Protein carbamylation: In vivo modification or in vitro artefact?
Protein carbamylation: In vivo modification or in vitro artefact?
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DOI：
10.1002/pmic.201200452
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发表时间：
2013-03-01
期刊：
影响因子：
3.4
通讯作者：
Zahedi, Rene P.
中科院分区：
文献类型：
--
作者：
Kollipara, Laxmikanth;Zahedi, Rene P.
赖氨酸残基和蛋白质 N 末端的氨甲酰化 (carbamoylation) 是一种非酶 PTM，与蛋白质老化有关。与其他 PTM（例如磷酸化）相比，氨甲酰化可以在用尿素制备样品期间人为引入，从而影响针对体内氨甲酰化的研究。在水溶液中，常用于使蛋白质变性的尿素与铵和异氰酸酯处于平衡状态。在碱性条件下，后者可与游离N端的伯胺和赖氨酸的ε胺基反应生成氨甲酰衍生物。尽管这是一个相对缓慢的过程（在高温下会加速），但在尿素缓冲液中长时间孵育蛋白质/肽样品会诱导不需要的氨甲酰化，不仅妨碍胰蛋白酶的蛋白水解消化和 MS 进行的肽鉴定，而且还会干扰稳定同位素标记技术，例如 iTRAQ、串联质量标签和同位素编码蛋白质标记。在这里，我们评估了常用样品制备条件下尿素诱导的氨甲酰化程度。从我们的结果中，我们可以推断出氨甲酰化发生在所有涉及尿素的情况下，但程度不同：例如8.0 M 尿素存在下的脲甲基化诱导 17% 的 N 末端和 4% 的 Lys 残基发生氨甲酰化。此外，对最近发表的大规模数据集的研究揭示了当前蛋白质组样本中尿素诱导的高度氨甲酰化。
Carbamylation (carbamoylation) of lysine residues and protein N-termini is a nonenzymatic PTM that has been related to protein ageing. In contrast to other PTM, such as phosphorylation, carbamylation can be artificially introduced during sample preparation with urea, thus affecting studies directed toward in vivo carbamylation. In aqueous solution, ureacommonly used for denaturing proteinsis in equilibrium with ammonium and isocyanate. Under alkaline conditions, the latter can react with primary amines of free N-termini and epsilon-amine groups of lysines to form carbamyl derivatives. Despite being a relatively slow process, which is accelerated at elevated temperatures, prolonged incubation of protein/peptide samples in urea buffers can induce undesired carbamylation, hampering not only the proteolytic digestion with trypsin and peptide identification by MS, but also interfering with stable isotope-labeling techniques such as iTRAQ, tandem mass tags, and isotope-coded protein labeling. Here, we evaluated the extent of urea-induced carbamylation under commonly used sample preparation conditions. From our results, we can deduce that carbamylation occurs in all cases involving urea, however with varying degree: e.g. carbamidomethylation in the presence of 8.0 M urea induced carbamylation of 17% of N-termini and 4% of Lys residues. Additionally, researching a recently published large-scale dataset revealed a high degree of urea-induced carbamylation in current proteomic samples.