Phospholipid Extraction and Mass Spectrometry Analysis

Classification of Phospholipids

Structural characteristics of phospholipids: They have a hydrophilic head composed of substituent groups (containing amino or alcohol groups) connected by phosphoric acid, and a hydrophobic tail composed of fatty acid chains. Phospholipids are classified into glycerophospholipids and sphingolipids (SM) based on the different alcohol components.

Glycerophospholipids are the most abundant class of phospholipids in organisms. In addition to forming biological membranes, they are also components of bile and surfactants on membrane surfaces, and they participate in cell membrane recognition of proteins and signal transduction. Glycerophospholipids are further classified into six major categories: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), and phosphatidic acid (PA). Within each major category, there are various subtypes with similar structures due to differences in fatty acid chains, such as lysophospholipids and lysolecithins. Cardiolipin (CL) is a diphosphatidylglycerol and a complex phospholipid widely found in the inner mitochondrial membrane. It can regulate the activity of oxidative phosphorylation-related enzymes and plays an important role in maintaining mitochondrial function and membrane integrity.

Sphingolipids, also known as sphingomyelins, are widely present in biological tissues, especially in the brain, where they are particularly abundant.

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Phospholipids: Structure, Functions, and Applications

Extraction of Phospholipids

Tissue phospholipid extraction

Take 40 mg of tissue and add 0.8 mL of cold CH₃OH-0.1mol/L HCl (1:1, v/v) in an ice bath. Homogenize the mixture for 1 minute. Transfer the resulting suspension to a cold centrifuge tube. Add 0.4 mL of ice-cold chloroform, vortex for 1 minute, and centrifuge (4 °C, 18,000 × g, 5 minutes) to separate the layers. Transfer the lower organic phase to the centrifuge tube and dry it with nitrogen gas. Store at -20 °C. Prior to injection, resolubilize the sample with the initial mobile phase and add 10 µL of NH₃H₂O (1.8 mol/L) to enhance ionization efficiency.

Cell phospholipid extraction

Start by collecting approximately 1 × 10^7 cells and subject them to two washes using 5 mL of ice-cold 1× PBS. Next, take 1.5 mL of the cell suspension and transfer it to a centrifuge tube. Centrifuge the tube at 4 °C and 600 × g for a duration of 10 minutes to remove the supernatant.

Proceed by adding 0.8 mL of cold CH₃OH -0.1mol/L HCl (1:1, v/v) and 0.4 mL of ice-cold chloroform to the tube. To ensure proper mixing, vortex the contents for 1 minute. Afterward, centrifuge the tube again, but this time at 4 °C and 18,000 × g for 5 minutes, which will effectively separate the layers.

Carefully transfer the lower organic phase to the centrifuge tube. Subsequently, use nitrogen gas to dry the transferred sample. For storage, maintain the sample at -20 °C. When you are ready to proceed with the injection, resolubilize the sample using the initial mobile phase. Additionally, add 10 µL of NH₃H₂O (1.8 mol/L) to enhance the ionization efficiency before conducting further analysis.

Plasma Phospholipid Extraction

After centrifugation of whole blood, take 300 µL of plasma and add 8 mL of CH₃Cl-CH₃OH (2:1, v/v) containing 0.01% 2,6-di-tert-butyl-4-methylphenol. Sonicate for 60 seconds, vortex for 30 seconds, and let it sit at room temperature for 0.5 hours. Add 1.3 mL of 50 mmol/L KCl solution, centrifuge (2000 × g, 15 minutes), and separate the layers. Extract the upper aqueous phase solution once with 2 mL of CH₃Cl, and combine the organic phase solutions obtained from two extractions. Dry the combined organic phase with nitrogen gas at -20 °C. Prior to injection, resolubilize the sample with the initial mobile phase.

Phospholipid Mass Spectrometry Analysis

The increasing diversity of phospholipid structures has posed challenges in their analysis. However, with the advancement of modern analytical techniques, there are now numerous feasible methods available, such as shotgun lipidomics and high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS), among others.

