title: Chemical modifications in aggregates of recombinant human insulin induced by metal-catalyzed oxidation: covalent cross-linking via michael addition to tyrosine oxidation products.
abstract: OBJECTIVE
  To elucidate the chemical modifications in covalent aggregates of recombinant human insulin induced by metal catalyzed oxidation (MCO).
  
  
  METHODS
  Insulin 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.
  
  
  RESULTS
  The 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.
  
  
  CONCLUSIONS
  MCO of insulin leads to covalent aggregation through cross-linking via Michael addition to tyrosine oxidation products.
authors.affiliation: Division of Drug Delivery Technology Leiden/Amsterdam Center for Drug Research (LACDR), Leiden University, P.O. Box 9502, 2300, Leiden, The Netherlands.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Torosantucci
authors.forename: Olivier
authors.initials: O
authors.lastname: Mozziconacci
authors.forename: Victor
authors.initials: V
authors.lastname: Sharov
authors.forename: Christian
authors.initials: C
authors.lastname: Schöneich
authors.forename: Wim
authors.initials: W
authors.lastname: Jiskoot
mesh_terms: D000595:Amino Acid Sequence; D001205:Ascorbic Acid / Q000737:chemistry; D003300:Copper / Q000737:chemistry*; D006801:Humans; D007004:Hypoglycemic Agents / Q000737:chemistry*; D007328:Insulin / Q000737:chemistry*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D007004:Hypoglycemic Agents; D007328:Insulin; D011994:Recombinant Proteins; D003300:Copper; D001205:Ascorbic Acid