| dct.abstract |
Lipidomics or global lipid profiling is a branch of metabolomics that aims to comprehensive analysis of lipids in a biological system. Lipidomics has become an important research field, since the increased awareness of lipid functions in cell and their role in many common diseases. Also the development of analytical methods, especially mass spectrometry has driven the progress of lipid profiling methods. Lipid profiling allows detection and quantitation of hundreds of intact lipid species in parallel. Challenges in lipidomics are the diversity of different lipid structures and varying lipid concentrations in biological samples. Despite of the development of lipidomics there is not yet a single analytical method to screen all lipids in a biological system. Therefore targeted lipidomics methods are needed in addition to global lipidomics.
The literature part of this study presents different analytical methods used in lipidomics studies. The most popular lipid profiling methods are liquid chromatography-mass spectrometry (LC-MS) and direct infusion mass spectrometry or so called shotgun analysis. LC-MS based methods usually utilize reversed phase columns including octadecylsilane stationary phases, but also normal phase chromatography has been applied. In shotgun lipidomics crude extracts are infused directly to mass spectrometer without prior separation. In lipidomics, both unit resolution mass spectrometers like triple quadrupole and ion trap as well as high mass accuracy mass spectrometers like Fourier transform ion cyclotron resonance, Orbitrap, and time of flight mass spectrometers have been used. Mass scanning methods have provided full scan acquisition with accurate mass as well as tandem mass spectrometry like precursor ion scan, neutral loss scan, and multiple reaction monitoring. From the atmospheric pressure ionization techniques, electrospray ionization has been the most applied ionization, but also other soft ionization techniques such as matrix-assisted laser desorption/ionization has been employed. Sample pretreatment in global lipid profiling relies on homogenization and extraction with chloroform-methanol. The most used extraction methods are Folch and Blight and Dyer extractions or modifications of these. In addition, other sample pretreatment methods like solid phase extraction, derivatization, and thin layer chromatography have been used, especially with targeted lipidomics methods. Lipidomics platforms have been applied to several studies of different diseases like diabetes, schizophrenia, and cancer.
Many bioactive signaling lipids cannot be detected with lipid profiling methods, since they exist at low concentration and have polar structure. In the experimental part a targeted method was developed for analysis of signaling lipids by ultrahigh-performance liquid chromatography-mass spectrometry. Selected compounds were lysophosphatidic acid, sphingosine-1-phosphate, and phosphoinositides. Method development was mainly done with triple quadrupole mass spectrometer, but also Orbitrap was applied. Several different columns, eluent systems, and pretreatment methods were tested, as well as direct infusion. The best chromatographic separation to signaling lipids was achieved with a reversed phase column at alkaline conditions. However, with this method also several drawbacks were encountered. Peaks were shifting and broadening, there were carryover effects, and problems with repeatability and sensitivity. Direct infusion on the contrary turned out to be problematic because of the unstable electrospray and formation of lysophosphatidic acid from lysophosphocholine in the ionization chamber. Development of a single analytical method for these signaling lipids turned out to be a challenging task and a complete working method for all the studied compounds was not attained. |
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