Extracellular vesicles (EVs) convey information used in cell‐to‐cell interactions. Lipid analysis of EVs remains challenging because of small sample amounts available. Lipid discovery using traditional mass spectrometry platforms based on liquid chromatography and high mass resolution typically employs milligram sample amounts. We report a simple workflow for lipid profiling of EVs based on multiple reaction monitoring (MRM) profiling that uses microgram amounts of sample. After liquid–liquid extraction, individual EV samples were injected directly into the electrospray ionization (ESI) ion source at low flow rates (10 μl/min) and screened for 197 MRM transitions chosen to be a characteristic of several classes of lipids. This choice was based on a discovery experiment, which applied 1,419 MRMs associated with multiple lipid classes to a representative pooled sample. EVs isolated from 12 samples of human lymphocytes and 16 replicates from six different rat cells lines contained an estimated amount of total lipids of 326 to 805 μg. Samples showed profiles that included phosphatidylcholine (PC), sphingomyelin (SM), cholesteryl ester (CE), and ceramide (Cer) lipids, as well as acylcarnitines. The lipid profiles of human lymphocyte EVs were distinguishable using principal component and cluster analysis in terms of prior antibody and drug exposure. Lipid profiles of rat cell lines EV's were distinguishable by their tissue of origin.

中文翻译：

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多反应监测分析作为研究细胞外囊泡中脂质的分析策略

细胞外囊泡 (EV) 传达用于细胞间相互作用的信息。由于可用的样本量很少，电动汽车的脂质分析仍然具有挑战性。使用基于液相色谱和高质量分辨率的传统质谱平台进行脂质发现通常使用毫克样品量。我们报告了基于多反应监测 (MRM) 分析的 EV 脂质分析的简单工作流程，该分析使用微克量的样品。液-液萃取后，将单个 EV 样品以低流速 (10 μl/min) 直接注入电喷雾电离 (ESI) 离子源，并筛选出 197 个 MRM 跃迁，这些跃迁被选为几类脂质的特征。这个选择是基于一个发现实验，它应用了 1，419 个与多个脂质类别相关的 MRM 与代表性混合样本相关。从 12 个人类淋巴细胞样本和 6 个不同大鼠细胞系的 16 个重复样本中分离出的 EV 含有估计量的 326 至 805 μg 的总脂质。样品显示的特征包括磷脂酰胆碱 (PC)、鞘磷脂 (SM)、胆固醇酯 (CE) 和神经酰胺 (Cer) 脂质，以及酰基肉碱。就先前的抗体和药物暴露而言，使用主成分和聚类分析可以区分人淋巴细胞 EV 的脂质谱。大鼠细胞系 EV 的脂质谱可通过它们的组织来源进行区分。样品显示的特征包括磷脂酰胆碱 (PC)、鞘磷脂 (SM)、胆固醇酯 (CE) 和神经酰胺 (Cer) 脂质，以及酰基肉碱。就先前的抗体和药物暴露而言，使用主成分和聚类分析可以区分人淋巴细胞 EV 的脂质谱。大鼠细胞系 EV 的脂质谱可通过它们的组织来源进行区分。样品显示的特征包括磷脂酰胆碱 (PC)、鞘磷脂 (SM)、胆固醇酯 (CE) 和神经酰胺 (Cer) 脂质，以及酰基肉碱。就先前的抗体和药物暴露而言，使用主成分和聚类分析可以区分人淋巴细胞 EV 的脂质谱。大鼠细胞系 EV 的脂质谱可通过它们的组织来源进行区分。