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.

中文翻译：

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在线二维色谱/质谱法对短链，中链和长链酰基辅酶A进行综合分析

酰基辅酶A（CoA）是许多生物学过程中的关键代谢中间体。然而，由于酰基辅酶A的性质随不同碳链的不同而有很大差异，因此对酰基辅酶A的全面分析仍具有挑战性。在这里，我们设计了一种二维液相色谱方法，并结合了高分辨率质谱（2D LC / HRMS），以在一次分析运行中涵盖所有短链，中链和长链酰基CoA。通过一维预分级分离，将复杂的酰基辅酶A根据其酰基链分为两个部分。然后，在第二维中通过两个平行柱进一步分离出包含短链酰基辅酶A或中链和长链酰基辅酶A的两个馏分。选择了19个代表性标准品以优化2D LC / HRMS方法的分析条件。分离度和灵敏度被证明可以大大提高，并且可以检测和区分出含量低的酰基CoA和酰基CoA异构体。通过使用2D LC / HRMS方法，从肝脏提取物中鉴定出90种酰基辅酶A（包括21种酰基脱磷辅酶CoA），这表明我们的方法是迄今为止获得酰基辅酶A全面分析的最有效方法之一。该方法进一步用于具有异柠檬酸脱氢酶1（IDH1）突变的恶性神经胶质瘤细胞的代谢组学研究，以探索其代谢差异。共检测到46种酰基辅酶A（包括2种酰基脱磷辅酶A），其中12种在IDH1突变的神经胶质瘤细胞中失调。这些结果证明了所建立方法的实用性和优越性。所以，