LC-quadrupole/Orbitrap high-resolution mass spectrometry enables stable isotope-resolved simultaneous quantification and ¹³C-isotopic labeling of acyl-coenzyme A thioesters.
LC-quadrupole/Orbitrap high-resolution mass spectrometry enables stable isotope-resolved simultaneous quantification and ¹³C-isotopic labeling of acyl-coenzyme A thioesters.
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DOI：
10.1007/s00216-016-9448-5
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发表时间：
2016-05
影响因子：
4.3
通讯作者：
Snyder NW
中科院分区：
文献类型：
--
作者：
Frey AJ;Feldman DR;Trefely S;Worth AJ;Basu SS;Snyder NW
酰基辅酶A硫代酯(acyl-COA)是进化上保守的、区隔的和能量激活的用于生化反应的底物。酰基-COAs在代谢中的广泛参与，包括三羧酸循环、脂肪酸代谢、氨基酸降解和胆固醇代谢，突出了酰基-COAs应用测量的广泛适用性。然而，酰基辅酶A水平的定量只提供了代谢信息的一个维度，更完整的代谢描述需要不同前体对个别底物和途径的相对贡献。使用两种不同的稳定同位素标记方法，可以用从泛酸衍生到辅酶A部分的固定的[13C315N1]标记酰基-COA，或者通过从代谢前体标记到酰链的可变[13C]标记。使用平行反应监测而不是单离子监测的液相色谱-混合四极杆/轨道捕获高分辨质谱仪，允许通过使用[13C315N1]标记的稳定同位素稀释法同时定量酰基-COAs，并测量来自[13C6]-葡萄糖、[13C515N2]-谷氨酰胺和[13C3]-丙酸的标记碳原子的掺入。作为原理的证明，我们将这种方法应用于培养中的人类B细胞淋巴瘤(WSU-DLCL2)细胞，以精确描述相对池大小和同位素示踪剂在乙酰基、琥珀酰基和丙酰辅酶A中的浓缩。这种方法将允许高精度、多重和稳定的同位素分解的代谢测定，以完善代谢模型，表征新的代谢，并测试涉及酰基COA的代谢途径的调节物。
Acyl-coenyzme A thioesters (acyl-CoAs) are evolutionarily conserved, compartmentalized, and energetically activated substrates for biochemical reactions. The ubiquitous involvement of acyl-CoAs in metabolism, including the tricarboxylic acid cycle, fatty acid metabolism, amino acid degradation, and cholesterol metabolism highlights the broad applicability of applied measurements of acyl-CoAs. However, quantitation of acyl-CoA levels provides only one dimension of metabolic information and a more complete description of metabolism requires the relative contribution of different precursors to individual substrates and pathways. Using two distinct stable isotope labeling approaches, acyl-CoAs can be labeled with either a fixed [13C315N1] label derived from pantothenate into the CoA moiety, or via variable [13C] labeling into the acyl-chain from metabolic precursors. Liquid chromatography-hybrid quadrupole/Orbitrap high resolution mass spectrometry using parallel reaction monitoring, but not single ion monitoring, allowed the simultaneous quantitation of acyl-CoAs by stable isotope dilution using the [13C315N1] label and measurement of the incorporation of labeled carbon atoms derived from [13C6]-glucose, [13C515N2]-glutamine and [13C3]-propionate. As a proof-of-principle we applied this method to human B-cell lymphoma (WSU-DLCL2) cells in culture, to precisely describe the relative pool size and enrichment of isotopic tracers into acetyl-, succinyl-, and propionyl-CoA. This method will allow highly precise, multiplexed, and stable isotope resolved determination of metabolism to refine metabolic models, characterize novel metabolism, and test modulators of metabolic pathways involving acyl-CoAs.