Identification of the site of glycation of human insulin

Peptides. 1996;17(8):1323-30. doi: 10.1016/s0196-9781(96)00231-8.


This study evaluates the nature of glycated human insulin formed following exposure to hyperglycemic conditions in vitro. Glycated insulin was purified by RP-HPLC and its molecular mass (5971.3 Da) determined by plasma desorption mass spectrometry (MS). The difference in mass (163.7 Da) from nonglycated insulin (5807.6 Da) corresponds to a single reduced glucose (glucitol) residue. Following reduction of insulin disulfide bridges, MS confirmed that the B-chain was glycated. Enzymatic digestions with trypsin, endoproteinase Glu-C, and thermolysin, followed by RP-HPLC and identification of fragments by MS, localized glycation to the B-chain (1-5) region. Electrospray tandem MS identified the site of glycation as the B-chain NH2-terminal Phe1 residue. This was confirmed by automated Edman degradation with glycated human insulin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Chromatography, High Pressure Liquid
  • Glycosylation
  • Humans
  • Hyperglycemia / metabolism
  • In Vitro Techniques
  • Insulin / analogs & derivatives*
  • Insulin / chemistry*
  • Insulin / metabolism*
  • Molecular Structure
  • Peptide Fragments / chemistry
  • Peptide Fragments / isolation & purification
  • Protein Conformation


  • Insulin
  • Peptide Fragments
  • insulin, glycosylated