Quantitative analysis of WRN exonuclease activity by isotope dilution mass spectrometry
Quantitative analysis of WRN exonuclease activity by isotope dilution mass spectrometry
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DOI：
10.1016/j.mad.2012.06.005
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发表时间：
2012-08-01
影响因子：
5.3
通讯作者：
Buerkle, Alexander
中科院分区：
文献类型：
--
作者：
Mangerich, Aswin;Veith, Sebastian;Buerkle, Alexander
沃纳综合征是一种以过早衰老表型为特征的疾病。该疾病是由WRN基因突变引起的，该基因编码DNA解旋酶/核酸外切酶，其参与DNA代谢的多个方面。目前的方法主要依赖于放射性技术来评估WRN核酸外切酶活性。在这里，我们提出了一种替代的，基于非放射性同位素稀释质谱法（LC-MS/MS）的定量方法。模拟端粒序列的寡聚双链体底物用于方法开发。释放的核苷酸，这与寡聚双链体降解的程度，脱磷酸化，纯化，并通过LC-MS/MS定量。重同位素标记的内标物被用来解释技术的可变性。在反应的重现性、时间过程和浓度依赖性方面对该方法进行了验证。如本研究所示，LC-MS/MS方法可以评估WRN突变体的核酸外切酶活性、WRN的底物和链特异性以及WRN相互作用伴侣和翻译后修饰的调节作用。此外，它可用于分析WRN核酸外切酶的选择性和持续合成能力，并允许筛选WRN核酸外切酶抑制剂的小分子。重要的是，这种方法可以很容易地适用于研究核酸酶以外的WRN。这是普遍感兴趣的，因为核酸外切酶是DNA代谢和衰老机制的关键参与者。(c)2012爱思唯尔爱尔兰有限公司保留所有权利。
Werner syndrome is a disorder characterized by a premature aging phenotype. The disease is caused by mutations in the WRN gene which encodes a DNA helicase/exonuclease which is involved in multiple aspects of DNA metabolism. Current methods mostly rely on radiometric techniques to assess WRN exonuclease activity. Here we present an alternative, quantitative approach based on non-radioactive isotope dilution mass spectrometry (LC-MS/MS). A oligoduplex substrate mimicking the telomeric sequence was used for method development. Released nucleotides, which correlate with the degree of oligoduplex degradation, were dephosphorylated, purified, and quantified by LC-MS/MS. Heavy-isotope-labeled internal standards were used to account for technical variability. The method was validated in terms of reproducibility, time-course and concentration-dependency of the reaction. As shown in this study, the LC-MS/MS method can assess exonuclease activity of WRN mutants, WRN's substrate and strand specificity, and modulatory effects of WRN interaction partners and posttranslational modifications. Moreover, it can be used to analyze the selectivity and processivity of WRN exonuclease and allows the screening of small molecules for WRN exonuclease inhibitors. Importantly, this approach can easily be adapted to study nucleases other than WRN. This is of general interest, because exonucleases are key players in DNA metabolism and aging mechanisms. (c) 2012 Elsevier Ireland Ltd. All rights reserved.