Abstract
PurposeTo elucidate the chemical modifications in covalent aggregates of recombinant human insulin induced by metal catalyzed oxidation (MCO).MethodsInsulin was exposed for 3 h at room temperature to the oxidative system copper(II)/ascorbate. Chemical derivatization with 4-(aminomethyl) benzenesulfonic acid (ABS) was performed to detect 3,4-dihydroxyphenylalanine (DOPA) formation. Electrospray ionization-mass spectrometry (ESI-MS) was employed to localize the amino acids targeted by oxidation and the cross-links involved in insulin aggregation. Oxidation at different pH and temperature was monitored with size exclusion chromatography (SEC) and ESI-MS analysis to further investigate the chemical mechanism(s), to estimate the aggregates content and to quantify DOPA in aggregated insulin.ResultsThe results implicate the formation of DOPA and 2-amino-3-(3,4-dioxocyclohexa-1,5-dien-1-yl) propanoic acid (DOCH), followed by Michael addition, as responsible for new cross-links resulting in covalent aggregation of insulin during MCO. Michael addition products were detected between DOCH at positions B16, B26, A14, and A19, and free amino groups of the N-terminal amino acids Phe B1 and Gly A1, and side chains of Lys B29, His B5 and His B10. Fragments originating from peptide bond hydrolysis were also detected.ConclusionMCO of insulin leads to covalent aggregation through cross-linking via Michael addition to tyrosine oxidation products.Electronic supplementary materialThe online version of this article (doi:10.1007/s11095-012-0755-z) contains supplementary material, which is available to authorized users.
Highlights
During pharmaceutical production and storage, therapeutic proteins can be exposed to components that are able to induceElectronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.R
Complementary detection of DOPA and DOCH was achieved using mass spectrometry and fluorogenic tagging with 4benzene sulfonic acid (ABS) (Scheme 1), according to the method reported recently by Sharov et al [19,20] Our results indicate that all aromatic amino acids of insulin, i.e. His, Tyr and Phe, are subject to metal catalyzed oxidation (MCO), predominantly yielding mono- and/or dihydroxylation products
Digested and non digested peptides were analyzed by means of an LTQ-FT hybrid linear quadrupole ion trap Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer (Thermo-Finnigan, Bremen, Germany) [26] and a SYNAPT-G2 (Waters Corporation, Milford, MA), both located in the Mass Spectrometry Laboratory of the University of Kansas, under the conditions as described by Ikehata et al [26]
Summary
Recombinant human insulin containing 0.4 % (w/w) zinc was provided by Merck (Oss, The Netherlands). L-ascorbic acid, ethylenediaminetetraacetic acid sodium salt (EDTA), copper dichloride, arginine, monobasic and dibasic sodium hydrogen phosphate, ammonium bicarbonate (ABI), dithiothreitol (DTT), iodoacetamide (IAM) and the solvents glacial acetic acid and acetonitrile were purchased from Sigma–Aldrich (St.Louis, MO, USA). Millipore Q water was used for the preparation of all the formulations and solutions. All chemicals were of analytical grade and used without further purification. ABS was synthesized according to a published procedure [21]. Centrifugal filter units with a volume capacity of 4 mL and a molecular weight cut-off of 3 kDa were purchased from Millipore (Billerica, MA, USA). Cassette dialysis slides with a 2 kDa cut-off were purchased from Thermo Scientific (Asheville, NC, USA)
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