Guedes Sofia, Vitorino Rui, Domingues M Rosário M, Amado Francisco, Domingues Pedro
Department of Chemistry, University of Aveiro, Aveiro, Portugal.
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.
牛胰岛素在高血糖还原条件和非还原条件下进行糖基化。通过高效液相色谱（HPLC）纯化可分离出胰岛素的糖基化形式，并纯化出一种新型三糖基化形式（6224.5 Da）。内肽酶Glu-C消化结合质谱（基质辅助激光解吸电离飞行时间/串联飞行时间质谱，MALDI-TOF/TOF）可确定每种分离出的糖化胰岛素中糖基化位点的确切位置。首次分离出胰岛素的三糖基化形式，并根据其糖基化位点进行了表征。这些葡萄糖结合位点被确定为两条链的N端（Gly1和Phe1）以及B链的Lys29残基。此外，在双糖基化胰岛素中，我们发现一种在两条链的N端（Gly1和Phe1）糖基化的形式与另一种在B链中含有两个葡糖醇加合物（Phe1和Lys29）的形式共存。同样，在还原和非还原条件下生成的单糖基化胰岛素中，一种在Phe1糖基化的形式与另一种在Lys29糖基化的形式（均为B链残基）共存。
