Quantitative Analysis of Yeast Phospholipids and Sterols by High-Performance Liquid Chromatography-Evaporative Light-Scattering Detection.

Normal-phase high-performance liquid chromatography (HPLC) is a standard method for separating the major lipid classes in an extract. Owing to the absence of a common property like light absorbance in the various lipid classes, evaporative light-scattering detection (ELSD) is the method of choice for qualitative and quantitative lipid detection. In most cases, neutral lipids and polar lipids are separated by different solvent systems, making it necessary to perform multiple analyses.

Derivatization and Gas Chromatography of Fatty Acids from Yeast.

Analysis of fatty acids by gas chromatography (GC) is facilitated by the generation of volatile fatty acid methyl ester (FAME) derivatives. Here we describe the esterification procedure and a typical program for separating FAMEs using a gas chromatograph equipped with a flame ionization detector or a dual stage quadrupole-mass spectrometry detector. GC is a rather simple technique that provides quantitative information on cellular fatty acid content and composition by use of an internal fatty acid standard.

Thin-Layer Chromatography to Separate Phospholipids and Neutral Lipids from Yeast.

Thin-layer chromatography (TLC) is a versatile technique for the separation of lipid classes. It provides excellent resolution, can be used for both preparative and analytical applications, and does not require expensive equipment. Here we describe the use of different solvent systems to separate yeast phospholipids and neutral lipids by TLC in one dimension.

Lipid Extraction from Yeast Cells.

The diversity of lipid molecules in biological tissues makes their analysis an experimental challenge. Not only do lipids differ greatly in their chemical structures and biophysical properties, but they also occur in greatly varying concentrations in living cells. Accordingly, even for an organism with a relatively simple lipidome such as yeast, multiple extraction and analysis protocols have been developed because none of them allows comprehensive and quantitative determination of all the diverse molecular lipid species.

Seipin is involved in the regulation of phosphatidic acid metabolism at a subdomain of the nuclear envelope in yeast.

Yeast Fld1 and Ldb16 resemble mammalian seipin, implicated in neutral lipid storage. Both proteins form a complex at the endoplasmic reticulum-lipid droplet (LD) interface. Malfunction of this complex either leads to LD clustering or to the generation of supersized LD (SLD) in close vicinity to the nuclear envelope, in response to altered phospholipid (PL) composition.

Regulation of gene expression through a transcriptional repressor that senses acyl-chain length in membrane phospholipids.

Membrane phospholipids typically contain fatty acids (FAs) of 16 and 18 carbon atoms. This particular chain length is evolutionarily highly conserved and presumably provides maximum stability and dynamic properties to biological membranes in response to nutritional or environmental cues. Here, we show that the relative proportion of C16 versus C18 FAs is regulated by the activity of acetyl-CoA carboxylase (Acc1), the first and rate-limiting enzyme of FA de novo synthesis.

A versatile ultra-high performance LC-MS method for lipid profiling.

A new UPLC-based untargeted lipidomic approach using a qTOF hybrid mass spectrometer is introduced. The applied binary gradient enables separations of lipid species including constitutional isomeric compounds and low abundant lipid classes such as phosphatidic acid (PA). Addition of phosphoric acid to the solvents improves peak shapes for acidic phospholipids.