Formation and reshuffling of disulfide bonds in bovine serum albumin demonstrated using tandem mass spectrometry with collision-induced and electron-transfer dissociation.
Formation and reshuffling of disulfide bonds in bovine serum albumin demonstrated using tandem mass spectrometry with collision-induced and electron-transfer dissociation.
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DOI：
10.1038/srep12210
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发表时间：
2015-07-20
影响因子：
4.6
通讯作者：
Delcour JA
中科院分区：
文献类型：
--
作者：
Rombouts I;Lagrain B;Scherf KA;Lambrecht MA;Koehler P;Delcour JA
使用交替电子转移解离 (ETD) 和碰撞诱导解离 (CID)，通过液相色谱 (LC) 电喷雾电离 (ESI) 串联质谱 (MS/MS) 分析新鲜分离、长期储存（6 年，6°C，干燥）和加热（60 分钟，90°C，过量水中）牛血清白蛋白 (BSA) 样品的嗜热菌蛋白酶水解物。将不同样品中二硫键和游离硫醇基团的位置与从天然 BSA 晶体结构推导的位置进行比较。结果显示，在分离、大量干燥储存和加热过程中，半胱氨酸发生非酶促翻译后修饰。热诱导的 BSA 可提取性损失与蛋白质解折叠对参与分子间和分子内硫醇-二硫键交换和硫醇氧化反应的特定半胱氨酸残基的影响有关。这里开发的方法有望在化学、生化、制药和食品加工相关条件下探索各种蛋白质中二硫键的形成和重组。
Thermolysin hydrolyzates of freshly isolated, extensively stored (6 years, 6 °C, dry) and heated (60 min, 90 °C, in excess water) bovine serum albumin (BSA) samples were analyzed with liquid chromatography (LC) electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using alternating electron-transfer dissociation (ETD) and collision-induced dissociation (CID). The positions of disulfide bonds and free thiol groups in the different samples were compared to those deduced from the crystal structure of native BSA. Results revealed non-enzymatic posttranslational modifications of cysteine during isolation, extensive dry storage, and heating. Heat-induced extractability loss of BSA was linked to the impact of protein unfolding on the involvement of specific cysteine residues in intermolecular and intramolecular thiol-disulfide interchange and thiol oxidation reactions. The here developed approach holds promise for exploring disulfide bond formation and reshuffling in various proteins under conditions relevant for chemical, biochemical, pharmaceutical and food processing.