Lipidomics, a branch of metabolomics, is a systems-based study of all lipid molecules (>30,000 individual species) within a biological system, tissue, fluid or cell. Lipids are the main structural components of biological membranes, a major form of energy storage in living cells, but are also well-known mediators of cell signaling. The lipidome is highly complex, consisting of eight major categories, over 80 major classes, 300 sub-classes, and thousands of lipid species[1] spanning a wide range of concentrations. In order to understand cellular physiology and pathophysiology, comprehensive identification and precise quantification of lipids is crucial in lipidomics research.[2]
Recent advances in high-performance liquid chromatography-mass spectrometry (HPLC-MS) platforms allow for rapid and sensitive detection of a variety of lipid species. One of the primary strategies undertaken in MS-based workflows is referred to as untargeted or discovery lipidomics. This strategy involves unbiased qualitative and quantitative analysis of a lipidome by analyzing an entire lipid extract by either LC-MS or direct infusion-MS without prerequisite targets.
An untargeted lipidomics approach makes it possible to analyze several hundreds to thousands of individual lipid species that may be valuable to assess an individual’s health status. Such detailed lipid profiles are very useful for assessing medical risks, monitoring and diagnosing patient treatments and are the basis for the concept of personalized medicine. Applications of untargeted lipidomics include agro science; biomarkers; Alzheimer's, atherosclerosis, cardiovascular, cancer, diabetes and obesity disease research; clinical diagnostics; drug discovery; food safety; neonatal screening; nutrition; plant science and systems biology.
1 Fahy, E. et al. LIPID MAPS comprehensive classification system for lipids. J. Lipid Res. 2009, 50, S9-S14. doi: 10.1194/jlr.R800095-JLR200.
2 Watson, A.D. Lipidomics: a global approach to lipid analysis in biological systems. J. Lipid Res. 2006, 47, 2101
For additional resources, search the Orbitrap Science Library >
overview
Workflow Overview of Untargeted Lipidomics using HRAM HPLC-MSn Approach
Improved lipidomic technologies greatly enhance the knowledge gained about lipid functions at the individual species level. Thin layer chromatography (TLC), the classical standard in lipid analysis, is cheap and fast, but it is very limited when it comes to identification issues below the level of lipid classes. Mass spectrometry (MS) determines the accurate molecular weight and reveals detailed structural information of lipid mixtures with high sensitivity and specificity. Due to its sensitivity and selectivity MS has become the method of choice for qualitative and quantitative lipidomic analysis.
High resolution accurate mass (HR/AM) HPLC-MSn approaches are often used to separate many overlapping isomeric or isobaric molecular ions in order to simultaneously identify and quantify the thousands of cellular lipid molecular species and their interactions with other lipids, proteins, and other metabolites. The identification of each lipid species is carried out by using the HR/AM MS/MS and/or MSn data and the simultaneous quantitation of identified lipids is carried out by using accurate mass of each lipid within a +/- 5 ppm mass tolerance window.
It is critical that the lipidomics LC-MSn platform offers high resolving power on both HPLC separation and MS detection to resolve the many isobaric and isomeric species from biological lipid extracts. It is also critical that the LC/MS platform offers fast effective MS/MS scan speed and excellent mass accuracy to identify and quantify as many of the lipid species from the lipid extracts. The LC/MS platform also needs to offer high sensitivity and wide dynamic range for both MS and MS/MS in order to detect and quantify both low abundance and high abundance lipid species. The high resolving power offered by the Orbitrap analyzer in combination with HR/AM enables a wide range of lipid identification and precise quantitation from complex biological samples. Dedicated software is also needed for high throughput lipid identification and quantification.
sample Preparation
Sample Preparation
One of most popular procedures to extract the total lipids from any kind of organism, tissue or cell types is using a mixture of chloroform/methanol/water. [1,2]
Recently, a simple methyl tert-butyl ether-based extraction for high-throughput lipid extraction has been validated. [3]
references
1. A simple method for the isolation and purification of total lipides from animal tissues
J. Biol. Chem. 1957, 226: 497-509.
Folch, J., Lees, M. and Sloane Stanley, G.H.
2. A rapid method of total lipid extraction and purification
Bligh, E.G. and Dyer, W.J.
Can J Biochem Physiol. 1959, 37(8):911-917
3. D. Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics
J. Lipid Res. 2008, 49:(5) 1137-1146
Matyash, V., Liebisch, G., Kurzchalia, T.V., Shevchenko, A. and Schwudke,
ADDITIONAL RESOURCES
Thermo Scientific protocol for lipid extraction with chlorofoam (serum, plasma)
Mass Spectrometry
Mass Spectrometry
The characterization of lipids by mass spectrometry (MS) is influenced by their ionization efficiency and fragmentation patterns. Different ion polarities and activation modes are required for successful structural elucidation. For most lipid classes, higher-energy collisional dissociation (HCD) in the positive mode is more than sufficient for structural identification. However, for certain lipids species there is insufficient information within the HCD fragmentation for identification. In such cases, alternative fragmentation such as low energy collisional activated dissociation (CID) would be better suited as it can fill in the missing fragment ion information that is lacking in HCD. For certain lipid classes positive ion fragmentation, whether it is CID or HCD, might not be suitable and negative ion HCD or CID is required. Ideally, complete characterization of lipid species requires data acquisition in both positive and negative polarity modes. Furthermore, in the situation where isomeric lipid structures exist additional stages of multistage fragmentation (MSn) data are needed to determine each isomer’s unique molecular composition.
The recent development of a novel intelligent acquisition strategy that combines both modes of polarity and the ability to trigger multiple lipid precursor ion dissociation techniques (HCD MS2, CID MS2 and CID MS3) within a single LC-MS run presents a powerful approach for lipid characterization. In this method, alternating data-dependent positive ion mode HCD MS2 and negative ion mode HCD MS2 data are acquired. A single additional positive ion CID MS2 spectrum is collected if a diagnostic fragment ion (184.0733) from a phosphocholine is detected in the positive HCD MS2 scan. This fragment ion is indicative of phosphocholine head group and CID fragmentation can provide the information that is lacking in HCD spectrum. Additionally, further CID MS3 data are collected for top three HCD fragment ions if fatty acid neutral losses are detected from the HCD MS2 spectrum of ammoniated triglycerides. The primary advantage of this new intelligent workflow is that it maximizes the complete identification of lipid species in a single analysis.
ADDITIONAL RESOURCES
Increased Throughput, Sensitivity, and Confidence for Metabolomics Research Using a New Tribrid Orbi...
Increased Identification Coverage and Throughput for Complex Lipidomes
Data Analysis
Data Analysis
Thermo Scientific LipidSearch software enables analysis of lipid data acquired on Thermo Scientific mass spectrometers. Candidate molecular species are identified by searching a large database > 10E+7 entries of accurate masses (lipid precursor and fragment ions) predicted from each potential lipid structure and positive/negative ion adducts. The search results obtained for each individual sample are aligned within a time window and the results are merged into a single report. Additionally, LipidSearch software enables relative quantitation as well as estimated quantitation provided samples are spiked with internal standard.
ADDITIONAL RESOURCES
A demonstration version of LipidSearch software can be downloaded from here.
Demonstration of lipidomics platform including LipidSearch software for simultaneous profiling and identification of lipids obtained from mouse liver treated with Fumonisin B1 vs. control can be accessed here.
Thermo Fisher Scientific
United States
