Analysis of Phospholipids, Lysophospholipids, and Their Linked Fatty Acyl Chains in Yellow Lupin Seeds (Lupinus luteus L.) by Liquid Chromatography and Tandem Mass Spectrometry.

Hydrophilic interaction liquid chromatography (HILIC) and electrospray ionization (ESI) coupled to either Fourier-transform (FT) orbital-trap or linear ion-trap tandem mass spectrometry (LIT-MS/MS) was used to characterize the phospholipidome of yellow lupin (Lupinus luteus) seeds. Phosphatidylcholines (PC) were the most abundant species (41 ± 6%), which were followed by lyso-forms LPC (30 ± 11%), phosphatidylethanolamines (PE, 13 ± 4%), phosphatidylglycerols (PG, 5.1 ± 1.7%), phosphatidic acids (PA, 4.9 ± 1.8%), phosphatidylinositols (PI, 4.7 ± 1.1%), and LPE (1.2 ± 0.5%). The occurrence of both isomeric forms of several LPC and LPE was inferred by a well-defined fragmentation pattern observed in negative ion mode. An unprecedented characterization of more than 200 polar lipids including 52 PC, 42 PE, 42 PA, 35 PG, 16 LPC, 13 LPE, and 10 PI, is reported. The most abundant fatty acids (FA) as esterified acyl chains in PL were 18:1 (oleic), 18:2 (linoleic), 16:0 (palmitic), and 18:3 (linolenic) with relatively high contents of long fatty acyl chains such as 22:0 (behenic), 24:0 (lignoceric), 20:1 (gondoic), and 22:1 (erucic). Their occurrence was confirmed by reversed-phase (RP) LC-ESI-FTMS analysis of a chemically hydrolyzed sample extract in acid conditions at 100 °C for 45 min.

Identification and quantification of phospholipids in strawberry seeds and pulp (Fragaria × ananassa cv San Andreas) by liquid chromatography with electrospray ionization and tandem mass spectrometry.

An extensive characterization and quantification of intact phospholipids (PLs) in strawberry (Fragaria × ananassa cv San Andreas) seed and pulp was carried out by hydrophilic interaction liquid chromatography (HILIC) and electrospray ionization (ESI) coupled to either Fourier-transform (FT) orbital-trap or linear ion-trap tandem mass spectrometry (LIT-MS/MS). More than 150 intact polar lipids including phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), phosphatidylglycerols (PGs), phosphatidic acids (PAs), phosphatidylinositols (PIs), lysophosphatidylcholines (LPCs), and lysophosphatidylethanolamines (LPEs) were identified in negative ESI mode. PC 18:2/18:2 and 18:2/18:3 were found to be the major components of strawberry lipid extracts at concentrations of 230 ± 36 and 189 ± 32 µg/g, respectively, in seeds and at concentrations of 330 ± 50 and 140 ± 22 µg/g, respectively, in pulp. The lipidic extracts of both strawberry seeds and pulp exhibited the dominance of LPC 16:0/0:0 at a content of 132 ± 19 and 114 ± 16 µg/g, respectively, and LPC 0:0/18:2 at 236 ± 20 and 150 ± 20 µg/g, respectively. The other most abundant species of strawberry seeds and pulp were PE 18:2/18:2, 40 ± 9 and 190 ± 40 µg/g, followed by PI 16:0/18:2, 51 ± 15 and 24 ± 8 µg/g, respectively, while PG, PA, and LPE show comparable abundance below 10 µg/g. The most recurrent fatty acyl substituents of PLs were C18:3 (α-linolenic acid), C18:2 (linoleic acid), C18:1 (oleic acid), C18:0 (stearic acid), C16:0 (palmitic acid), and relatively high contents of a shorter chain such as C14:0 (myristic acid).

Lipidomic analysis of Botryococcus (Trebouxiophyceae, Chlorophyta) - Identification of lipid classes containing very long chain fatty acids by offline two-dimensional LC-tandem MS.

Very long chain fatty acids (VLCFAs) were identified in four strains of the green alga Botryococcus braunii (Trebouxiophyceae). The algae contained a series of monoenoic fatty acids up to triacontenoic acid and further VLCFAs in amounts around 1% of total fatty acids. The separation of lipid classes using hydrophilic interaction chromatography revealed that the most abundant VLCFAs (28:2, 28:1 and 28:0) were contained in neutral lipids (triacylglycerols and/or diacylglycerols) and in phospholipids (phosphatidic acid and/or phosphatidylcholine). Using non-aqueous reversed-phase liquid chromatography tandem mass spectrometry (NARP-LC/MS2) of the appropriate collected fractions, molecular species of triacylglycerols containing one or two VLCFAs were described and phosphatidylcholines containing VLCFAs were separated for the first time. Because the presence of Botryosphaerella sudetica (Chlorophyceae) as contaminant of Botryococcus braunii strain Droop 1950/807-1 placed some doubts on the results of previous studies, a strain of this green alga of was also analyzed. In contrast to Botryococcus, C16, a substantially lower proportion of C18 polyunsaturated fatty acids and no VLCFAs were detected in Botryosphaerella.

Assessment of HDACi-Induced Acetylation of Nonhistone Proteins by Mass Spectrometry.

Posttranslational acetylation of lysine residues has been discovered as multifaceted regulatory modification for various nuclear, cytoplasmic, and mitochondrial proteins. The implementation of high-resolution and high-throughput mass spectrometry (MS) approaches has led to the identification of a hitherto underappreciated, large number of acetylation sites for a broad spectrum of cellular proteins. In this chapter, we describe a comprehensive protocol for the purification of an in vivo-acetylated, ectopically expressed, FLAG-epitope tagged nonhistone protein through immunoprecipitation (IP). The protocol also covers the sample preparation by SDS-PAGE, proteolytic digestion, and the analysis by LC-ESI MS. The success of this methodology, however, strongly depends on the physico-chemical properties of the respective protein(s) and the quality of selected peptide mass spectra.