title: Identification of oxidation sites and covalent cross-links in metal catalyzed oxidized interferon Beta-1a: potential implications for protein aggregation and immunogenicity.
abstract: Oxidation via Cu(2+)/ascorbate of recombinant human interferon beta-1a (IFNβ1a) leads to highly immunogenic aggregates, however it is unknown which amino acids are modified and how covalent aggregates are formed. In the present work we mapped oxidized and cross-linked amino acid residues in aggregated IFNβ1a, formed via Cu(2+)/ascorbate catalyzed oxidation. Size exclusion chromatography (SEC) was used to confirm extensive aggregation of oxidized IFNβ1a. Circular dichroism and intrinsic fluorescence spectroscopy indicated substantial loss of secondary and tertiary structure, respectively. Derivatization with 4-(aminomethyl)benzenesulfonic acid was used to demonstrate, by fluorescence in combination with SEC, the presence of tyrosine (Tyr) oxidation products. High performance liquid chromatography coupled to electrospray ionization mass spectrometry of reduced, alkylated, and digested protein was employed to localize chemical degradation products. Oxidation products of methionine, histidine, phenylalanine (Phe), tryptophan, and Tyr residues were identified throughout the primary sequence. Covalent cross-links via 1,4- or 1,6-type addition between primary amines and DOCH (2-amino-3-(3,4-dioxocyclohexa-1,5-dien-1-yl)propanoic acid, an oxidation product of Phe and Tyr) were detected. There was no evidence of disulfide bridge, Schiff base, or dityrosine formation. The chemical cross-links identified in this work are most likely responsible for the formation of covalent aggregates of IFNβ1a induced by oxidation, which have previously been shown to be highly immunogenic.
authors.affiliation: Division of Drug Delivery Technology, Leiden/Amsterdam Center for Drug Research, Leiden University, 2300 RA Leiden, The Netherlands.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Torosantucci
authors.forename: Victor S
authors.initials: VS
authors.lastname: Sharov
authors.forename: Miranda
authors.initials: M
authors.lastname: van Beers
authors.forename: Vera
authors.initials: V
authors.lastname: Brinks
authors.forename: Christian
authors.initials: C
authors.lastname: Schöneich
authors.forename: Wim
authors.initials: W
authors.lastname: Jiskoot
mesh_terms: D001205:Ascorbic Acid / Q000737:chemistry; D001555:Benzene Derivatives / Q000737:chemistry; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry; D006801:Humans; D016899:Interferon-beta / Q000737:chemistry* / Q000276:immunology; D008744:Methylamines / Q000737:chemistry; D015394:Molecular Structure; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: C581528:4-(aminomethyl)benzene sulfonate; D001555:Benzene Derivatives; D008744:Methylamines; D016899:Interferon-beta; D003300:Copper; D001205:Ascorbic Acid