Shotgun Lipidomics

Lipidomics provides a systematic analysis of the lipids in an organism, tissue or cell and the molecules that interact with them. Shotgun lipidomics tracks lipid networks in biological systems on a larger scale, allowing lipid recognition, quantification and interaction to be studied.

Lipidomics studies focus on changes in lipid interactions. This includes information such as lipid component species, lipid content, and understanding their movement within and outside the cell lumen. Shotgun lipidomics was first proposed by Han and Gross in 2003. They accomplished a qualitative and quantitative analysis of the complex lipid composition in organisms through a combination of cutting-edge technologies and further explored the molecular mechanisms of lipids by studying their involvement in different intracellular functions. Since then, larger-scale lipidomics has followed. By 2005, hundreds of lipid molecules belonging to more than 20 different lipid classes could be analyzed using the more mature Shotgun lipidomics technology, which covers more than 95% of intracellular lipid components. Despite the high coverage, there are still many low-abundance lipid molecules that are not well localized by current Shotgun lipidomics techniques.

Shotgun lipidomics utilizes conventional lipid extraction methods for biological samples, and based on effective intrasource separation, appropriate multidimensional array analysis methods are selected according to the different intrinsic charged properties of lipid ions or fragments to achieve the characterization and quantification of the target lipid molecules.

Lipidomics workflow.Lipidomics workflow.

The combination of intrasource separation, multidimensional mass spectrometry and computer analysis has become one of the most representative analytical techniques in lipidomics research.

Intrasource separation

The basic principle of intrasource separation is to use an electrospray ion source to dissociate biomolecules in a sample into differently charged ions under the action of a high potential (typically 4 kV), which are then selectively resolved in a process similar to electrophoresis. Since different lipid molecules have different ionization properties, the degree of charge depends largely on the chemical characteristics of their polarhead groups. Therefore, the initial separation of lipid molecules in lipid extracts according to their intrinsic charge can be performed by intrasource separation, and the subsequent analytical steps can be used to obtain a mass spectral map of the composition of a series of lipid molecules in lipid biological samples.

Multidimensional mass spectrometric identification of lipid molecules

Mass spectrometry is the qualitative analysis of a substance by measuring the ion mass-to-charge ratio (m/z). The basic process is to ionize the components of the sample under the action of an ion source to generate positively charged ions with different mass-to-charge ratios. The ion beam is formed in an accelerating electric field and then enters the mass analyzer. The velocity dispersion effects of the electric and magnetic fields in different directions of action are used to effectively differentiate the ions with different mass-to-charge ratios in the mass spectrometry spectrum and thus determine their masses. Typically, the primary ion mass spectrum is used as the one-dimensional chromatogram. On this basis, a series of progressive changes in the experimental conditions by means of the test equipment is used to introduce additional dimensions.

Mass spectrometric quantification of lipid molecules

After qualitative identification by mass spectrometry, quantitative analysis can further provide more valuable information about lipid molecules. Given the wide variety of lipid molecules and their close elemental composition, the quantification usually requires a two-step process. First, select the molecular components with high abundance and non-overlapping mass spectrum peaks in the lipid extract to be isotopically removed at 13C, and quantify the molecules based on the ratio of the preselected internal reference standard. The other lipid molecules with relatively low abundance and overlapping mass spectral peaks are then traced by one- or multi-dimensional mass spectrometry by adding the pre-selected internal standard as a new reference to all or some of the quantified molecular species. By eliminating background noise and filtering out overlapping molecular signals, the two-step quantification process allows for a significant extension of the linear measurement range of the analysis.

Data processing and informatics analysis

Shotgun lipidomics analysis based on multi-dimensional mass spectrometry can generate a large amount of fragment information about the identification of lipid molecules. The bioinformatics processing of these results also needs to combine the physical characteristics, chemical properties and other information of various lipids.

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