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What is Phospholipids
Phospholipids refer to lipids containing phosphate groups. They belong to complex lipids and can be divided into two categories: glycerophospholipids and sphingomyelin, which are composed of glycerol and sphingomyelin respectively. The chemical structure of phospholipids includes a phospholipid bond formed by removing an OH from a phosphate group and removing H from an alcohol. The molecular structure of phospholipids is composed of two hydrophobic long hydrocarbon chains and a hydrophilic phosphate group. The constituent elements of phospholipids are usually C, H, O, N, and P. Due to the structural characteristics of the polar head of phospholipids, the hydrophilic ends of phospholipid molecules are close to each other, and the hydrophobic ends are close to each other to form a lipid bilayer. Molecules such as proteins, glycolipids, and cholesterol are connected through phospholipid linkage to form cell membranes with biological functions.
What Is Phospholipidomics
In 2008, Jan and colleagues from the University of Bremen, Germany, proposed the concept of phospholipidomics. Phospholipidomics is an emerging discipline that involves the systematic analysis of the entire phospholipid profile. It is a branch of lipidomics that aims to identify key phospholipid biomarkers involved in metabolic regulation by comparing changes in phospholipid metabolic networks under different physiological conditions. Ultimately, phospholipidomics aims to uncover the mechanisms of phospholipids in various biological processes.
Electrospray Ionization-Mass Spectrometry (ESI-MS) is the core research technique in the field of phospholipidomics, enabling high-resolution, high-sensitivity, and high-throughput analysis of phospholipids. With the development of liquid chromatography-mass spectrometry (LC-MS), phospholipidomics has shown promising applications in the identification of phospholipid biomarkers for diseases, disease diagnosis, discovery of drug targets and lead compounds, and the study of drug action mechanisms. It holds significant importance for biomedical research and has broad prospects for application.
Classification of Phospholipids
Phospholipids possess a characteristic structure, consisting of a hydrophilic head (containing amino or alcohol groups) linked to a hydrophobic tail (composed of fatty acid chains) through a phosphate group. Phospholipids can be classified into two main categories based on the different alcohol components: glycerophospholipids and sphingolipids (SM).
Glycerophospholipids are the most abundant type of phospholipids in organisms. Apart from forming biological membranes, they also serve as components of bile and membrane surfactants. Additionally, they participate in cell membrane recognition and signal transduction of proteins. Glycerophospholipids can be further categorized into six classes based on the different polar heads: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), and phosphatidic acid (PA). Within each class, there are numerous structurally similar subclasses distinguished by different fatty acid chains, such as plasmalogens and lysolecithins. Cardiolipin (CL) is a diphosphatidylglycerol, a complex phospholipid widely present in the inner mitochondrial membrane. It can regulate the activity of oxidative phosphorylation enzymes and plays a crucial role in maintaining mitochondrial function and membrane integrity.
Sphingolipids, also known as sphingomyelins, are widely distributed in various biological tissues, especially in high quantities within brain tissue.
Phospholipid Structure Diagram
Functions of Phospholipids
Phospholipids serve multiple critical physiological functions, and extensive research has shown that disruptions in phospholipid metabolism can lead to various diseases, such as diabetes, obesity, atherosclerosis, coronary heart disease, Alzheimer's disease, brain injuries, cancer, fatty liver, and Barth syndrome, among others. Consequently, the study of phospholipids and their metabolic processes in living organisms has become a key focus in understanding disease mechanisms, diagnosis, treatment, and pharmaceutical research.
In order to obtain a comprehensive profile of phospholipids in biological samples, better elucidate the mechanisms of phospholipid substances within the organism, and identify biomarkers or metabolic patterns associated with diseases, scientists have made phospholipid analysis a prominent area of research. This approach aims to provide a scientific basis for early disease diagnosis and further advance our understanding of the roles played by phospholipids in biological systems.
Phospholipid Mass Spectrometry Analysis Methods
Traditional analysis methods are complex and have low sensitivity, such as HPLC, TLC, and GC-MS. When using GC-MS analysis, phospholipids need to be hydrolyzed and derivatized first, and it can only provide information about the fatty acyl chains, lacking precise determination of the phospholipid structure.
