ArticlePharmaceutical research2012
Chemical modifications in aggregates of recombinant human insulin induced by metal-catalyzed oxidation: covalent cross-linking via michael addition to tyrosine oxidation products.
Article in Pharmaceutical research, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 25 papers.
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Who cites it
25 citing papers in PubMed, 54 citations in OpenAlex.
- Oxidative Post-Translational Modifications of Insulin in Type 1 Diabetes Autoimmunity.Immunological reviews · 2026Review
- Synthesis of ZIF-67 Nanoparticles for Camel Whey Protein Delivery: Promising Antioxidant, Anti-inflammatory, Anticancer Effects, and Anti-biofilm Activity.Molecular biotechnology · 2026Article
- The role of oxidative post-translational modifications in type 1 diabetes pathogenesis.Frontiers in immunology · 2025Review
- Autoantibody and T cell responses to oxidative post-translationally modified insulin neoantigenic peptides in type 1 diabetes.Diabetologia · 2023Article
- Assessment of Therapeutic Antibody Developability by Combinations of In Vitro and In Silico Methods.Methods in molecular biology (Clifton, N.J.) · 2022Article
- Oxidative Crosslinking of Peptides and Proteins: Mechanisms of Formation, Detection, Characterization and Quantification.Molecules (Basel, Switzerland) · 2021Review
- Machine learning prediction of methionine and tryptophan photooxidation susceptibility.Molecular therapy. Methods & clinical development · 2021Article
- Top-Down ETD-MS Provides Unreliable Quantitation of Methionine Oxidation.Journal of biomolecular techniques : JBT · 2019Article
- Effect of Iron Oxide Nanoparticles on the Oxidation and Secondary Structure of Growth Hormone.Journal of pharmaceutical sciences · 2019Article
- Identification of Lysine Misincorporation at Asparagine Position in Recombinant Insulin Analogs Produced in E. coli.Pharmaceutical research · 2019Article
- Identification of N-Terminally Truncated Derivatives of Insulin Analogs Formed in Pharmaceutical Formulations.Pharmaceutical research · 2018Article
- Advancements in Free-Radical Pathologies and an Important Treatment Solution with a Free-Radical Inhibitor.SF journal of biotechnology and biomedical engineering · 2018Article
- Chemical and Biophysical Characteristics of Monoclonal Antibody Solutions Containing Aggregates Formed during Metal Catalyzed Oxidation.Pharmaceutical research · 2017Article
- Nanoparticulate Impurities Isolated from Pharmaceutical-Grade Sucrose Are a Potential Threat to Protein Stability.Pharmaceutical research · 2017Article
- Free-radicals and advanced chemistries involved in cell membrane organization influence oxygen diffusion and pathology treatment.AIMS biophysics · 2017Article
- Mouse Models for Assessing Protein Immunogenicity: Lessons and Challenges.Journal of pharmaceutical sciences · 2016Review
- Protein oxidation and peroxidation.The Biochemical journal · 2016Review
- Immunogenicity of Biotherapeutics: Causes and Association with Posttranslational Modifications.Journal of immunology research · 2016Review
- Purification and identification of high molecular weight products formed during storage of neutral formulation of human insulin.Pharmaceutical research · 2015Article
- Titanium Implant Osseointegration Problems with Alternate Solutions Using Epoxy/Carbon-Fiber-Reinforced Composite.Metals · 2014Article
Corrections and comments
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Authors and funding
5 authors at 2 institutions in 2 countries.
Funding
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.
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