A relative quantitative positive/negative ion switching method for untargeted lipidomics via high resolution LC-MS/MS from any biological source
A relative quantitative positive/negative ion switching method for untargeted lipidomics via high resolution LC-MS/MS from any biological source
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DOI：
10.1007/s11306-016-1157-8
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发表时间：
2017-03-01
期刊：
影响因子：
3.6
通讯作者：
Asara, John M.
中科院分区：
文献类型：
--
作者：
Breitkopf, Susanne B.;Ricoult, Stephane J. H.;Asara, John M.
引言高分辨率质谱技术的进步为研究疾病和生物系统中的全球脂质生物化学产生了新的兴趣。我们提出了一种非靶向的30分钟LC-MS/MS平台，该平台利用正/负极性切换，通过更高能量的碰撞解离（HCD）碎片化进行无偏的数据依赖性采集（DDA），以分析超过1000- 1500个主要来自甲基叔丁基醚（MTBE）或氯仿的脂离子：方法该平台使用C-18反相色谱法与混合QExactive Plus/HF Orbitrap质谱仪偶联，并且对于包含12个样品的数据集，从脂质提取到鉴定/定量的整个过程需要近似10小时（类似于单个样品的4小时）。脂质是由两个准确的前体离子质量和碎片的功能和定量使用脂质搜索和元素software.Results使用这种方法，我们能够配置完整的脂质离子从多达18个不同的主要脂质类和66个子类。我们展示了几项来自不同生物来源的研究，包括培养的癌细胞，小鼠切除的组织，如肺和乳腺肿瘤，以及生物液体，如血浆和尿液。我们发现TSC 2（-/-）KD显著消除脂质生物合成，雷帕霉素可以拯救甘油三酯（TG）脂质，我们发现SREBP-/-通过mTORC 1信号通路显著关闭脂质生物合成。我们发现，在小鼠EGFR驱动的肺肿瘤中，大量TG和磷脂酰甲醇（PMe）脂质升高，而一些磷脂（PL）显示脂质水平从2000种已鉴定的脂质离子中最大幅度降低。此外，我们还从人血浆中鉴定了1500多种独特的脂质。
Introduction Advances in high-resolution mass spectrometry have created renewed interest for studying global lipid biochemistry in disease and biological systems.Objectives Here, we present an untargeted 30 min. LC-MS/MS platform that utilizes positive/negative polarity switching to perform unbiased data dependent acquisitions (DDA) via higher energy collisional dissociation (HCD) fragmentation to profile more than 1000-1500 lipid ions mainly from methyl-tert-butyl ether (MTBE) or chloroform: methanol extractions.Methods The platform uses C-18 reversed-phase chromatography coupled to a hybrid QExactive Plus/HF Orbitrap mass spectrometer and the entire procedure takes similar to 10 h from lipid extraction to identification/quantification for a data set containing 12 samples (similar to 4 h for a single sample). Lipids are identified by both accurate precursor ion mass and fragmentation features and quantified using Lipid-Search and Elements software.Results Using this approach, we are able to profile intact lipid ions from up to 18 different main lipid classes and 66 subclasses. We show several studies from different biological sources, including cultured cancer cells, resected tissues from mice such as lung and breast tumors and biological fluids such as plasma and urine.Conclusions Using mouse embryonic fibroblasts, we showed that TSC2(-/-) KD significantly abrogates lipid biosynthesis and that rapamycin can rescue triglyceride (TG) lipids and we show that SREBP-/- shuts down lipid biosynthesis significantly via mTORC1 signaling pathways. We show that in mouse EGFR driven lung tumors, a large number of TGs and phosphatidylmethanol (PMe) lipids are elevated while some phospholipids (PLs) show some of the largest decrease in lipid levels from similar to 2000 identified lipid ions. In addition, we identified more than 1500 unique lipid species from human blood plasma.