Although the structural diversity of phospholipids increases the analytical challenges, with the advancement of modern analytical techniques, there are now various feasible methods available, such as shotgun lipidomics and High-Performance Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (HPLC-ESI-MS). Electrospray Ionization-Mass Spectrometry (ESI-MS) offers advantages of simple sample preparation, high resolution, and easy automation, making it particularly suitable for rapid, sensitive, and high-throughput qualitative and quantitative analysis of phospholipid mixtures. The hyphenation of liquid chromatography and mass spectrometry has significantly advanced phospholipidomics, with ESI-MS as the core technology, enhancing the separation and identification of phospholipids with high throughput, sensitivity, and efficiency. Moreover, multidimensional mass spectrometry techniques have shown new progress in phospholipidomics research.
Shotgun lipidomics provides high sensitivity, speed, and ease of automation, but it has some limitations when analyzing phospholipid isomers. The hyphenation of liquid chromatography and mass spectrometry effectively improves the deficiencies in shotgun lipidomics, such as ion suppression effects on low-abundance phospholipids and the inability to accurately analyze isomers.
Currently, liquid chromatography-mass spectrometry (LC-MS) has become the most widely used technology in phospholipid analysis.
Workflow for Phospholipid LC-MS Analysis Platform
Application of Phospholipid Mass Spectrometry Analysis
With the advancement of phospholipidomics research, the functions of phospholipids within living organisms will be further revealed. The application of mass spectrometry technology to analyze changes in the phospholipid profile in biological systems and identify potential biomarkers will find widespread applications in the following areas:
Early diagnosis: By identifying reliable biomarkers and correlating them with prognosis, mass spectrometry analysis can provide a basis for selecting appropriate treatment plans.
Disease monitoring: Monitoring changes in the types or quantities of phospholipid biomarkers can reflect the progression of diseases.
Drug development: Based on insights into the pathogenesis, mass spectrometry analysis can provide potential targets for drug design.
Studying the overall differences in phospholipid metabolism between normal and diseased states, identifying disease-specific phospholipid biomarkers, and combining them with enzyme research can lead to in-depth investigations of metabolic pathways or pathogenic mechanisms, ultimately leading to the discovery of effective diagnostic and therapeutic approaches.
It is foreseeable that as phospholipidomics research continues to progress, our understanding of the structure and mechanisms of phospholipids will deepen. The accuracy of diagnosing diseases and monitoring disease progression at the level of phospholipid metabolism will further improve. Through the regulation of the phospholipid metabolic network within the body, it is hoped that the treatment of diseases can be achieved.
Our Phospholipid LC-MS Analysis Platform
Currently, a reliable and reproducible method using highly sensitive LC-MS/MS platform for the identification and quantification of diverse phospholipid species in different sample types has been established by the scientists at Creative Proteomics, which can satisfy the needs of academic and industrial study in your lab.
Platform
- LC-MS/MS
Summary
Identification and quantification of diverse phospholipids by mixed organic solvent extraction in cells or tissue. These phospholipid species can be measured in a Phospholipid Panel by targeted LC-MS/MS or by shotgun approaches.
Sample Requirement
| Sample Type | Lipidomics |
| Animal Tissue | 100-200 mg |
| Plant Tissue | 100-200 mg |
| Plasma/Serum | >100 μL |
| Urine | 200-500 μL |
| Saliva, Amniotic fluid, Bile, Tears, etc. | >200 μL |
| Cells | >1*107 |
| Culture Supernatant | >2 mL |
| Wastewater/Culture Medium | >2 mL |
| Microbial Culture | >2 mL |
| Feces/Intestinal Contents | 100-200 mg |
| Soil Sample | >1 g |
| Swab | 2 |
Customized Phospholipid Purity
Customizing phospholipid purity involves selecting high-quality raw materials, employing extraction and purification techniques, fractionating, and adjusting concentrations to meet specific application requirements.
Report
- A full report including all raw data, MS/MS instrument parameters and step-by-step calculations will be provided (Excel and PDF formats).