Electrospray ionization mass spectrometry (ESI-MS) offers various benefits, including simplified sample preparation, enhanced resolution, and automation convenience. This technique is particularly well-suited for rapid, sensitive, and high-throughput analysis of phospholipid mixtures, enabling both qualitative and quantitative assessments. The integration of liquid chromatography with mass spectrometry has played a pivotal role in advancing phospholipidomics. ESI-MS serves as the core technology in this approach, working in tandem with chromatographic methods for efficient phospholipid separation, enabling high-throughput analysis, exceptional sensitivity, and effective phospholipid identification. Moreover, multidimensional mass spectrometry has contributed to groundbreaking advancements in phospholipidomics research.

Shotgun lipidomics provides advantages of high sensitivity, speed, and automation. However, it has certain limitations when analyzing phospholipid isomers. Liquid chromatography-mass spectrometry (LC-MS) can effectively overcome the drawbacks of shotgun lipidomics, such as ion suppression effects on low-abundance phospholipids and the inability to accurately analyze isomeric species. Currently, LC-MS has become the most widely applied technique in phospholipid analysis.

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Shotgun Mass Spectrometry

Shotgun MS utilizes mass spectrometry techniques to systematically analyze individual or all phospholipids and their related molecules, studying their changes and exploring possible mechanisms of action in biological systems. The bottleneck issues in traditional phospholipid analysis have been overcome with the emergence of electrospray ionization mass spectrometry-based methods, ushering in an era of high-throughput, high-precision, and high-efficiency phospholipid analysis.

Phospholipids are widely distributed and diverse in biological systems, and they are closely associated with human diseases. Applying phospholipidomics analysis methods to discover disease-specific phospholipid biomarkers and elucidate their roles in complex processes of disease development can provide new insights and strategies for diagnosis and treatment.

By employing conventional methods for extracting phospholipids from biological samples and incorporating in-source separation, suitable multidimensional array analysis techniques can be selected based on the inherent charged properties of phospholipid ions or fragments. This facilitates the qualitative and quantitative analysis of specific target phospholipid molecules. The fundamental principle of in-source separation involves utilizing electrospray ionization sources at high potentials (typically 4 kV) to dissociate biomolecules in the sample into ions with varying charges. These ions subsequently undergo selective resolution akin to electrophoresis. Given that different phospholipid molecules exhibit distinct ionization characteristics, their charge states predominantly depend on the chemical attributes of their polar head groups. Hence, in-source separation allows for initial separation of phospholipid molecules based on their inherent charge, followed by subsequent analytical steps to obtain mass spectra encompassing a range of phospholipid compositions present within the biological sample.

Mass spectrometry-based shotgun lipidomics (Hsu et al., 2018)

High-Performance Liquid Chromatography-Electrospray Ionization-Mass Spectrometry

Based on the structural features of different phospholipids, it can be said that acidic glycerophospholipids like PS, PG, PI, PA, and CL display good responses in negative ion mode, whereas neutral phospholipids like PC, PE, and SM demonstrate good responses in positive ion mode. Based on retention time and mass-to-charge ratio (m/z), preliminary analysis of phospholipid structures in two-dimensional mass spectra can be carried out. Efficient separation of phospholipid isomers is possible using a high-performance liquid chromatography column. The investigation of phospholipid profiles in tissues, cells, and plasma is made easier by using high-performance liquid chromatography-electrospray ionization-mass spectrometry.

LC-MS spectra of phospholipid classes (Da et al., 2018)

References

Hsu, Fong-Fu. "Mass spectrometry-based shotgun lipidomics–a critical review from the technical point of view." Analytical and bioanalytical chemistry 410 (2018): 6387-6409.
Da Costa, Elisabete, et al. "High-resolution lipidomics of the early life stages of the red seaweed Porphyra dioica." Molecules 23.1 (2018): 187.

* For Research Use Only. Not for use in diagnostic procedures.

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