Wancewicz Benjamin, Pergande Melissa R, Zhu Yanlong, Gao Zhan, Shi Zhuoxin, Plouff Kylie, Ge Ying
Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, Wisconsin, 53705, USA.
Human Proteomics Program, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, 53705, USA.
bioRxiv. 2023 Sep 16:2023.09.15.558013. doi: 10.1101/2023.09.15.558013.
The heart contracts incessantly and requires a constant supply of energy, utilizing numerous metabolic substrates such as fatty acids, carbohydrates, lipids, and amino acids to supply its high energy demands. Therefore, a comprehensive analysis of various metabolites is urgently needed for understanding cardiac metabolism; however, complete metabolome analyses remain challenging due to the broad range of metabolite polarities which makes extraction and detection difficult. Herein, we implemented parallel metabolite extractions and high-resolution mass spectrometry (MS)-based methods to obtain a comprehensive analysis of the human heart metabolome. To capture the diverse range of metabolite polarities, we first performed six parallel liquid-liquid extractions (three monophasic, two biphasic, and one triphasic extractions) of healthy human donor heart tissue. Next, we utilized two complementary MS platforms for metabolite detection - direct-infusion ultrahigh-resolution Fourier-transform ion cyclotron resonance (DI-FTICR) and high-resolution liquid chromatography quadrupole time-of-flight tandem MS (LC-Q-TOF MS/MS). Using DI-FTICR MS, 9,521 metabolic features were detected where 7,699 were assigned a chemical formula and 1,756 were assigned an annotated by accurate mass assignment. Using LC-Q-TOF MS/MS, 21,428 metabolic features were detected where 626 metabolites were identified based on fragmentation matching against publicly available libraries. Collectively, 2276 heart metabolites were identified in this study which span a wide range of polarities including polar (benzenoids, alkaloids and derivatives and nucleosides) as well as non-polar (phosphatidylcholines, acylcarnitines, and fatty acids) compounds. The results of this study will provide critical knowledge regarding the selection of appropriate extraction and MS detection methods for the analysis of the diverse classes of human heart metabolites.
心脏持续收缩，需要持续的能量供应，它利用多种代谢底物，如脂肪酸、碳水化合物、脂质和氨基酸来满足其高能量需求。因此，迫切需要对各种代谢物进行全面分析以了解心脏代谢；然而，由于代谢物极性范围广泛，使得提取和检测困难，完整的代谢组分析仍然具有挑战性。在此，我们采用平行代谢物提取和基于高分辨率质谱（MS）的方法来全面分析人类心脏代谢组。为了捕获不同极性范围的代谢物，我们首先对健康人类供体心脏组织进行了六次平行液 - 液萃取（三次单相萃取、两次双相萃取和一次三相萃取）。接下来，我们利用两个互补的MS平台进行代谢物检测——直接进样超高分辨率傅里叶变换离子回旋共振（DI - FTICR）和高分辨率液相色谱四极杆飞行时间串联质谱（LC - Q - TOF MS/MS）。使用DI - FTICR MS，检测到9521个代谢特征，其中7699个被分配了化学式，1756个通过精确质量分配被注释。使用LC - Q - TOF MS/MS，检测到21428个代谢特征，其中基于与公开可用库的碎片匹配鉴定出626种代谢物。本研究共鉴定出2276种心脏代谢物，它们涵盖了广泛的极性范围，包括极性化合物（苯类、生物碱及其衍生物和核苷）以及非极性化合物（磷脂酰胆碱、酰基肉碱和脂肪酸）。本研究结果将为分析人类心脏不同类别代谢物时选择合适的提取和MS检测方法提供关键知识。