- Analytes are reported as uM or ug/mg (tissue), and CV's are generally<10%
| Phospholipid Classes | Bioactive Phospholipids |
|---|---|
| Phosphatidic acids (PA) | PAF (platelet activating factor) |
| Bismonoacylglycerophosphates (BMP) | Lyso-PA |
| Phosphatidylglycerols (PG) | Lyso-PS |
| Phosphatidylethanolamines (PE) | Lyso-PC |
| Phosphatidylcholines (PC) | S-1-P (Sphingosine-1-Phosphate) |
| Phosphatidylinositols (PI) | |
| Phosphatidylserines (PS) | |
| Cardiolipins (CL) | |
| Sphingomyelins (SM) |
| Phospholipids | ||
|---|---|---|
| Phosphatidic acid (PA) | Phosphatidylethanol (PEtOH) (or phosphatidylbutanol, PBuOH) | Phosphatidylglycerol (PG) |
| Phosphatidylcholine (PC) | Platelet-activating factor (PAF) | Phosphatidylethanolamine (PE) |
| Phosphatidylinositol (PI) | Monophosphorylated phosphatidylinositol (PIP) | Bisphosphorylated phosphatidylinositol (PIP2) |
| Trisphosphorylated phosphatidylinositol (PIP3) | Phosphatidylserine (PS) | Cardiolipin (CL) |
| The corresponding lyso-phospholipids (LPL) | The corresponding ether lipids | |
Ordering Procedure:
*If your organization requires signing of a confidentiality agreement, please contact us by email.
Staffed by experienced biological scientists, Creative Proteomicscan provide a wide range of services ranging from the sample preparation to the lipid extraction, characterization, identification and quantification. We promise accurate and reliable analysis, in shorter duration of time! You are welcome to discuss your project with us.
Reference
- Sen Yang, Jingyuan Xue, Cunqi Ye.Protocol for rapid and accurate quantification of phospholipids in yeast and mammalian systems using LC-MS.STAR Protocols. 2022
PCA plot
Heatmap plot
Volcano plot
Correlation dot plot
Title: Plasmalogen Profiling in Porcine Brain Tissues by LC-MS/MS
Journal: Foods
Published: 2023
Background
This study explores the profiling of plasmalogens, a unique class of glycerophospholipids, in porcine brain tissues using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Plasmalogens are known for their health benefits, including anti-oxidative and anti-inflammatory properties. The study investigates the potential of plasmalogen extraction from porcine brain for developing health food products, focusing on how different industrial processing steps impact plasmalogen content and composition.
Materials & Methods
Sample Collection and Processing
Various porcine brain products were collected and processed using methods including freeze-drying, acetone precipitation, and ethanol extraction to yield different types of glycerophospholipid products. Comparative analysis included commercially available egg and soy-derived lecithin samples.
Chemical Analysis
LC-MS/MS analysis was performed using an Atlantis T3 C18 column coupled with an LTQ Orbitrap mass spectrometer. Solvents and reagents were sourced from reputable suppliers, and plasmalogen species were identified based on their unique fragmentation patterns in negative ion mode.
Statistical and Nutritional Analysis
The nutritional value was assessed using indexes such as the PUFA/SFA ratio, n-6/n-3 ratio, and thrombogenicity index, calculated from the fatty acyl composition of the plasmalogens.
Results
LC-MS/MS Analysis and Identification of Plasmalogen Species
Through LC-MS/MS analysis, the authors observed the major ion peaks of different plasmalogen species in porcine brain tissue, demonstrating effective separation of these molecules. They further used high-resolution mass spectrometry and tandem mass spectrometry to identify plasmalogen species, using PlsEtn 34:2 as an example to detail the fragmentation patterns of the fatty acyl chains. These analyses showcase the capability of high-resolution mass spectrometry to effectively identify and quantify plasmalogen molecules in porcine brain tissue.
Figure 6.Identification of plasmalogen species in porcine brain tissue using high-resolution Orbitrap MS.
