Glycerophospholipid Identification and Quantitation by Electrospray Ionization Mass Spectrometry

Specific roles of phosphatidylglycerols in hosts and microbes

“…At neutral pH, PG is in the anionic form, so that mild acidification of the extraction solvent allows the protonation of PG lipids and a better extraction yield. The modified Bligh and Dyer method developed by Ivanova et al with mild acidification (0.1N HCl:methanol (1:1)) of the methanol is therefore very efficient for acidic lipids such as PG (4). As for all other glycerolipids, PGs polarity allows their detection using electrospray ionization, but the fast ESI-MS/MS shotgun approach from crude extracts is however not appropriate for minor lipids such as PG whose signal gets easily suppressed by more abundant lipids in the ESI source.…”

“…As for all other glycerolipids, PGs polarity allows their detection using electrospray ionization, but the fast ESI-MS/MS shotgun approach from crude extracts is however not appropriate for minor lipids such as PG whose signal gets easily suppressed by more abundant lipids in the ESI source. PG analysis using MS/MS therefore requires prior separation by liquid chromatography, that can be achieved with hydrophobicity-based separation (chain length and unsaturation) using reversed-phase LC columns (5) or polarity-based separation depending on M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT headgroup using normal phase columns (4). The latter chromatography uses however hazardous apolar solvents such as chloroform or hexane and tends to get replaced by hydrophilic interaction chromatography (HILIC), which allows a normal-phase type separation using reversed-phase type solvents at a relatively fast speed with the development of core-shell technology (6).…”

Regulation of Phospholipase D Activity and Phosphatidic Acid Production after Purinergic (P2Y6) Receptor Stimulation

“…Here UDP was used to stimulate the P2Y 6 receptor in a dose-dependent manner. After ligand stimulation, PA formation was measured using quantitative lipidomic analysis developed in our laboratory (25,27). PLD activation was also determined using a PLDspecific transphosphatidylation reaction with MS detection (29).…”

“…Glycerophospholipids were extracted using a modified Bligh and Dyer procedure as previously described (25). Briefly, cells were plated in 6-well tissue culture plates at 300,000 cells/well 48 h before the experiment in complete growth medium.…”

“…Glycerophospholipids (including phosphatidylbutanol) were analyzed on an Applied Biosystems/MDS SCIEX 4000 Q TRAP hybrid triple quadrupole/linear ion trap mass spectrometer (Applied Biosystems, Foster City, CA) equipped with a Shimadzu high pressure liquid chromatography system with a Phenomenex Luna Silica column (2 ϫ 250 mm, 5-m particle size) using a gradient elution as previously described (25,27). The identification of the individual species, accomplished by LC/MS/MS, was based on their chromatographic and mass spectral characteristics.…”