Targeted Determination of Tissue Energy Status by LC-MS/MS
Targeted Determination of Tissue Energy Status by LC-MS/MS
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DOI：
10.1021/acs.analchem.9b00217
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发表时间：
2019-05-07
影响因子：
7.4
通讯作者：
Burgess, Shawn C.
中科院分区：
文献类型：
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作者：
Fu, Xiaorong;Deja, Stanislaw;Burgess, Shawn C.
细胞内核苷酸和酰基辅酶A是调节能量代谢的核心代谢物。它们设置细胞能量电荷和氧化还原状态，作为变构调节剂，调节信号传导和转录因子，并介导激活氧化或生物合成底物。不幸的是，没有方法可以同时定量组织提取物中的这些生物分子。采用离子对反相高效液相色谱-电喷雾串联质谱法（HPLC-ESI-MS/MS）同时定量腺嘌呤核苷酸（AMP、ADP和ATP）、吡啶二核苷酸（NAD+和NADH）和短链酰基辅酶A（乙酰基、丙二酰基、琥珀酰基和丙酰基）。这些分子在小鼠肝脏中的定量分析，实现了使用稳定同位素标记的内标。通过测定线性、准确度、重复性和含量测定稳定性，对该方法进行了广泛验证。生物反应性在具有可变缺血持续时间的肝组织的测定中得到证实，其对组织能量电荷和氧化还原状态具有实质性影响。我们的结论是，该方法提供了一个简单，快速，可靠的方法，同时分析核苷酸和短链酰基辅酶A。
Intracellular nucleotides and acyl-CoAs are metabolites that are central to the regulation of energy metabolism. They set the cellular energy charge and redox state, act as allosteric regulators, modulate signaling and transcription factors, and thermodynamically activate substrates for oxidation or biosynthesis. Unfortunately, no method exists to simultaneously quantify these biomolecules in tissue extracts. A simple method was developed using ion-pairing reversed-phase high-performance liquid chromatography-electrospray-ionization tandem mass spectrometry (HPLC-ESI-MS/MS) to simultaneously quantify adenine nucleotides (AMP, ADP, and ATP), pyridine dinucleotides (NAD+ and NADH), and short-chain acyl-CoAs (acetyl, malonyl, succinyl, and propionyl). Quantitative analysis of these molecules in mouse liver was achieved using stable-isotope-labeled internal standards. The method was extensively validated by determining the linearity, accuracy, repeatability, and assay stability. Biological responsiveness was confirmed in assays of liver tissue with variable durations of ischemia, which had substantial effects on tissue energy charge and redox state. We conclude that the method provides a simple, fast, and reliable approach to the simultaneous analysis of nucleotides and short-chain acyl-CoAs.