T, Iodo-Gen((R)) and lactoperoxidase was investigated by experimental design. Different responses, i.e. species percentage and yield, peptide recovery and iodination yield were evaluated. Mono-up till tetra-iodinated species are possible depending on the reaction conditions with electrophilic substitutions occurring at Tyr(16) and His(19) as confirmed by LC/MS/MS. The two possible mono-iodinated obestatin isomers, i.e. [I(1)-Tyr(16)]-obestatin and [I(1)-His(19)]-obestatin, could be chromatographically separated. Several significant main and quadratic effects, and interaction of factors were observed from which optimum conditions for a specific response could be derived. The highest impact on the response surface diagrams was overall attributed to the amount of iodide added. Synthesis methods were compared relative to the different response factors: lactoperoxidase was found to be the overall most robust iodination technique, and also gave the highest mono-iodinated species yield. The applicability of our research was demonstrated by non-carrier-added (125)I-radioiodination. To our knowledge, this is the first time an LC separation of mono-iodinated peptide isomers has been reported.
authors.affiliation: Drug Quality and Registration (DruQuaR) group, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium.
authors.forename: Valentijn
authors.initials: V
authors.lastname: Vergote
authors.forename: Bram
authors.initials: B
authors.lastname: Baert
authors.forename: Eva
authors.initials: E
authors.lastname: Vandermeulen
authors.forename: Kathelijne
authors.initials: K
authors.lastname: Peremans
authors.forename: Henri
authors.initials: H
authors.lastname: van Bree
authors.forename: Guido
authors.initials: G
authors.lastname: Slegers
authors.forename: Christian
authors.initials: C
authors.lastname: Burvenich
authors.forename: Bart
authors.initials: B
authors.lastname: De Spiegeleer
mesh_terms: D000818:Animals; D002700:Chloramines / Q000737:chemistry / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D054439:Ghrelin / Q000737:chemistry* / Q000378:metabolism; D006632:Histamine / Q000737:chemistry / Q000378:metabolism; D007457:Iodine Radioisotopes / Q000737:chemistry; D007536:Isomerism; D007553:Isotope Labeling / Q000379:methods; D007784:Lactoperoxidase / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D015203:Reproducibility of Results; D012107:Research Design; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013678:Technology, Pharmaceutical / Q000379:methods; D014105:Tosyl Compounds / Q000737:chemistry / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
chemical_list: D002700:Chloramines; D054439:Ghrelin; D007457:Iodine Radioisotopes; D010455:Peptides; D014105:Tosyl Compounds; C016300:chloramine-T; D014443:Tyrosine; D006632:Histamine; D007784:Lactoperoxidase