Quantitative Lipidomics - IMBIO Institute, University of Bonn
The IMBIO Institute, (Prof. Dr. Dörmann), provides a lipid analytical platform for the campus of the University of Bonn. We measure a large variety of lipids in a quantitative manner employing state-of-the art analytical equipment, including fluorescence HPLC, gas chromatography (GC), GC-MS and Q-TOF (quadrupole time of flight) mass spectrometry.
Expertise
Lipids are nonpolar metabolites that require specialized extraction protocols employing organic solvents. The core facility Quantitative Lipidomics provides services for the measurement of a large variety of lipids and other small molecules in a quantitative manner employing state-of-the-art analytical equipment, including Q-TOF (quadrupole time of flight) mass spectrometry and QQQ (triple quadrupole) mass spectrometry, gas chromatography (GC) with flame ionization (FID) or mass spectrometric detection (MSD) and HPLC with fluorescence or UV detection.
We include internal standards for the absolute measurements for the respective lipid classes (Table 1). In addition to the major classes of glycerolipids (phospholipids, galactolipids, triacylglycerol, diacylglycerol), sphingolipids (GIPC, gangliosides, hexosylceramides, ceramides, sphingomyelin) and sterol lipids (free sterols, steryl esters, steryl glucosides, acylated steryl glucosides), we also provide protocols for the measurements of fatty acids, lipid vitamins (tocochromanols, phylloquinone, carotenoids). We focus on plants and bacteria, but also work with animal/human samples on a collaborative basis.
Quantification Workflows
To ensure accurate quantification, we employ optimized sample preparation workflows (see Table 1) as well as internal standards for each individual lipid class.
Data storage
The data and results are recorded by the lipidomics facility and stored digitally. The
measurement data are made available to the users. Users are instructed to transfer
the data to another location as soon as possible.
Together with the network administration, the lipidomics facility ensures the operation of the IT infrastructure and the availability of the data within the scope of the technical and personnel resources.
The archiving of the data remains with the responsibility of the user, as the owner of the data.
The data will not be passed on to third parties.
Instrumentation
| Instrument | Comment | Commissioning |
| Agilent 6530 Q-TOF | LC-ESI-MS und nanospray-MS | 2008 |
| AB/Sciex 6500+ Q-Trap | LC-ESI-MS | 2017 |
| Agilent 6546 Q-TOF | LC-ESI-MS und CE (capillary electrophoresis)-MS | 2023 |
| Agilent 7100 Capillary Electrophoresis (CE) | CE-MS | 2023 |
| Agilent ChipCube | Nanospray source | 2008 |
| TriVersa NanoMate | Nanoflow and nanospray source | 2017 |
| Agilent GC 7890A | GC, split/splitless, flame ionization etector | 2008 |
| Agilent GC 7890A with 5975 MSD | GC-MS, split/splitless, quadrupole mass detector | 2008 |
| Agilent 1200 HPLC | Diode array detector (DAD) and fluorescence detector (FLD) | 2008 |
Safety instructions
The following safety and health-related issues must be observed:
- Health and safety regulations (also with regard to the use of vacuum pumps, etc.),
- Laws relevant to genetic engineering (S1 Gentechnik),
- Disposal of chemical waste,
- Before booking and using, the user must ensure that special legal requirements for the intended use must be observed.
Confidentiality and protection of intellectual property
The work of the facility and their users is considered their intellectual property and must therefore be treated confidentially, unless agreed otherwise.
Unless otherwise agreed, all rights, titles and interests in the self-developed technology (e.g. experiments) remain with the lipidomics facility, including all copyrights and patent rights, rights to trade secrets and other intellectual property in connection with ideas, concepts, methods, procedures, techniques and inventions or copyrighted works (including algorithms, programs and documentation) developed by the facility.
Methods
Lipids measured by the Lipidomics Platform.
(Letters in brackets indicate cost calculation).
