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Optimization of lipid extraction and analytical protocols for UHPLC-ESI-HRMS-based lipidomic analysis of adherent mammalian cancer cells

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Abstract

Lipidomics, which reveals comprehensive characterization of molecular lipids, is a rapidly growing technology used in biomedical research. Lipid extraction is a critical step in lipidomic analysis. However, the effectiveness of different lipid extract solvent systems from cellular samples still remains unclear. In the current study, the protocol of reverse-phase liquid chromatography mass spectrometry (LC/MS)-based lipidomics was optimized for extraction and detection of lipids from human pancreatic cancer cell line PANC-1. Four different extraction methods were compared, including methanol/methyl-tert-butyl ether (MTBE)/H2O, methanol/chloroform, methanol/MTBE/chloroform, and hexane/isopropanol. Data were acquired using high-resolution mass spectrometry in positive and negative ion modes respectively. The number of total detected and identified lipids was assessed with the aid of automated lipid identification software LipidSearch. Results demonstrated that methanol/MTBE/H2O provided a better extraction efficiency for different lipid classes, which was chosen as the optimized extraction solvent system. This validated method enables highly sensitive and reproducible analysis for a variety of cellular lipids, which was further applied to an untargeted lipidomic study on human pancreatic cancer PANC-1 cell lines. Moreover, this optimized extraction solvent system can be further applied to other cancer cell lines with similar chemical and physical properties.

Optimized UHPLC-ESI-HRMS-based lipidomic analysis of cancer cells

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Abbreviations

CE:

Cholesteryl ester

CER:

Ceramide

CL:

Cardiolipin

DG:

Diacylglycerols

FA:

Fatty acid

LPC:

Lysophosphatidylcholine

LPE:

Lysophosphatidylethanolamine

MTBE:

Methyl-tert-butyl ether

PA:

Phosphatidic acid

PC:

Phosphatidylcholine

PCA:

Principal component analysis

PE:

Phosphatidylethanolamine

PG:

Phosphatidylglycerol

PI:

Phosphatidylinositol

PS:

Phosphatidylserine

SM:

Sphingomyelin

TG:

Triacylglycerol

UHPLC-ESI-HRMS:

Ultra-high-performance liquid chromatography electrospray ionization high-resolution mass spectrometry

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Acknowledgements

The work was supported by the Natural Science Foundation of China (Grants: 81522047, 81573489, 81373470, 81320108027), the 111 project (Grant: B16047), the Key Laboratory Foundation of Guangdong Province (Grant: 2011A060901014), the Natural Science Foundation of Guangdong (Grant: 2015A030313124), and the Guangzhou Health Care Collaborative Innovation Program (Grant: 201508020250).

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Authors and Affiliations

  1. School of Pharmaceutical Sciences, Sun Yat-sen University, 132# Waihuandong Road, Guangzhou University City, Guangzhou, 510006, China

    Huizhen Zhang, Yue Gao, Jiahong Sun, Shicheng Fan, Xinpeng Yao, Min Huang & Huichang Bi

  2. Thermo Fisher Scientific, Shanghai, China

    Xiaorong Ran & Chuanqi Zheng

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  1. Huizhen Zhang
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  2. Yue Gao
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Correspondence to Huichang Bi.

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Zhang, H., Gao, Y., Sun, J. et al. Optimization of lipid extraction and analytical protocols for UHPLC-ESI-HRMS-based lipidomic analysis of adherent mammalian cancer cells. Anal Bioanal Chem 409, 5349–5358 (2017). https://doi.org/10.1007/s00216-017-0483-7

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