Characteristics of Plasmalogen Profile
By combining hierarchical cluster analysis (HCA) and principal component analysis (PCA), the authors identified significant differences in the composition of sphingolipids among different samples. The results showed that egg-derived lecithin products were clearly distinguished from those derived from pig brain, with pig brain products being richer in various unsaturated sphingolipid species. Furthermore, different processing methods have a certain impact on the composition of sphingolipids; for instance, ethanol extraction and concentration steps may alter the proportions of sphingolipid molecular species. These analytical results suggest that industrial processing can stabilize the sphingolipid composition in pig brain products, contributing to improved consistency and quality control of the products.
Figure 7.Multivariate statistics of dietary phospholipid samples containing plasmalogens.
Conclusion
The study confirms the feasibility of extracting plasmalogens from porcine brain tissues for health product development. These findings suggest strategies for enhancing the quality and nutritional value of plasmalogen-based supplements.
Reference
- Wu, Y., Chen, Y., Zhang, M., Chiba, H., & Hui, S.-P. (2023). Plasmalogen Profiling in Porcine Brain Tissues by LC-MS/MS. Foods, 12(8), 2990. https://doi.org/10.3390/foods12162990
How to test the phosphate in lipids?
Testing for phosphate in lipids can be conducted using various methods, including colorimetric assays like Mohr's method and the ascorbic acid method, which measure the absorbance of colored complexes formed with molybdate. Spectrophotometry, particularly UV-Vis spectrophotometry, is used to quantify phosphate after hydrolysis of the lipids. Chromatographic techniques such as high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), and ion chromatography allow for the separation and quantification of phosphates, while enzymatic methods can also be employed for analysis. Nuclear magnetic resonance (NMR) spectroscopy provides structural insights into phosphate-containing lipids. Effective testing requires lipid extraction, potential hydrolysis to release phosphates, and calibration with known standards for accurate quantification.
How do phospholipids and amino acids linked together？
Certain phospholipids can form covalent bonds with amino acids. For example, phosphatidylserine (PS) includes the amino acid serine as its head group. In this case, the hydroxyl group (-OH) of serine is linked to the phosphate group of the phospholipid. In membrane proteins, hydrophobic amino acids can interact with the hydrophobic fatty acid tails of phospholipids, anchoring the protein within the lipid bilayer without the need for direct chemical bonds.
What categories are there for phospholipid MS analysis?
Phospholipid analysis using mass spectrometry (MS) can be divided into two main categories: targeted lipidomics and untargeted lipidomics analysis. Targeted analysis focuses on specific phospholipid species, employing techniques such as Selected Reaction Monitoring (SRM), Multiple Reaction Monitoring (MRM), and Tandem MS (MS/MS) for quantification and identification. This approach is particularly useful for studying lipid metabolism, cell signaling, and monitoring specific lipid biomarkers. In contrast, untargeted analysis aims to profile all phospholipid species in a sample without prior knowledge of their identities, utilizing techniques like Shotgun Lipidomics and LC-MS. This method is ideal for comprehensive lipid profiling and discovering novel phospholipid species. Overall, both targeted and untargeted MS approaches are essential for phospholipid analysis, with the choice depending on the research goals and the complexity of the sample.
What is phospholipid quantification?
Phospholipid quantification is the measurement of phospholipid concentrations and compositions in biological samples.Accurate quantification requires proper sample preparation and calibration with known standards. This process is important for studying lipid metabolism, drug effects, and cellular signaling.
How can phospholipids be separated experimentally?
Phospholipids can be separated experimentally through various methods, including extraction and analysis techniques. The extraction process typically involves using solvents like chloroform and methanol to isolate phospholipids from biological tissues, cells, or plasma. Then the phospholipid analysis mass spectrometrys are used for separation
Annexin A2 modulates phospholipid membrane composition upstream of Arp2 to control angiogenic sprout initiation
Timothy M. Sveeggen, Colette A. Abbey, et al
Journal:The FASEB Journal
Year：2022
Loss of G0/G1 switch gene 2 (G0S2) promotes disease progression and drug resistance in chronic myeloid leukaemia (CML) by disrupting glycerophospholipid metabolism
Gonzalez, M. A., Olivas, I. M., et al.
Journal:Clinical and Translational Medicine
Year：2022
