Mass spectrometry characterization of the glycation sites of bovine insulin by tandem mass spectrometry

J Am Soc Mass Spectrom. 2009 Jul;20(7):1319-26. doi: 10.1016/j.jasms.2009.03.004. Epub 2009 Mar 13.


Bovine insulin was glycated under hyperglycemic reducing conditions and in nonreducing conditions. Purification through HPLC allowed isolating glycated forms of insulin and a novel triglycated form (6224.5 Da) was purified. Endoproteinase Glu-C digestion combined with mass spectrometry (MALDI-TOF/TOF) allowed determining the exact location of the glycation sites in each of the isolated glycated insulins. For the first time, a triglycated form of insulin was isolated and characterized accordingly to its glycation sites. These glucose binding sites were identified as the N-terminals of both chains (Gly1 and Phe1) and residue Lys29 of B-chain. Moreover, in diglycated insulin we found the coexistence of one specie glycated at the N-terminals of both chains (Gly1 and Phe1) and another specie containing the two glucitol adducts in B-chain (Phe1 and Lys29). Also, in monoglycated insulin generated in reducing and nonreducing conditions, one specie glycated at Phe1 and another specie glycated at Lys29, both B-chain residues coexist.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cattle
  • Chromatography, High Pressure Liquid
  • Glucose / chemistry*
  • Glucose / metabolism
  • Glycosylation
  • Insulin / analogs & derivatives
  • Insulin / chemistry*
  • Insulin / metabolism
  • Molecular Sequence Data
  • Peptide Fragments / chemistry
  • Peptide Fragments / metabolism
  • Serine Endopeptidases / chemistry
  • Serine Endopeptidases / metabolism
  • Tandem Mass Spectrometry / methods*


  • Insulin
  • Peptide Fragments
  • Serine Endopeptidases
  • glutamyl endopeptidase
  • Glucose