Complementary Square-Wave Voltammetry and LC-MS/MS Analysis to Elucidate Induced Damaged and Mutated Mitochondrial and Nuclear DNA from in Vivo Knockdown of the BRCA1 Gene in the Mouse Skeletal Muscle.
Complementary Square-Wave Voltammetry and LC-MS/MS Analysis to Elucidate Induced Damaged and Mutated Mitochondrial and Nuclear DNA from in Vivo Knockdown of the BRCA1 Gene in the Mouse Skeletal Muscle.
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DOI：
10.1021/acs.analchem.1c02249
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发表时间：
2021-08-24
影响因子：
7.4
通讯作者：
Hvastkovs EG
中科院分区：
文献类型：
--
作者：
LaFave ER;Tarpey MD;Balestrieri NP;Spangenburg EE;Hvastkovs EG
乳腺癌1基因（BRCA 1）DNA突变影响骨骼肌功能。诱导性骨骼肌特异性Brca 1纯合子敲除（Brca 1 KOsmi，KO）小鼠积累线粒体DNA（mtDNA）突变，导致肌肉质量损失。双电化学-质谱分析用于快速评估直接从小鼠骨骼肌中提取的mtDNA或核DNA（nDNA）。用方波伏安法（SWV）监测了Ru（bpy）32+电催化下DNA层层固定（LbL）的氧化峰电流（Ip）。与杂合子KO（Het）或野生型（WT）相比，KO mtDNA的Ip显著降低（p < 0.05），表明鸟嘌呤含量降低。与WT相比，KO中的nDNA Ip显著增加（p < 0.05），表明损伤的nDNA的积累。使用液相色谱-串联质谱法（LC-MS/MS）通过适当的m/z质量转换监测鸟嘌呤或氧化损伤的鸟嘌呤含量。与WT相比，KO mtDNA和nDNA中的鸟嘌呤显著降低，而KO nDNA中氧化损伤的鸟嘌呤显著升高。这些数据表明鸟嘌呤含量的损失与线粒体DNA突变的积累一致。KO nDNA中的氧化损伤表明与Brca 1相关的修复过程受到影响。总的来说，电化学和LC-MS/MS分析可以为生物模型表型反应提供化学水平的答案，作为已建立测定的快速且具有成本效益的分析替代方案。
Breast cancer 1 gene (BRCA1) DNA mutations impact skeletal muscle function. Inducible skeletal muscle specific Brca1 homozygote knockout (Brca1KOsmi, KO) mice accumulate mitochondrial DNA (mtDNA) mutations resulting in loss of muscle quality. Dual electrochemical-mass spectrometry analysis was utilized to rapidly assess mtDNA or nuclear DNA (nDNA) extracted directly from mouse skeletal muscle. Oxidative peak currents (Ip) from DNA immobilized layer-by-layer (LbL) were monitored using square wave voltammetry (SWV) via Ru(bpy)32+ electrocatalysis. Ip significantly decreased (p < 0.05) for KO mtDNA compared to heterozygous KO (Het) or wild type (WT), indicative of decreases in guanine content. nDNA Ip significantly increased in KO compared to WT (p < 0.05), suggesting an accumulation of damaged nDNA. Guanine or oxidatively damaged guanine content was monitored via appropriate m/z mass transitions using liquid chromatography-tandem mass spectroscopy (LC-MS/MS). Guanine in both KO mtDNA and nDNA was significantly lower, while oxidatively damaged guanine in KO nDNA was significantly elevated versus WT. These data demonstrate a loss of guanine content consistent with mtDNA mutation accumulation. Oxidative damage in KO nDNA suggests that repair processes associated with Brca1 are impacted. Overall, electrochemical and LC-MS/MS analysis can provide chemical level answers to biological model phenotypic responses as a rapid and cost-effective analysis alternative to established assays.
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影响因子：
4.7
作者：
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