Techniques Used in Lipidomics Analysis

The concept of lipidomics was proposed with the rise of genomes and proteomics research, which was proposed by Chinese scientist Han Xianlin in 2003. Lipidomics is a comprehensive systematic analysis and identification of lipids and molecules interacting with organisms, tissues or cells, which aims at understanding the structure and function of lipids, and revealing lipid metabolism and cells, organs and even the body. It is a subject of the relationship between physiological and pathological processes.

Lipid is a large class of compounds in nature that is insoluble in water and easily soluble in organic solvents. It is chemically and structurally heterogeneous, mainly including fatty acids and their naturally occurring derivatives (such as esters or amines), as well as biosynthesis and functionally related compounds. Lipid can participate in a variety of biological metabolism, such as energy conversion, material transport, information recognition and transmission, cell development and differentiation, apoptosis, and so on. Abnormal lipid metabolism can lead to various human diseases, including diabetes, obesity, cancer, neurodegenerative diseases, etc. Lipidomics has been widely used in important areas, including drug discovery, molecular physiology, molecular pathology, functional genomics, nutrition, environment, health, etc. Studies have shown that there are about 1000-2000 lipids in mammals, and with the development of lipid research technology, various new lipid molecules are still being discovered.

Due to its great variety, the National Institutes of Health (NIH) proposed to classify lipids into the following eight categories, fatty acids, glycerolipds, glycerophospholipids, sphingolipdis, sterol lipids, prenolipids, saccharolipids and polyketides. 

lipidomics 

distribution of common lipids in cells

Lipidomics Identification Techniques

Lipid metabolites are complex in structure and can be classified into polar lipids (such as glycerophospholipids, sphingolipids) and non-polar lipids (such as glycerides, cholesterol) by polarity. Different lipids can also be separated on the chromatogram depending on the difference in functional groups, carbon chain length, number of double bonds, and the like. Techniques for the identification of commonly used lipids include Shotgun, GC-MS, LC-MS and HILIC-MS.

Shotgun Method

The "Shotgun method" based on electrospray ionization mass spectrometry ESI-MS is currently a commonly used analytical method for studying lipidomics. After the lipid sample is purified, the ion source is separated according to the difference in charge under different pH values of the lipid molecule after direct injection without separating by chromatographic column. Different lipid molecules are detected separately in positive and negative ion modes. The Shotgun method has the characteristics of simple sample preparation, small sample consumption and short analysis time. The disadvantage is that it is more difficult to analyze low abundance lipids.

shotgun lipidomics

 GC-MS

Most of the lipid small molecules are of high volatility. GC-MS can effectively separate lipids from other macromolecules, and then identify small lipid molecules by mass spectrometry. However, the requirements for analyte volatility and derivatization restrict the application of GC-MS in lipidomics.

LC-MS

Compared with GC-MS, lc-ms lipid analysis has no volatility requirements for analytes and does not require derivatization. It can achieve good separation on the chromatographic column while retaining the complete information of lipids. It is widely used in lipidomics analysis. The liquid chromatography mobile phase and column type can be divided into two separation modes, Normal Phase Liquid Chromatography (NPLC) and Reversed Phase Liquid Chromatography (RPLC).

HILIC-MS

Hydrophilic interaction liquid chromatography (HILIC) is a new chromatographic separation mode with a separation mechanism between NPLC and RPLC that integrates the advantages and disadvantages of RPLC and NPLC. HILIC uses unbonded silica polar stationary phase and water-water-soluble organic solvent as mobile phase, which is beneficial to the separation of polar lipids and overcomes the shortcomings of NPLC and RPLC in the separation of polar lipids.

Further separation within the class is achieved based on the separation between classes. Since the mobile phase contains a high concentration of organic phase, it can enhance the efficiency of lipid ionization, improve the sensitivity of lipid detection, and has good compatibility with mass spectrometry.

characteristics comparison of HILIC and NPLC, RPLC


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