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. © 2009 American Society for Mass Spectrometry.
Guedes, S., Vitorino, R., Domingues, M. R. M., Amado, F., & Domingues, P. (2009). Mass Spectrometry Characterization of the Glycation Sites of Bovine Insulin by Tandem Mass Spectrometry. Journal of the American Society for Mass Spectrometry, 20(7), 1319–1326. https://doi.org/10.1016/j.jasms.2009.03.004