Department of Clinical and Experimental Medicine, Division of Metabolic Disease, University of Padova.
Rapid Commun Mass Spectrom. 2009 Dec;23(23):3837-42. doi: 10.1002/rcm.4290.
Homocysteinylation is a post-translational protein modification which involves homocysteine-thiolactone and may be responsible for many pathophysiological changes secondary to hyperhomocysteinemia. Therefore, methods to measure protein homocysteinylation in intact biological samples are required. We tested whether matrix assisted-laser/desorption ionization mass spectrometry (MALDI-MS) can detect time- and dose-dependent changes in in vitro homocysteine-thiolactone binding to human serum albumin. We have compared this method with a 35S-thiolactone radioactive binding assay. Incubations with and without dithiothreitol allowed measurement of the amide-linked and disulfide-linked thiolactone-protein adducts, respectively. A good correspondence in time- and dose-dependent protein-thiolactone formation was observed between the two methods. A maximum of 9 to 12 thiolactone residues were bound to each albumin molecule. The 35S-thiolactone bound albumin tightly, particularly at the lowest concentrations, with approximately 70% of the binding amide-linked. Although the results of the two methods were rather similar, the radioactive method appears to be more sensitive than the MALDI-MS technique.
同型半胱氨酸酰基化是一种翻译后蛋白质修饰，涉及同型半胱氨酸-硫内酯，可能是高同型半胱氨酸血症引起许多病理生理变化的原因。因此，需要能够在完整生物样本中检测蛋白质同型半胱氨酸酰基化的方法。我们测试了基质辅助激光解吸电离质谱（MALDI-MS）是否可以检测体外同型半胱氨酸-硫内酯与人血清白蛋白之间的时间和剂量依赖性结合变化。我们将这种方法与 35S-硫内酯放射性结合测定进行了比较。有和没有二硫苏糖醇的孵育分别允许测量酰胺键和二硫键连接的硫内酯-蛋白质加合物。两种方法都观察到时间和剂量依赖性蛋白质-硫内酯形成之间的良好相关性。每个白蛋白分子最多结合 9 到 12 个硫内酯残基。35S-硫内酯与白蛋白紧密结合，尤其是在最低浓度下，约 70%的结合是酰胺键连接的。尽管两种方法的结果相当相似，但放射性方法似乎比 MALDI-MS 技术更灵敏。
