基于LC-MS/MS技术的胶质瘤细胞酰基肉碱代谢轮廓分析研究

建立了胶质瘤细胞样本中酰基肉碱类化合物的LC-MS/MS代谢轮廓分析方法。首先，采用Q-Exactive四极杆-轨道阱高分辨串联质谱的全扫描（Full MS Scan）和平行反应监测（PRM）模式对细胞样本中酰基肉碱进行定性分析。根据一级和二级高分辨质谱数据，并结合酰基肉碱类化合物的特征裂解规律，在U87MG胶质瘤细胞、胶质瘤干细胞样细胞和胶质瘤干细胞分化细胞3种干性不同的胶质瘤细胞样品中鉴定出17种酰基肉碱类化合物。采用Qtrap 5500四极杆-线性离子阱串联质谱的多反应监测（MRM）模式，建立了细胞样本中17种酰基肉碱化合物的代谢轮廓分析方法，并对比分析了3种胶质瘤细胞中酰基肉碱化合物的差异。结果表明，该方法的线性关系良好，线性相关系数大于0.99，准确度与精密度均符合要求。细胞样本中酰基肉碱的定量分析结果表明，与胶质瘤干细胞相比，胶质瘤干细胞分化细胞和胶质瘤细胞中的肉碱和酰基肉碱含量均明显上调。此研究可为细胞样本中酰基肉碱类化合物的代谢轮廓分析提供方法参考。

Metabolic Profiling of Acylcarnitines in Glioma Cells Based on LC-MS/MS

Cell metabolomics is now becoming an invaluable tool to gain mechanistic insight into biological processes. Carnitine and acylcarnintines play important roles in fatty acid oxidation. A method for metabolic profiling of acylcarnitines in glioma cells was developed herein by liquid chromatography tandem mass spectrometry (LC-MS/MS). The cell samples (5×106 ) were quenched by -80 ℃ 100% methanol and extracted with 80% methanol aqueous solution by freeze-thaw cycles. Separation and identification were performed using UPLC/Q-Exactive HRMS with electrospray ionization in positive mode. Firstly, the acylcarnitines reported in the HMDB database and literatures were selected from the full scan data and then chosen as precursor ions to be performed by parallel reaction monitoring (PRM) acquisition. The characteristic product ions of acylcarnitines at m/z 60.080 8, 85.028 4, 144.101 9, and the neutral loss of 59.073 5 Da were used to screen potential acylcarnitines. The metabolites identified as acylcarnitines should fulfill the following conditions: (1) The mass tolerance of precursor ion is within 5×10^-6. (2) More than three fragment features are detected at mass tolerance of 10×10^-6, and retention behavior of these fragment ions should match with the precursor ion. (3) The characteristic product ion of m/z 85.028 4 is prerequisite as it is the most stable fragment for acylcarnitines. As a result, a total of 17 acylcarnitines were identified in the U87MG malignant cells (GCs), U87MG stem-like cells (SLCs) and U87MG stem-like cell differentiation cells (SLCDCs), including seven short-, four medium and six long-chain acylcarnitines. Then 17 acylcarnitines was profiled using UPLC/Qtrap operated in the multiple reaction monitoring (MRM) mode, which enabled rapid quantification of 14 acylcarnitines within 15 min. The method afforded satisfactory results in terms of sensitivity, specificity, precision as well as accuracy. The results showed that the levels of carnitine and most acylcarnitines in SLCDCs and GCs were up-egulated compared with SLCs, which suggest that mitochondrial energy metabolism alterations associated with SLC self-renewal and differentiation. This study can promote the discovery of acylcarnitine-related metabolic reprogramming processes at the cellular level and may provide perspectives for anti-tumor research based on cellular energy metabolism.