2018 Fiscal Year Research-status Report
Development of a novel technique for direct analysis of single lipid droplets in mammalian cells by nanoESI-MS
| Project/Area Number |
18K14247
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| Research Institution | Hokkaido University |
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Principal Investigator |
趙 瑤瑤 北海道大学, 保健科学研究院, 助教 (50800702)
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| Project Period (FY) |
2018-04-01 – 2020-03-31
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| Keywords | single lipid droplet / Mass spectrometry / solvent microextraction |
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| Outline of Annual Research Achievements |
lipid droplets (LDs) were successfully induced by oleic acid and palmitic acid in HepG2 cells. By using multifaceted nanomanipulator, we can choose the LDs in the same size. Herein, we developed a novel technique termed in-tip solvent microextraction mass spectrometry (ITSME-MS) to separate phosphatidylcholines and triglycerides in a single cellular lipid droplet. A single lipid droplet with a small amount of buffer solution was sucked into a nanotip using a three-dimensional micromanipulator. The nanotip was subsequently backfilled with the organic solvent suitable for lipid extraction and subjected to nanoESI-MS analysis. A gradient solvent system at the tip zone was formed, from aqueous buffer solution to increasingly hydrophobic organic solvent. As a result, phosphatidylcholines were detected earlier than triglycerides as it dissolved better in water. Besides, we obtained the structural information of all lipid components by using LC-MS/MS. This method has been successfully applied for the analysis of LDs in HepG2 cells by mass spectrometry and to compare the lipid profiles in three different kinds of LDs at single LD level. The developed method should find wide applications, such as lipid biology and disease diagnosis, and allows a better understanding of the mechanisms of lipid production, packaging into cytosolic LDs, and pathophysiology.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
The novel technique was developed for the profiling of phosphatidylcholines and triglycerides at the single lipid droplet level. This method was used to analyze lipid droplets in mammalian cells and allowed comparison of lipid profiles among three different kinds of lipid droplets.The method has been published in Analytical Chemistry (2019, 91 (7), 4466-4471) and it was selected as front cover.
The method is the core part of this research. Based on the single lipid droplets analysis method, we can do the further research, for example, test lipid droplets induced by fatty acids with different carbon numbers or compare lipid droplets with different size.
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| Strategy for Future Research Activity |
Diets rich in long-chain fatty acids (LCFAs, 14 or more carbon atoms) promote lipid accumulation and insulin resistance, whereas medium-chain fatty acids (MCFAs) are associated with increased oxidative metabolism and reduced adiposity, with few deleterious effects on insulin action. Lipid droplets (LDs) emerge as an important intracellular organelle relevant for lipid homeostasis and development of metabolic diseases. However, the relationship between MCFAs and the LDs composition has been poorly investigated. Next, we aims to investigate the effect of medium-chain fatty acid on triglyceride profile of LDs in HepG2 cells by using the method we have developed.
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| Causes of Carryover |
The incurring amount can be used for article costs.
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