The IMBIO Institute, AG Dörmann, provides a lipid analytical platform for the campus of the University of Bonn in the framework of the SFB645. 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 (Figure 1).
Figure 1: Equipment of the lipidomics platform at the IMBIO Institute, University of Bonn
From left to right: Agilent 1100 HPLC, Agilent GC and GC-MS, Agilent Q-TOF (quadrupole time-of flight) mass spectrometer equipped with Agilent Chipcube nanospray, APCI and ESI source.
Lipids measured by the lipidomics platform (Letters in brackets indicate cost calculation)
Lipid isolation (A)
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.
Solid phase extraction (SPE) on silica columns (B)
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.
Phospholipids/Galactolipids (membrane glycerolipids) from plants, bacteria and fungi (A, C)
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 MS/MS). Lipids are quantified by MS/MS experiments with internal standards following the strategy developed by Ruth Welti (Kansas State University) (also see: Gasulla et al., 2013).
Lyso-phospholipids (A, B, C)
After lipid isolation, lyso-phospholipids (lyso-PC) are purified by SPE on silica columns prior to measurement by direct infusion Q-TOF mass spectrometry.
Sphingolipids from animals (please contact us)
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.
Sphingolipids from Plants (please contact us)
Plant sphingolipids in crude lipid extracts are purified by alkaline hydrolysis of phospholipids and galactolipids. Glucosylceramides from plants can be measured by direct infusion with the Q-TOF mass spectrometer after SPE on silica columns.The full spectrum of sphingolipids (ceramide, glucosylceramide, sphingobase, GIPC) can be measured by LC-MS/MS following a protocol developed by Jonathan Markham (University of Lincoln Nebraska).
Triacylglycerol, Diacylglycerol (A, B, C)
Triacylglycerol and diacylglycerol are isolated from the sample 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; vom Dorp et al., 2013).
Fatty acids (E, F)
Total fatty acids in biological samples can be quantified after conversion of all acyl groups into fatty acid methyl esters. The methyl esters are quantified by gas chromatography (GC) with flame ionization detector using pendadecanoic acid (15:0) as internal standard. For structural identification, fatty acid methyl esters are analyzed by gas chromatography-mass spectrometry (GC-MS).
Sterol lipids (A, B, C, E, F)
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).
vom Dorp K., Briesen I. and Dörmann P. (2013) Quantification of diacylglycerol by mass spectrometry (Chapter 5). T. Munnik and I. Heilmann, eds., Plant Lipid Signaling Protocols, Methods in Molecular Biology Vol. 1009, pp. 43-54
Gasulla F., vom Dorp K., Dombrink I., Zähringer U., Gisch N., Dörmann P. and Bartels D. (2013) The role of lipid metabolism in the acquisition of desiccation-tolerance in Craterostigma plantagineum: A comparative approach. Plant J. 75, 726-741
Lippold F., vom Dorp K., Abraham M., Hölzl G., Wewer V., Lindberg Yilmaz J., Lager I., Montandon C., Besagni C., Kessler F., Stymne S. and Dörmann P. (2012). Fatty acid phytyl ester synthesis in chloroplasts of Arabidopsis thaliana. Plant Cell 24, 2001–2014.
Wewer V., Dombrink I., vom Dorp K. and Dörmann P. (2011) Quantification of sterol lipids in plants by quadrupole time of flight mass spectrometry. J. Lipid Res. 52, 1039-1054
Wewer V., Brands M. and Dörmann P. (2014) Fatty acid synthesis and lipid metabolism in the obligate biotrophic fungus Rhizophagus irregularis during mycorrhization of Lotus japonicus. Plant J. 79, 398-412
Zbierzak A.M., Kanwischer M., Wille C., Vidi P.A., Giavalisco P., Lohmann A., Briesen I., Porfirova S., Bréhélin C., Kessler F. and Dörmann P. (2010) Intersection of the tocopherol and plastoquinol metabolic pathways at the plastoglobule. Biochem J. 425, 389-399