Sample preparation
Microbial cultures were grown in specific media and placed on an orbital shaker. Cell shape and sizes may be observed using a light microscope. Usually, cells were harvested in early or mid-stationary phase by centrifugation. Hence, the turbidity of the media (reflecting cell density) was measured at a certain wavelength. Following, the cell pellets were washed with basal salt, vortexed, centrifuged and supernatant discarded a second time. Finally, the cell pellets were frozen overnight and subsequently lyophilized prior lipid extraction. Lipids were extracted according to a modified Bligh and Dyer extraction protocol. An aliquot of the total lipid extract (TLE) was dissolved in organic solvents and analyzed via HPLC-ESI-MS. Lipid identification was achieved by analyzing the exact masses of possible precursor ions in combination with their characteristic fragmentation patterns.
State-of-the-art techniques on lipid analysis
Thousands of different lipids are found in eukaryotic and prokaryotic cells. This data suggests that there must exist evolutionary advantages linked to this complex lipid repertoire and that the diversity of lipid structures may support diverse cellular functions. In recent years, advances in mass spectrometry (MS) have been one of the major drivers that have propelled the field of lipidomics. Notably, the application of electrospray ionization (ESI) to crude lipid extracts without prior fractionation—the so-called shotgun approach—is one major example of these advances.
The field of lipidomics is concerned with the identification and quantification of lipids within a given biological system, but ultimately also seeks to elucidate how individual molecular species affect lipid metabolism and the function (or dysfunction) of the system as a whole.
Mass spectra
Here we showcase the identification of compounds by MS/MS during HPLC-ESI-MS. Although, MS/MS mass spectra of several lipids are available elsewhere (http://www.lipidmaps.org or http://fiehnlab.ucdavis.edu/projects/LipidBlast) our focus in this section is the fragmentation patterns of particularly archaeal polar lipids, membrane-associated lipids (e.g. quinones, squalenes, carotenoids, etc) and storage lipids (TAGs, cholesteryl-esters, etc.), which are rarely reported in the literature. Our goal is to update the list of compounds as soon as novel lipids are identified.
