Citation: WANG Xue-Ying, ZONG Zhao-Yun, JIAO Yu-Pei, XU Li-Na, WANG Yu-Song, LI Lin, LIU Xiao-Hui. Methylation Assisted High Throughput Analysis of Phosphatidic Acid by High Resolution Mass Spectrometry[J]. Chinese Journal of Analytical Chemistry, ;2019, 47(2): 249-255. doi: 10.19756/j.issn.0253-3820.181614 shu

Methylation Assisted High Throughput Analysis of Phosphatidic Acid by High Resolution Mass Spectrometry

WANG Xue-Ying ^1,2, ,
ZONG Zhao-Yun ² ,
JIAO Yu-Pei ^1,2 ,
XU Li-Na ^1,2 ,
WANG Yu-Song ^1,2 ,
LI Lin ² ,
LIU Xiao-Hui ^1,2

1.
National Protein Science Facility(Beijing), Tsinghua University, Beijing 100084, China;
2.
School of Life Sciences, Tsinghua University, Beijing 100084, China

Received Date: 26 September 2018
Revised Date: 2 November 2018

Fund Project: This work was supported by the National Natural Science Foundation of China (Nos. 81501776, 81871093), and we are grateful for the help of Metabolomics Center and Animal Core Facility at Tsinghua University.

A methylation-based approach for analysis of phosphatidic acids (PAs) using ultra-high performance liquid chromatography-Orbitrap mass spectrometry with incorporation of in-house database was established. This method allowed high throughput analysis with confident molecular identification and quantitation. Phosphatidic acids were extracted using the mixture of chloroform and methanol with subsequent derivatization using (trimethylsilyl) diazomethane. Derivatized PAs (Me-PA) were analyzed in data dependent acquisition (Full MS/dd-MS²) mode of high resolution mass spectrometer. In-house database compatible with Tracefinder was derived allowing lipid identification based on MS/MS spectra. In this work, to achieve confident molecular identification and quantitation, combinatorial analysis of positive and negative ion modes was performed. The method was evaluated by the linearity, lower limit of detection (LLOQ), inter-and intra-day precision using standard molecule PA(14:0/14:0), and R²>0.99, LLOD of 0.125 μg/mL, inter-and intra-day precision<8.47% were achieved. PAs in aged mice brains were analyzed using this high throughput approach. A total of 14 phosphatidic acid molecules were identified and quantified in C57BL/6 mice brain. Moreover, decreased level of phosphatidic acid containing polyunsaturated fatty acid chain was observed in aged brains. Conclusively, this orbitrap-based platform with methylation provides a sensitive and accurate approach to analyze phosphatidic acid quantitatively.
- Phosphatidic acid,
- Lysophosphatidic acid,
- Methylation,
- High resolution mass spectrometry,
- High throughput
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