Department of Chemistry, Yonsei University, 50 Yonsei-Ro, Seoul, 03722, Republic of Korea.
Department of Chemistry, Yonsei University, 50 Yonsei-Ro, Seoul, 03722, Republic of Korea.
Anal Chim Acta. 2019 Sep 27;1073:79-89. doi: 10.1016/j.aca.2019.04.069. Epub 2019 May 1.
We investigated the effect of oxidative stress (OS) on lipidomic perturbations in the subcellular fractions and exosomes of human embryonic kidney (HEK) 293 cells using asymmetrical flow field-flow fractionation (AF4) and nanoflow ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry (nUHPLC-ESI-MS/MS). We treated HEK 293 cells with hydrogen peroxide (HO) and fractionated the cell lysates using AF4 to determine the change in size and population of the subcellular fractions and exosomes, and to obtain narrow size fractions for lipid analysis. A total of 438 lipids from 642 identified species-including oxidized lipids-were quantified. The relative amount of secreted exosomes increased by 28% during OS, whereas the amount of subcellular species decreased by 35%. There was a significant increase in the level of oxidized phospholipids in the mitochondrion-enriched subcellular fractions, but not in the exosomes. Most high-abundance triacylglycerol (TG) species increased in the stressed cells, whereas they decreased in the exosomes. During OS, ceramides involved in the apoptotic mitochondrial pathway were accumulated in the subcellular fractions, whereas their levels were unaffected in the exosomes. The present study demonstrated that AF4 and nUHPLC-ESI-MS/MS can be used to investigate lipid alterations in subcellular and extracellular species during OS, and the pathological relationships in diseases caused by reactive oxygen species.
我们使用不对称流场流分离（AF4）和纳流超高效液相色谱-电喷雾电离-串联质谱（nUHPLC-ESI-MS/MS）研究了氧化应激（OS）对人胚肾（HEK）293 细胞亚细胞成分和外泌体中脂质组学变化的影响。我们用过氧化氢（HO）处理 HEK 293 细胞，并用 AF4 对细胞裂解物进行分级，以确定亚细胞成分和外泌体的大小和群体变化，并获得用于脂质分析的窄粒径分数。共鉴定出 642 种脂质，包括氧化脂质，定量了其中的 438 种。在 OS 期间，分泌的外泌体的相对量增加了 28%，而亚细胞物种的量减少了 35%。富含线粒体的亚细胞成分中氧化磷脂的水平显著增加，但外泌体中没有增加。大多数高丰度三酰基甘油（TG）物种在应激细胞中增加，而在外泌体中则减少。在 OS 期间，参与凋亡线粒体途径的神经酰胺在亚细胞成分中积累，而在外泌体中其水平不受影响。本研究表明，AF4 和 nUHPLC-ESI-MS/MS 可用于研究 OS 期间亚细胞和细胞外物种中脂质的变化，以及活性氧引起的疾病中的病理关系。
