O'Harte F P, Højrup P, Barnett C R, Flatt P R
School of Biomedical Sciences, University of Ulster, Coleraine, N. Ireland, UK.
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.
本研究评估了体外暴露于高血糖条件下形成的糖化人胰岛素的性质。糖化胰岛素通过反相高效液相色谱法（RP-HPLC）纯化，其分子量（5971.3道尔顿）通过等离子体解吸质谱法（MS）测定。与未糖化胰岛素（5807.6道尔顿）的质量差异（163.7道尔顿）对应于一个单一的还原葡萄糖（山梨醇）残基。胰岛素二硫键还原后，质谱证实B链被糖化。用胰蛋白酶、谷氨酸蛋白酶Glu-C和嗜热菌蛋白酶进行酶切，然后进行RP-HPLC并通过MS鉴定片段，将糖化定位到B链（1-5）区域。电喷雾串联质谱确定糖基化位点为B链NH2末端的苯丙氨酸1残基。用糖化人胰岛素进行自动Edman降解证实了这一点。