Glycerolipids are isolated from leaves, bacteria and fungi using the boiling water protocol, or from animal tissues or from plant roots with acidic chloroform/methanol extraction. Lipid isolation should in principle be performed by the collaborating scientists with assistance by the core facility.
SPE on silica columns is used to separate neutral lipids (triacylglycerol, diacylglycerol), glycolipids (galactolipids, hexosylceramides), phospholipids and lyso-phospholipids, and for the separation of sterol lipid classes from plants. SPE separation should alsp be performed by the collaborating scientists with assistance by the core facility.
Phospholipid and galactolipid from plants, bacteria and fungi are isolated using the boiling water protocol or the acidic chloroform/methanol extraction and measured by tandem mass spectrometry (Q-TOF or QQQ). Lipids are quantified by MS/MS experiments with internal standards following the strategy developed by Ruth Welti (Kansas State University) (see: Gasulla et al., 2013, doi:10.1111/tpj.12241)).
After lipid isolation, lysophospholipids (e.g. lyso-PC) are purified by SPE on silica columns prior to measurement by direct infusion Q-TOF MS/MS.
The sphingolipid composition from animals (ceramide, hexosylceramide, sphingomyelin, gangliosides, sulfatides) can be measured after alkaline hydrolysis of phospholipids.
Neutral and acidic sphingolipids are separated by solid phase extraction and quantified by direct infusion Q-TOF MS/MS.
Plant sphingolipids in crude lipid extracts are purified by alkaline hydrolysis of phospholipids and galactolipids. Glucosylceramides from plants can be measured by direct infusion Q-TOF MS/MS after SPE on silica columns.
The full spectrum of sphingolipids (ceramides, glucosylceramides, sphingobases, GIPC) can be measured by LC-MS/MS following a protocol developed by Jonathan Markham (University of Lincoln Nebraska).
Triacylglycerol and diacylglycerol are isolated from the tissue using the boiling water protocol or the acidic chloroform/methanol extraction and then separated from polar lipids by SPE on silica columns.
Triacylglycerol and diacylglycerol are quantified by direct infusion mass spectrometry (Q-TOF) (Lippold et al., 2012, doi:10.1105/tpc.112.095588; vom Dorp et al., 2013, doi:10.1007/978-1-62703-401-2_5).
Total fatty acids in biological samples can be quantified after conversion of all acyl groups into fatty acid methyl esters (FAMEs). The methyl esters are quantified by gas chromatography (GC) with flame ionization detector using pentadecanoic acid (15:0) as internal standard. For structural identification, fatty acid methyl esters are analyzed by gas chromatography-mass spectrometry (GC-MS).
Free sterols can be measured by GC-MS after silylation, and total sterols (free and conjugated) sterols can be measured in the same way after acidic/alkaline hydrolysis and silylation (A, E, F)
In order to analyze the full spectrum of free and conjugated sterols, the sterol lipid classes are separated by SPE on silica columns into a polar fraction (only present in plants and some fungi: sterol glucosides, acylated sterol glucosides) and a non-polar fraction (free sterols, sterol esters). These fractions are measured separately by direct infusion Q-TOF MS (A, B, C).
Free sterols need to be derivatized e.g. with chlorobetainyl chloride prior to quantification (Wewer et al., 2010).
Usage costs
The use of the lipidomics facility is chargeable. Costs for the use of devices for mass spectrometry are based on the flat rates from the DFG form 55.04. "Notes on device
usage costs and device centers".
Lipidomics Publications
Publications (starting 2019) with participation of the Core Facility Quantitative Lipidomics at the Institute of Molecular Physiology and Biotechnology of Pants (IMBIO).
Localization and structure
Institute of Molecular Physiology and Biotechnology of Plants (IMBIO)
Karlrobert-Kreiten-Straße 13
53115 Bonn (Germany)
Location
Professor Dr. Peter Dörmann
Dr. Katharina Gutbrod
Helga Peisker