Comprehensive Analysis of Short-, Medium-, and Long-Chain AcylCoenzyme A by Online Two-Dimensional Liquid Chromatography/Mass Spectrometry
Comprehensive Analysis of Short-, Medium-, and Long-Chain AcylCoenzyme A by Online Two-Dimensional Liquid Chromatography/Mass Spectrometry
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通过在线二维液相色谱/质谱法综合分析短链、中链和长链酰基辅酶 A
DOI：
10.1021/acs.analchem.7b03659
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发表时间：
2017
影响因子：
7.4
通讯作者：
Xu Guowang
中科院分区：
文献类型：
--
作者：
Wang Shuangyuan;Wang Zhichao;Zhou Lina;Shi Xianzhe;Xu Guowang
酰基辅酶A（CoA）是许多生物过程中的关键代谢中间体。然而，酰基辅酶A的综合分析仍然具有挑战性，因为酰基辅酶A的性质随不同碳链而变化很大。在这里，我们设计了一个二维液相色谱法结合高分辨率质谱（2D LC/HRMS），以涵盖所有的短，中，长链酰基辅酶A在一个分析运行。复合酰基辅酶A根据其酰基链通过第一维预分级分离成两个级分。然后，含有短链酰基辅酶A或中链和长链酰基辅酶A的两个级分通过两个平行柱在第二维中进一步分离。选择19个代表性标准品以优化2D LC/HRMS方法的分析条件。结果表明，该方法的分离度和灵敏度都有很大提高，可以检测和区分低丰度的酰基辅酶A和酰基辅酶A异构体。通过使用二维LC/HRMS方法，从肝脏提取物中鉴定了90种酰基辅酶A（包括21种酰基脱磷酸辅酶A），这表明我们的方法是迄今为止获得酰基辅酶A综合谱的最有效方法之一。该方法进一步用于异柠檬酸脱氢酶1（IDH 1）突变的恶性胶质瘤细胞的代谢组学研究，以探讨其代谢差异。共检测到46个酰基辅酶A（包括2个酰基脱磷酸辅酶A），其中12个在IDH 1突变的胶质瘤细胞中表达异常。结果表明了该方法的实用性和优越性。因此，2D LC/HRMS方法为组织、细胞和其他生物样品中酰基辅酶A的综合分析提供了一种稳健且可重现的方法。
Acyl-coenzyme A (CoA) is a pivotal metabolic intermediate in numerous biological processes. However, comprehensive analysis of acyl-CoAs is still challenging as the properties of acyl-CoAs greatly vary with different carbon chains. Here, we designed a two-dimensional liquid chromatography method coupled with high-resolution mass spectrometry (2D LC/HRMS) to cover all short-, medium-, and long-chain acyl-CoAs within one analytical run. Complex acyl-CoAs were separated into two fractions according to their acyl chains by the first dimensional prefractionation. Then, two fractions containing short-chain acyl-CoAs or medium- and long-chain acyl-CoAs were further separated by the two parallel columns in the second dimension. Nineteen representative standards were chosen to optimize the analytical conditions of the 2D LC/HRMS method. Resolution and sensitivity were demonstrated to be improved greatly, and lowly abundant acyl-CoAs and acyl-CoA isomers could be detected and distinguished. By using the 2D LC/HRMS method, 90 acyl-CoAs (including 21 acyl-dephospho-CoAs) were identified from liver extracts, which indicated that our method was one of the most powerful approaches for obtaining comprehensive profiling of acyl-CoAs so far. The method was further employed in the metabolomics study of malignant glioma cells with an isocitrate dehydrogenase 1 (IDH1) mutation to explore their metabolic differences. A total of 46 acyl-CoAs (including 2 acyl-dephospho-CoAs) were detected, and 12 of them were dysregulated in glioma cells with the IDH1 mutation. These results demonstrated the practicability and the superiority of the established method. Therefore, the 2D LC/HRMS method provides a robust and reproducible approach to the comprehensive analysis of acyl-CoAs in tissues, cells, and other biological samples.