mp300665u_si_001.pdf (3.16 MB)
Identification of Oxidation Sites and Covalent Cross-Links in Metal Catalyzed Oxidized Interferon Beta-1a: Potential Implications for Protein Aggregation and Immunogenicity
journal contribution
posted on 2013-06-03, 00:00 authored by Riccardo Torosantucci, Victor S. Sharov, Miranda van Beers, Vera Brinks, Christian Schöneich, Wim JiskootOxidation
via Cu2+/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 Cu2+/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.