改进的^18O同位素标记法标记蛋白质多肽

Peptide Labeling with Improved ^(18)O Incorporation Method

针对18O同位素标记反应两个重要影响因素——肽段分散度和胰酶灭活方法,进行了标记条件的改进和灭活方法的优化。在H218O中加入RapigestTM SF助溶剂并微波辅助加热,使α-酪蛋白胰酶酶切肽段的标记效率得到明显改进(18O/16O峰面积比值>99%)。标记后,对胰酶进行还原烷基化化学修饰彻底灭活,使标记后的肽段稳定性显著提高,放置6d不发生回交反应。对标准蛋白质甲状腺球蛋白酶切肽段混合物标记后的质谱实验结果表明:优化的标记方法能快速稳定地标记蛋白质酶切多肽。

In order to optimize the 18O labeling method,two key aspects,peptide dispersion and trypsin deactivation were discussed.The addition of RapigestTM SF in H218O and microwave heating enhanced labeling efficiency of α-casein digested peptides（18O/16O ratio 〉 99%）.Chemical modification with tris（2-carboxyethyl）phosphine（TCEP） and iodoacetamide（IAA） resulted in trypsin deactivated completely.No significant back-exchange from 18O to 16O was observed after labeling in 6 days.The experiment result with peptide mixture from showed that the improved method could be effectively used to label protein and peptide.